Compositions and methods for inhibiting histone expression

Abstract

Histone H1.0 inhibitors, including combinations of siRNA, are described, along with their use for regulating chromatin condensation and treating fibrotic disease or disorder.

Description

COMPOSITIONS AND METHODS FOR INHIBITING HISTONE EXPRESSIONCROSS REFERENCE TO RELATED APPLICATIONSThis application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application No. 63 / 685, 431, filed August 21, 2024, entitled “COMPOSITIONS AND METHODS FOR INHIBITING HISTONE EXPRESSION”, which application is incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under HL150225, and HL105699 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0002] The varied physiological demands of different organ systems necessitate coping with a wide range of mechanical forces and extracellular signals. Fibroblasts are a specialized cell type present across most mammalian tissues that are responsible for synthesis of connective tissue. In adulthood, the actions of fibroblasts are essential to maintain tissue integrity and to respond to injury or cell death in various organs through a process that involves adoption of a myofibroblast phenoty pe. Activated myofibroblasts develop actin stress fibers, become contractile and synthesize extracellular matrix as part of a response that stiffens the tissue and heals wounds (Plikus et al., 2021, Cell, 184:3852-3872). The plasma membrane is connected to the extracellular matrix by a network of proteins that tether the cell within the organ, thereby facilitating communication between cells and relaying extracellular physical cues to the intracellular organelles. In situations of stress,extracellular cues in the form of physical forces, cytokines and hormones induce changes in transcription that alter the mechanical properties of the cell.

[0003] The nucleus itself has been shown to directly influence distensibility of the cell and respond to mechanical signals (Uhl er et al., 2017. Nature reviews. Molecular cell biology, 18:717-727). The nucleoskeleton is coupled to the cellular cytoskeleton, enabling force transduction and physical regulation of cellular compliance. Changes in nuclear deformability and histone post-translational modification have been show n to adaptively respond to mechanical stress, protecting the genome against aberrant gene expression (Nava et al., 2020, Cell, 181 :800-81). Changes in extracellular tension can directly impact nuclear flexibility and chromatin compaction in fibroblasts (Walker et al., 2021. Nat Biomed Eng, 12: 1485-1499) and the general compaction state of chromatin can influence the mechanical stability and activation state of the cell (Kalukula et al.. 2022, Molecular cell biology, 9:583-602). The functional unit of chromatin is an octamer of two copies each of histone H2A, H2B, H3 and H4 wrapped with -145-147 base pairs of DNA, together comprising a nucleosome (Kornberg et al.. 1974, Science, 184:868-871). Chromatin is then packaged into higher order structures, ranging from fibers comprised of dozens of nucleosomes and a few' kilobases of DNA, to topologically associated domains (thousands of nucleosomes and megabases of DNA) and nuclear territories (whole chromosomes) (Dekker et al., 2016, Cell, 164;1110-1121). The complex packaging rules that govern how the same genome is stored and retrieved differently across cells are known to involve the actions of histone modifying proteins, which post- translationally modify core histones, thereby priming the targeted regions of chromatin for tasks like DNA repair, replication, transcription and gene silencing (Wu et al., 2000, Trends in biochemical sciences, 25:619-623; Strahl et al., 2000, Nature, 403:41-45).

[0004] Because altered chromatin accessibility and gene expression have been shown to drive disease, there is a need in the art for improved methods of modulatinghistone abundance and chromatin condensation. This invention addresses this unmet need.SUMMARY OF THE INVENTION

[0005] Provided herein are histone Hl .0 inhibitors for preventing chromatin condensation and for treating or preventing fibrotic disease in a subject in need thereof.

[0006] In some embodiments, the present invention provides compositions comprising at least one histone H1.0 inhibitor.

[0007] In some embodiments, the composition comprises at least one antisense RNA molecule. In some embodiments, the antisense RNA molecule comprises an siRNA or shRNA molecule.

[0008] In some embodiments, the composition comprises an siRNA comprising the sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68, or a combination thereof.

[0009] In some embodiments, the composition comprises an shRNA comprising the sequence of SEQ ID NO:69.

[0010] In some embodiments, the present invention provides methods of modulating chromatin condensation in a cell, the method comprising administering a composition comprising at least one histone H1.0 inhibitor to the subject. In some embodiments, the histone H1.0 inhibitor comprises at least one antisense RNA molecule. In some embodiments, the antisense RNA molecule comprises an siRNA or shRNA molecule. In some embodiments, the histone H1.0 inhibitor comprises an siRNA comprising a sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:65. SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68, or a combination thereof. In some embodiments, the histone Hl .0 inhibitor comprises an shRNA comprising the sequence of SEQ ID NO:69.

[0011] In some embodiments, the method comprises decreasing the level of chromatin compaction in the cell.

[0012] In some embodiments, the cell comprises a fibroblast.

[0013] In some embodiments, the present invention provides methods of inhibiting stress-induced activation of fibroblasts, the method comprising administering a composition comprising at least one histone H1.0 inhibitor to the subject. In some embodiments, the histone H1.0 inhibitor comprises at least one antisense RNA molecule. In some embodiments, the antisense RNA molecule comprises an siRNA or shRNA molecule. In some embodiments, the histone H1.0 inhibitor comprises an siRNA comprising a sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO: 67, or SEQ ID NO: 68, or a combination thereof. In some embodiments, the histone H 1.0 inhibitor comprises an shRNA comprising the sequence of SEQ ID NO:69.

[0014] In some embodiments, the method comprises decreasing the level of chromatin compaction in the cell.

[0015] In some embodiments, the present invention provides methods of treating a disease or disorder in a subject in need thereof, the method comprising administering a composition comprising at least one histone H1.0 inhibitor to the subject. In some embodiments, the histone H1.0 inhibitor comprises at least one antisense RNA molecule. In some embodiments, the antisense RNA molecule comprises an siRNA or shRNA molecule. In some embodiments, the histone H1.0 inhibitor comprises an siRNA comprising a sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO: 67, or SEQ ID NO: 68, or a combination thereof. In some embodiments, the histone H1.0 inhibitor comprises an shRNA comprising the sequence of SEQ ID NO:69.

[0016] In some embodiments, the disease or disorder is a fibrotic disease or disorder.

[0017] In some embodiments, the disease or disorder is cardiac fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, lung fibrosis, asthma, COPD, Raynaud's phenomenon, pulmonary fibrosis, cirrhosis, liver cirrhosis, atrial fibrosis, endomyocardial fibrosis, arthrofi brosis, Crohn’s Disease, mediastinal fibrosis, myelofibrosis, tubulointerstitial fibrosis, hepatic fibrosis, premacular fibrosis, retinal fibrosis, dermal fibrosis, wound-associated fibrosis, Peyronie’s disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, fibroma, scleroderma, systemic scleroderma, Sjogren syndrome or kidney fibrosis.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0019] Figure 1, comprising Figure 1A through Figure IE, depicts data demonstrating that histone H1.0 is the principal histone Hl isoform in mouse fibroblasts. Heatmaps showing expression of Hl isoforms in fibroblasts from healthy murine tissue (Figure 1 A) and murine disease models (Figure IB). Data from fibroXplorer.com (Buechler et al., 2021). Figure 1C depicts a representative heatmap of single-cell RNA-seq data (Ren et al., 2020), showing average expression of each Hl isoform in murine cardiac cell types. Figure ID depicts representative aSMA and Periostin expression after TGF-P treatment as measured by Western blot (left). Quantification of Western blot (mean ± SD; Welch’s unpaired t-test) (right). Periostin and aSMA immunostaining in fibroblasts after TGF-P (10 ng / mL, 48h) (bottom) or vehicle control (nuclei stained with DAPI, scale bar=10pm; representative of n=4 biological replicates from separate isolations; mean ± SD; Welch’s unpaired t-test). Figure IE depicts a representative Western blot of histone Hl .0 protein abundance after fibroblast activation (24h, 48h, 72h, 96h 10 ng / mL TGF-P) (left). Immunoblot quantification (TGF-P signal as fold of Control; mean ± SEM; no significantdifferences; one replicate in d and e is a single isolation followed by treatment as indicated and western blotting) (right).

[0020] Figure 2, comprising Figure 2A through Figure 2G, depicts data demonstrating that histone H1.0 is necessary for stress-induced activation of fibroblast mechanical behaviors. Figure 2A depicts a representative treatment schematic (top left). aSMA and periostin protein levels after TGF-P treatment and effect of histone Hl.O KD (top right), along with quantification (bottom; one way ANOVA post-hoc Tukey test; mean ± SD; one replicate is a single isolation followed by treatment as indicated and western blotting). Figure 2B depicts representative immunofluorescence and quantification of periostin (top) and aSMA (bottom) protein localization in situ (DAPI stains cell nuclei; Scale bar=10pm; mean ± SD). Figure 2C depicts a representative traction force assay measuring force production at the single cell level (one replicate is an individual isolation and contraction force measurement). Figure 2D depicts a representative gel contraction assay (one replicate is an individual isolation and single well area calculation; one way ANOVA post-hoc Tukey test; mean ± SD). Figure 2E depicts a presentative Western blot demonstrating effect of histone Hl.O overexpression on periostin (left) and aSMA protein levels (right three panels, quantitation; Welch’s unpaired t-test; mean ± SD; one replicate is a single isolation followed by treatment as indicated and western blotting). Figure 2F depicts histone Hl.O overexpression and effect on gel contraction (left; quantification, right; Welch’s unpaired t-test; mean ± SD; one replicate is an individual isolation and single well area calculation). Figure 2G depicts representative absorbance measurements at 450 nm wavelength measure cell proliferation (mean ± SD with one-way ANOVA with a post-hoc Tukey test; one replicate is a single isolation followed by treatment as indicated and calculation of cell migration).

[0021] Figure 3, comprising Figure 3A through Figure 3D, depicts data demonstrating that histone H 1 .0 is necessary for transcriptional activation in response to TGF-p. Figure 3A depicts a representative heatmap of gene expression changes TGF-P vs. Scramble (left) and TGF-P + Hl.O KD vs. TGF-P (right; a subset of geneswith p-value < 0.01 were selected; Benjamin’ s-Hochberg adjusted Wald test p-value from the DESeq2 package). Figure 3B depicts Ingenuity Pathway Analysis (IP A) identifying genes significantly altered by TGF-P stimulation (left) and those whose expression is influenced by histone H1.0 KD prior to TGF-P (right). Upregulated genes are shown (left) (orange shading indicates activation, light / dark red indicate increased transcription following TGF-P treatment, orange lines represent predicted and measured activation). Expression of these same genes when histone H1.0 is depleted prior TGF-P versus TGF-P alone is shown on right, showing widespread inhibition of transcriptional changes (light / dark blue indicate decreased expression, light red indicates increased expression, and grey indicates no change). Blue lines indicate predicted and measured inhibition. Grey lines indicate no prediction of direction. Figure 3C depicts a representative heatmap showing effect of histone H1.0 KD on the expression pattern of extracellular matrix genes activated by TGF-P treatment. Figure 3D depicts a representative Western blot showing the effects of histone H1.0 KD on TGF-P-induced changes in RNA Pol II Ser2 phosphorylation (mean ± SD analyzed by one-way ANOVA with a post-hoc Tukey test; one replicate is a single isolation followed by treatment as indicated and western blotting).

[0022] Figure 4, comprising Figure 4A through Figure 4J, depicts data demonstrating that histone H1.0 depletion prevents TGF-P-induced histone H3K27Ac and modulates actions of HD AC 1 and BRD4. Figure 4A depicts a representative immunoblot showing changes in global H3K27Ac after histone H1.0 depletion (left) and quantification (right; mean ± SD, one-way ANOVA with a post-hoc Tukey test; one replicate is a single isolation followed by treatment as indicated and western blotting). Figure 4B depicts a stacked bar chart show ing direction of histone H1.0 KD-induced H3K27ac occupancy change (H1.0 KD + TGF-P relative to TGF-P treatment alone, y-axis) in regions undergoing significant (FDR < 0.05) change in H3K27ac with TGF-P alone (left). Black and grey coloring indicate regions undergoing reversed or enhanced H3K27ac occupancy in the knockdown condition, respectively. Heatmap depicting the log2FoldChange in H3K27ac in regions thatundergo a significant increase in H3K27ac occupancy with TGF- and are prevented with histone H1.0 KD (right, top). A visualization for the opposite behavior, regions with decreased H3K27ac occupancy that is prevented by histone H1.0 KD (right, bottom). Figure 4C depicts a representative Gene Ontology analysis of 300 unique genes closest to the regions whose increases in H3K27ac occupancy and increases in transcription after TGF-P are histone Hl.O-dependent (Fischer’s one tailed exact test, corrected by g:SCS algorithm and multiple comparison adjustment). Figure 4D depicts a representative Western blot of HDAC1 showing effect of histone H1.0 KD on TGF-P-induced Hdacl upregulation (left) and quantified (right; mean ± SD, oneway ANOVA with a post-hoc Tukey test). Figure 4E depicts fibroblasts were transfected with human Adv-HDACl or Adv-GFP for 48 hours and Hdacl protein levels were detected by Western blot (top) and quantified (bottom; mean ± SD, Welch’s unpaired t-test). Figure 4F depicts a representative Western blot showing changes in H3K27ac abundance after Hdacl overexpression (top) and quantification (bottom; mean ± SD, Welch’s unpaired t-test). Figure 4G depicts a representative coIP assay, performed with anti-FLAG antibody using lysates from Adv-GFP or Adv- GFP-H1.0-FLAG transfected fibroblasts confirms histone H1.0 interaction with Hdacl (representative of 5 independent Co-IP experiments). Figure 4H depicts data demonstrating the effect of histone H1.0 knockdow n on transcript BRD4 transcript levels (RNA-seq counts with Benjamin’ s-Hochberg adjusted Wald test p-value from the DESeq2 package), BRD4 protein (mean ± SD; one-way ANOVA with a post-hoc Tukey test) (Figure 41), and ChlP-qPCR against BRD4 in primary fibroblasts examining TGF-P-induced changes in BRD4 occupancy at the promoters of Sertad4 (left), Acta2, and Postn (right) and the effects of histone H1.0 depletion (mean ± SD; one-way ANOVA post-hoc Tukey test) (Figure 4 J).

[0023] Figure 5, comprising Figure 5A through Figure 5D, depicts data demonstrating that histone Hl .0 levels control chromatin fiber compaction. Figure 5 A depicts representative ChlP-seq examining histone H1.0 occupancy at transcription start sites (TSS) relative to other genomic regions (y-axis indicates log2(IP / Input)signal) at differentially transcribed genes: upregulated (green), down regulated (orange) or not differentially expressed (NDE, grey) (left). Labeling in bottom right of inset panel indicates the RNA-seq dataset comparisons. Quantification of the local minimum for each condition within each inset graph from ChlP-seq profiles on left (right). Figure 5B depicts representative ChlP-qPCR performed in isolated murine cardiac fibroblasts transfected with Adv-GFP (control) or Adv-GFP-Hl.O examining histone Hl .0 occupancy in the promoter region of the Periostin (top) Acta2, (middle) and Gapdh (bottom); mean ± SD, Welch’s unpaired t-test; one replicate is a single isolation followed by treatment as indicated and qPCR. Figure 5C depicts representative gel images of nuclease digested genomic DNA (wherein more compact chromatin will experience less digestion and migrate higher on the gel) from fibroblasts transfected with Adv-GFP-Hl.O or Adv-GFP control (first panel), histone H1.0 siRNA or scrambled siRNA control (second panel), treated with TGF-P (third panel), or treated with TGF-P in presence or absence of histone H1.0 (fourth panel). Quantification of genomic DNA in the 100-300bp range is shown next to each gel image (mean ± SD, one-way ANOVA with a post-hoc Tukey test; one replicate is a single isolation followed by treatment as indicated and gel densitometry). Figure 5D depicts the location of primers used for qPCR on DNA from fibroblasts after histone H1.0 overexpression or knockdown prior to TGF-P treatment (top). qPCR measured amount of DNA (less signal indicates less DNA and thus greater compaction of region in question; mean ± SEM, Welch’s unpaired t-test; one replicate is a single isolation followed by treatment as indicated, MNase digestion and qPCR) (bottom).

[0024] Figure 6, comprising Figure 6A through Figure 6F, depicts data demonstrating that histone H1.0 levels directly influence cellular stiffness and nuclear condensation. Figure 6A depicts a representative diagram of the cellular filtration assay (left) and effects of histone H1.0 depletion levels and TGF-P on cellular retention (right). 2 min applied pressure for KD and TGF-P; for Hl .0 overexpressing cells, >4 min were required because of increased stiffness (mean ± SD, one-way ANOVA with a post-hoc Tukey test [knockdown and TGF-P groups] or Welch’s t-test[H1.0 overexpression]; one replicate is a single isolation followed by treatment as indicated and cellular filtration measurement). Figure 6B depicts a representative heatmap depicting actin cytoskeleton genes and their expression after TGF-P in the presence or absence of histone H1.0. Figure 6C depicts DAPI staining of primary fibroblasts incubated under different osmotic environments for Ih (bar=l Opm) (left). Chromatin condensation parameter quantifies condensation induced by hypotonic conditions and decondensation induced by hypertonic conditions (violin plots indicate median and quartiles; one-way ANOVA with post-hoc Tukey test, representative of 3 biological replicates) (right). Figure 6D depicts DAPI staining of primary fibroblasts depleted of histone Hl.0 (left). Chromatin condensation parameter, same as in c (n=70 nuclei / group) (right). Figure 6E depicts DAPI staining of primary fibroblasts transfected with Adv-GFP-Hl.O or Adv-GFP control (left). Chromatin condensation parameter, same as in Figure 6C (right) (n=70 nuclei / group; violin plots mean and quartiles for Figures 6C-6E). Figure 6F depicts a representative schematic illustration of the effect of histone H1.0 abundance on chromatin compaction.

[0025] Figure 7, comprising Figure 7A through Figure 7F, depicts data demonstrating that histone H1.0 depletion prevents disease-associated cardiac fibrosis in vivo. Figure 7A depicts representative heart weight to body weight ratios (left) and E / A ratios (right). Figure 7B depicts a representative Western blot from whole heart showing histone H1.0 KD effect on ISO-induced periostin activation. Figure 7C depicts data demonstrating the quantification of Figure 7B. A positive Pearson's correlation was observed between histone H1.0 and periostin levels in hearts from C57BL / 6J mice. Figure 7D depicts representative RT-qPCR showing the effect of histone H1.0 KD on ISO-induced changes in Collal transcript abundance. Figure 7E depicts representative gel images of genomic DNA digested from hearts of C57BL / 6J mice. Figure 7F depicts Masson’s trichrome staining of heart sections to measure fibrosis (C3H / HeJ mice; bar in whole heart images = 1 mm; bar in zoomed images = 100pm; square indicates region from which high magnification images wereacquired) (left). Quantification of fibrotic area. All data are presented as mean ± SD, analyzed by one-way ANOVA with post-hoc Tukey test (right).

[0026] Figure 8, comprising Figure 8A through Figure 8F, depicts data demonstrating the relationship between histone H1.0 and periostin expression in human datasets. Figure 8A depicts representative heatmaps showing expression of Hl isoforms in fibroblasts from human disease models. Data from fibroXplorer.com (Buechler, et al. 2021. Nature. 593:575-579). Figure 8B depicts representative heatmaps showing the average expression of each Hl isoform in macrophages (MP), fibroblasts (FB), endothelial cells (EC) and cardiomyocytes (CM) from single-cell RNA-seq analysis of healthy human hearts (Wang et al. 2020, Nat. Cell Biol. 22: 108- 119). Figure 8C depicts data demonstrating a significant positive correlation was observed between H1F0 (histone H1.0) and POSTN (periostin) mRNA expression in the GTEx database, as calculated using the GEPIA web server (Tang et al.. Nucleic Acids Res. 2017;45:W98-W102) in both the human left ventricle (left) and left atrial appendage (right). Spearman correlation coefficients and p-values are shown. Figure 8D depicts data demonstrating a positive spearman correlation was also observed between H1F0 and POSTN mRNA expression in the fibroblast subset of a single nucleus RNA-seq dataset from human myocardial infarcted tissue (Kuppe et al.. Nature 2022;608:766-777). Normalized counts of H1F0 and POSTN are plotted from the 345 cardiac fibroblasts that express non-zero transcripts of both genes, colored by region of origin (BZ = Border Zone, CTRL = Control, FZ = Fibrotic Zone, IZ = Ischemic Zone, RZ = Remote Zone). Figure 8E depicts Spearman correlation between H1F0 and POSTN mRNA expression as calculated from the fibroblast subset of a single cell RNA-seq dataset from dilated cardiomyopathy or donor human hearts (Koenig et al. Nat Cardiovasc Res. 2022;1:263-280). Figure 8F depicts Spearman correlation between POSTN and H1F0 mRNA expression calculated from the fibroblast subset of a single nucleus RNA-seq dataset from human heart failure patients or healthy controls (Wang et al. Nat Cell Biol. 2020;22: 108-119). Linearregression line is shown in blue, and p-value was calculated using cor.test() in R for panels (Figure 8C through Figure 8F).

[0027] Figure 9, comprising Figure 9A through Figure 9L, depicts data demonstrating histone H1.0 is enriched in fibroblasts, associated with cardiac dysfunction and the only isoform whose depletion influences fibroblast activation. Figure 9A depicts a representative bar chart showing significant Pearson correlation (indicated on y-axis) between cardiac H1.0 transcription in the Hybrid Mouse Diversity Panel (Rau et al. Circ CV Genetics, 2015) and left ventricular weight, E amplitude, and A amplitude. For all three phenotypes, p-values (top of bars) were calculated using cor.test() in R. Transcription and phenotype data were measured in hearts from control mice. Figure 9B depicts representative RNA-seq data from adult mouse hearts, examining transcription in isolated myoc tes, fibroblasts, and endothelial cells. Figure 9C depicts representative RT-qPCR on histone Hl isoforms performed in isolated murine cardiac fibroblasts activated by TGF-P (48 h, 10 ng / mL; mean ± SEM, Welch’s unpaired t-test). Figure 9D depicts representative RNA-seq showing that siRNA knockdow n of histone H1.0 significantly inhibits TGF- induced aSMA (left) and periostin (right) transcription (mean ± SD, Benjamin’ s-Hochberg adjusted Wald test p-value from the DESeq2 package is shown). Figure 9E depicts a representative Western blot showing histone H1.0 KD does not affect histone H3 protein level (representative of n = 3 biological replicates). Figure 9F depicts a graph showing RT-qPCR of all remaining Hl isoforms after H1.0 siRNA depletion (mean± SD, Welch’s unpaired t-test for each Hl isoform). Figure 9G through Figure 9K depict representative Western blots and quantitation of periostin, aSMA and the indicated histone Hl isoforms following TGF-P in the presence or absence of knockdown of the various histone Hl family members (mean± SD, one-way ANOVA with a post-hoc Tukey test). Figure 9L depicts the quantification of collagen gel contraction (one replicate is an individual isolation and single well area calculation; same as in Figure 2D) following TGF-P treatment in the presence or absence of histone Hl.2 knockdown (mean ± SD, one-way ANOVA with a post-hoc Tukey test).

[0028] Figure 10, comprising Figure 10A through Figure 10E, depicts data demonstrating the effect of histone H1.0 depletion on TGF-P-dependent fibroblast activation. Figure 10A through Figure IOC depict representative Western blots and quantitation of the effect of TGF-P and histone H1.0 KD on histone H2A, H2B and H4. Figure 10D depicts data demonstrating that knockdown of histone Hl .0 after fibroblast activation with TGF-P fails to block aSMA and periostin induction (n = 3). Figure 10E depicts data demonstrating concomitant histone H1.0 KD and TGF-P treatment slightly attenuates aSMA induction. All data are mean ± SD, analyzed by one-way ANOVA with a post-hoc Tukey test; one replicate in Figure 10A through Figure 10E is a single isolation followed by treatment as indicated and western blotting.

[0029] Figure 11, comprising Figure 11 A through Figure 11H, depicts data demonstrating that depletion of histone H1.0 prevents distinct fibroblast mechanical behaviors in cells from different organs and in response to different stimuli. Figure 11 A depicts data demonstrating murine cardiac fibroblasts transfected with Adv-GFP- H1.0-FLAG of Adv-GFP control (48 h) were immunolabeled with an anti-FLAG antibody to show nuclear localization of histone H1.0 (red). DAPI (blue) stains cell nuclei. Scale bar=10pm (n = 3 independent experiments). Figure 11B depicts a representative Western blot in isolated murine lung fibroblasts transfected with histone H1.0 siRNA or scrambled negative control (48 h) and then treated with TGF-P shows upregulation of periostin and aSMA is histone H1.0 dependent in lung (left). Immunoblot quantification (center left). Efficiency of histone H1.0 KD is demonstrated by RT-qPCR (center right) (mean ± SD, one-way ANOVA with a post- hoc Tukey test). Figure 11C depicts a representative Western blot in isolated murine skin fibroblasts transfected with histone H1.0 siRNA or scrambled negative control (48 h) and then treated with TGF-P shows up-regulation of aSMA is histone H1.0 dependent in skin (left). Immunoblot quantification (right) (mean ± SD; one-way ANOVA with a post-hoc Tukey test). Figure 1 ID depicts a representative Western blot in isolated murine cardiac fibroblasts transfected with histone H1.0 siRNA orscrambled negative control (48 h) and then treated with Angiotensin II (48 h, 1 pM) shows that induction of periostin and aSMA upregulation by Angiotensin II is histone H1.0 dependent (left). Immunoblot quantification (middle and right) (mean± SD. oneway ANOVA with a post-hoc Tukey test; one replicate in b-d is a single isolation followed by treatment as indicated and western blotting). Figure 1 I E depicts a wound scratch assay that was conducted using murine cardiac fibroblasts, demonstrating the necessary role of histone H1.0 in TGF-P-induced cell migration and proliferation. A scratch wound was made across the cell layer as indicated by the guidelines. Images were captured along the scratch wound guidelines at 0 h and 24 h (left). Percentage of wound closure ([0 h wound area - 24 h wound area] / O h wound area) was measured using ImageJ (mean ± SD, one-way ANOVA with a post-hoc Tukey test) (right). Figure 1 IF depicts data demonstrating that histone H1.0 knockdown was confirmed by western blot in human skin fibroblasts (left). Quantification (mean±SD, Welch’s unpaired t-test) (right). Figure 11G depicts data demonstrating that histone H1.0 knockdown impairs human skin fibroblast migration (left), Quantification (right). Figure 11H depicts a representative wound scratch assay using murine cardiac fibroblasts transfected with Adv-GFP-Hl.O-FLAG or Adv-GFP control (left) (right, quantification: 48 h; mean ± SD, Welch’s unpaired t-test). Mean ± SD, Welch’s unpaired t-test; one replicate in e-h is a single isolation followed by treatment as indicated and calculation of cell migration).

[0030] Figure 12, comprising Figure 12A through Figure 121, depicts data demonstrating the effects of histone H1.0 depletion of transcriptional machinery and stress responsive genes. Figure 12A depicts a representative principal component analysis of RNA-seq data (3 biological replicates per condition). PCI captures the effect of TGF-P, PC2 of histone H1.0 KD. Figure 12B depicts a table summarizing ChlP-seq read pair counts and mapping rates for each replicate. Figure 12C depicts KEGG pathway analysis of TGF-|3-upregulated genes that were histone Hl .0 dependent (left). Western blot of p-Akt and total Akt in isolated murine cardiac fibroblasts following TGF-P treatment in the presence or absence of histone H1.0 KD(center left). Immunoblot quantification (mean ± SD, one-way ANOVA with a post- hoc Tukey test) (center right). Extended analysis of collagens and other ECM genes reveals dependency of their upregulation on histone H1.0 (right). Figure 12D depicts a representative Western blot showing that histone H1.0 KD abrogates TGF-0-induced upregulation of the extracellular matrix protein thrombospondin 4 (THBS4) (left). Immunoblot quantification (mean ± SD, one-way ANOVA with a post-hoc Tukey test) (right). Figure 12E depicts a representative Western blot confirming Thbs4 knockdown in primary cardiac fibroblasts. Right, Immunoblot quantification (mean n > 3 ± SD, one-way ANOVA with a post-hoc Tukey test) (left). Figure 12F depicts a representative Western blot showing that Thbs4 KD prevents induction of periostin and aSMA by TGF-P (left). Immunoblot quantification (right) (mean± SD, one-way ANOVA with a post-hoc Tukey test; one replicate in c-e is a single isolation followed by treatment as indicated and western blotting). Figure 12G depicts representative transcript levels of four RNA Polymerase II subunits that were analyzed by RNA-seq (mean± SD, Benjamin’ s-Hochberg adjusted Wald test p-value from the DESeq2 package is shown; one replicate is one RNA-seq experiment). Figure 12H depicts a representative Western blot demonstrating the effect of histone H1.0 KD on TGF-P induced RNA Pol II upregulation (left). Immunoblot quantification (right)(mean ± SD, one-way ANOVA with a post-hoc Tukey test; one replicate is a single isolation followed by treatment as indicated and western blotting). Figure 121 depicts representative histone H1.0 ChlP-seq, performed using an antibody against endogenous histone H 1.0 in isolated cardiac fibroblasts overexpressing histone H1.0- FLAG, reveals depletion of H1.0 occupancy at gene TSS, with stronger depletion at upregulated genes (green) when compared to downregulated (orange) or not differentially expressed (NDE, grey) genes (left panels). Each inset panel label indicates the RNA-seq dataset from which each gene subset was analyzed. Quantification of the local minimum for each condition within each inset graph from ChlP-seq profiles (right panels).

[0031] Figure 13, comprising Figure 13A through Figure 131, depicts data demonstrating that modulation of histone H1.0 levels can affect fibroblast viability or size. Figure 13A depicts data demonstrating cell viability (left) and cell size (right) analyses of isolated mouse cardiac fibroblasts transfected with histone H1.0 siRNA or scrambled negative control and treated with vehicle or TGF-P show no changes among groups (representative of 2 biological replicates; mean± SD plotted for cell viability, median and quartiles indicated for cell size). Figure 13B depicts data demonstrating cell viability (left) and cell size (right) analyses of fibroblasts transfected with Adv-GFP-Hl.O or Adv-GFP control show no changes among groups (representative of 2 biological replicates; mean ± SD plotted for cell viability, median and quartiles indicated for cell size). Figure 13C depicts a representative heatmap depicting several myosin and focal adhesin genes whose increased expression after TGF-P is prevented by histone H1.0 knockdown. Figure 13D depicts data demonstrating that fibroblasts treated with vehicle or TGF-P were immunolabeled with anti-Vimentin (green) and anti-Lamin A / C (red) antibodies (left). DAPI (blue) stains cell nuclei. Scale bar- 1 Oprn. Cell and nuclear area quantification (right) (mean± SD, Welch’s unpaired t-test). Figure 13E depicts data demonstrating that fibroblasts transfected with histone H1.0 siRNA or scrambled negative control were immunolabeled with anti-Vimentin (green) and anti-Lamin A / C (red) antibodies. DAPI (blue) stains cell nuclei (left). Scale bar=10pm. Histone H1.0 KD promoted a reduction in nuclear area without altering cell size. Cell and nuclear area quantification (right) (mean ± SD, Welch’s unpaired t-test). Figure 13F depicts data demonstrating that fibroblasts transfected with Adv-GFP-Hl.O or Adv-GFP control were immunolabeled with anti-Vimentin (red) and Lamin A / C (green) antibodies. DAPI (blue) stains cell nuclei (left). Scale bar=10pm. Histone H1.0 overexpression resulted in increased cell and nuclear size. Cell and nuclear area quantification (mean ± SD, Welch’s unpaired t-test) (right). Figure 13G depicts the examination of the fibrotic area in genetically distinct mouse strains undergoing isoproterenol treatment (Rau et al. Circ CV Genetics, 2015); asterisks indicate strains used in thisstudy. Figure 13H depicts a representative Pearson’s correlation between histone H1.0 protein level and fibrotic area in hearts treated with ISO in the presence or absence of histone H1.0 depletion by siRNA. Figure 131 depicts data demonstrating that neither ISO nor histone H1.0 KD affected ventricular ejection fraction in C57BL / 6 J and C3H / HeJ mice (mean ± SD, one-way ANOVA with a post-hoc Tukey test within strains).

[0032] Figure 14. comprising Figure 14A through Figure 14C, depicts data demonstrating that depletion of histone Hl.O prevents fibrosis in vivo. Figure 14A depicts data demonstrating the efficiency of histone Hl.O KD examined by Western blot in hearts of C3H / HeJ mice (left). Immunoblot quantification (right) (mean ± SD, one-way ANOVA with post-hoc Tukey test). Figure 14B depicts a representative Western blot showing histone Hl.O protein levels from C57BL / 6 J kidney after ISO treatment and depletion of histone Hl.O (left). Immunoblot quantification (right) (mean± SD one-way ANOVA with a post-hoc Tukey test). Figure 14C depicts a representative Masson trichrome staining of C57BL / 6 J kidney, showing that histone Hl.O KD reduces kidney fibrosis in the ISO-treated group (left). Scale bars: 100 pm. Image quantification (median and quartiles shown; one-way ANOVA with post-hoc Tukey test; n > 5 sections / group, >14 images per group as indicated in figure) (right).

[0033] Figure 15, comprising Figure 15A through Figure 15D, depicts data demonstrating the echocardiography measurements after in vivo histone Hl.O knockdown and ISO treatment. Figure 15A depicts data demonstrating mice from both C57BL / 6 J and C3H / HeJ backgrounds subjected to tail vein injection of scramble siRNA or siRNA against histone Hl.O, concurrent with PBS or isoproterenol treatment. Echocardiography was performed to measure stroke volume (left), fractional shortening (FS, middle), and global circumferential strain (GCS, right). Figure 15B depicts data demonstrating left ventricular anterior wall thickness (LVAW) in systole (left) and diastole (right). Figure 15D depicts data demonstrating left ventricular posterior wall thickness (LVPW) in systole (left) and diastole (right).Figure 15D depicts data demonstrating cardiac index (CI, left), and cardiac output (right). Mean ± SD displayed in all panels; no significant differences were observed.

[0034] Figure 16, comprising Figure 16A through Figure 16G, depicts data demonstrating that AAV9 delivery of shRNA in vivo depletes histone H1.0 and prevents isoproterenol -induced fibrosis. Figure 16A depicts data demonstrating in vivo histone H1.0 knockdown attenuates ISO-induced cardiac hypertrophy as measured by heart weight to body weight ratio (HW / BW) after an AAV9-based in vivo knockdown approach targeting shRNA against histone H1.0 (AAV9-Tcf21-GFP- shRNAmir-Hl.O shRNA or AAV9-CMV-GFP-shRNAmir-scramble shRNA). The AAV9 is taken up by fibroblasts and myocytes in heart: the fibroblast-specific Tcf21 promoter drives GFP and the shRNAmir promoter drives transcription of the shRNA. This strategy allows us to identify fibroblasts taking up the vector (they will express GFP. myocytes will not) and in those cells characterize the level of histone H 1.0 depletion. 3x 101 1 GC were injected. In the control vector, GFP is driven by CMV promoter and scramble shRNA (Scr) is driven by U6. 5 weeks post AAV9 injection, PBS or ISO (20 mg / kg / day) were injected for one week. Physiological endpoints were then examined via gross histology. ECHO, and PCR (n > 4 / group). Figure 16B depicts data demonstrating that ejection fraction (EF) is not significantly changed after in vivo shRNA injection and / or one week ISO treatment. Figure 16C depicts data demonstrating that histone H1.0 depletion attenuates ISO-induced diastolic dysfunction as measured by E / A ratio (n> 4 / group). Figure 16D depicts data demonstrating the quantification of histone H1.0 depletion by RT-qPCR (n> 3). Figure 16D depicts data demonstrating representative images of Masson’s trichrome staining to examine cardiac fibrosis after in vivo histone H1.0 knockdown and / or ISO treatment (n> 4 / group). Figure 16F depicts data demonstrating the quantitation of the fibrotic area showing that depletion of histone H1.0 significantly inhibits ISO-induced fibrosis. Mean ± SD, one-way ANOVA with post-hoc Tukey test for panels (Figure 16A through Figure 16D and Figure 16F). Figure 16G depicts data demonstrating antibody -based detection of GFP in hearts of animals receiving AAV9 oruntransfected controls. Scale bar = 10 iim (representative of n = 3 biological replicates).DETAILED DESCRIPTION

[0035] The present invention is based, in part, on the discovery that histone H1.0 has a role in regulating chromatin condensation. Provided herein are histone H1.0 inhibitors for preventing chromatin condensation and for treating or preventing fibrotic disease in a subject in need thereof. In certain embodiments, the invention relates to methods comprising administering histone H1.0 inhibitors for treating fibrotic disease.Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Any methods and materials similar or equivalent to those described herein can be used in the practice of and / or for the testing of the present invention. In describing and claiming the present invention, the following terminology will be used according to how it is defined, where a definition is provided.

[0037] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0038] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0039] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1 %, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0040] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0041] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0042] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency wi th which such a sign or symptom is experienced by a patient, or both, is reduced.

[0043] “Encoding” refers to the inherent property7of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0044] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cisacting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0045] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g.. if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between tw o sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0046] “Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as. for example, a host cell.

[0047] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0048] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0049] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In some embodiments, the patient,subject or individual is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkey and human). In certain non-limiting embodiments, the patient, subject or individual is a human.

[0050] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.

[0051] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0052] “Antisense” refers particularly to the nucleic acid sequence of the noncoding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary' solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.

[0053] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residuescovalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0054] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The an antibody in the present invention may exist in a variety of forms where the antigen binding portion of the antibody is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0055] The term “antibody fragment” refers to at least one portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, sdAb (either VL or VH), camelid Vini domains, scFv antibodies, and multi-specific antibodies formed from antibodyfragments. The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-Vn or may comprise Vn-linker-VL.

[0056] The term “isolated” when used in relation to a nucleic acid, as in “isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences (e.g., a specific mRNA sequence encoding a specific protein), are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid includes, by way of example, such nucleic acid in cells ordinarily expressing that nucleic acid where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide contains at a minimum, the sense or coding strand (i.e., the oligonucleotide may be singlestranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).

[0057] The term “isolated” when used in relation to a polypeptide, as in “isolated protein” or “isolated polypeptide” refers to a polypeptide that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated polypeptide is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated polypeptides (e g., proteins and enzymes) are found in the state they exist in nature.

[0058] By “expression cassette” is meant a nucleic acid molecule comprising a coding sequence operably linked to promoter / regulatory sequences necessary for transcription and, optionally, translation of the coding sequence.

[0059] The term “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of sequences encoding amino acids in such a manner that a functional (e g., enzymatically active, capable of binding to a binding partner, capable of inhibiting, etc.) protein or polypeptide is produced.

[0060] The term “promoter” as used herein is defined as a DNA sequence recognized by the translational machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0061] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0062] The phrase “inhibit,” as used herein, means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and / or a protein’s expression, stability , function or activity by a measurable amount or to prevent entirely. Inhibitors arecompounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.

[0063] By the term “modulating / ’ as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject (e.g., a human.)

[0064] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0065] As used herein, “treating a disease or disorder” means reducing the frequency with w hich a symptom of the disease or disorder is experienced by a patient. Disease and disorder are used interchangeably herein.

[0066] The phrase “therapeutically effective amount,” as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases.

[0067] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0068] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are know n in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectorsinclude, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0069] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4. 5, 5.3, and 6. This applies regardless of the breadth of the range.Description

[0070] The present invention provides compositions and methods for modulating chromatin condensation in cells. The present invention also provides compositions and methods for inhibiting histone H1.0 and for treating or preventing fibrosis or a cardiovascular disease or disorder. In certain embodiments, the invention relates to treating cardiac fibrosis.

[0071] In one aspect, the invention relates to the discovery that histone Hl levels directly influence cellular stiffness and nuclear condensation and that depletion of histone H1.0 prevents disease-associated cardiac fibrosis.

[0072] In one embodiment, the method comprises treating or preventing fibrosis by modulating histone H1.0 abundance, association with chromatin, or both.

[0073] In one embodiment, fibrosis is a disease or disorder eliciting abnormal formation, accumulation and precipitation of an extracellular matrix, caused by fibroblasts, and refers to abnormal accumulation of a collagen matrix due to injury or inflammation that changes the structures and functions of various types of tissue.Regardless of where fibrosis arises, most etiology of fibrosis includes excessive accumulation of a collagen matrix substituting normal tissue.

[0074] In certain embodiments, the method is used to treat or prevent fibrosis or a disease or disorder associated therewith in a subject. Non-limiting examples of fibrotic diseases and disorders that can be treated using the compositions and methods described herein include, but are not limited to, cardiac fibrosis, interstitial lung disease, idiopathic pulmonary' fibrosis, lung fibrosis, asthma, COPD, Raynaud’s phenomenon, pulmonary fibrosis, cirrhosis, liver cirrhosis, atrial fibrosis, endomyocardial fibrosis, arthrofibrosis, Crohn’s Disease, mediastinal fibrosis, myelofibrosis, tubulointerstitial fibrosis, hepatic fibrosis, premacular fibrosis, retinal fibrosis, dermal fibrosis, wound-associated fibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, fibroma, scleroderma, systemic scleroderma, Sjogren syndrome, Alport Syndrome, metabolic disorders, and kidney fibrosis.Compositions

[0075] In one embodiment, the invention provides an inhibitor of histone H1.0 to alter the condensation levels of chromatin. In one embodiment, the present invention includes compositions for altering the condensation levels of chromatin in a subject, a cell, a tissue, or an organ in need thereof. In one embodiment, the compositions of the invention inhibit the amount of polypeptide of histone H1.0, the amount of mRNA of histone H 1.0, the amount of activity of histone H1.0, or a combination thereof.

[0076] The compositions of the invention include compositions for treating or preventing fibrosis, and fibrosis-related diseases. In one embodiment, an inhibitor of histone H1.0 of the invention is useful for treating fibrosis, fibrosis-related diseases and cardiovascular diseases.

[0077] In one embodiment, the inhibitor is a small interfering RNA (siRNA), shRNA, a microRNA (miRNA), a guide RNA, a GapmeR, an antisense nucleic acid, aribozyme, an expression vector encoding a transdominant negative mutant, an intracellular antibody, a peptide or a small molecule.Nucleic Acids

[0078] In one embodiment, the composition of the invention comprises at least one antisense nucleic acid molecule.

[0079] Inhibiting the expression of a gene using an antisense oligonucleotide is well known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods of expressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No. 5.190,931). The methods of the invention include the use of antisense oligonucleotide to diminish the amount of histone H1.0. Contemplated in the present invention are antisense oligonucleotides that are synthesized and provided to the cell by way of methods well known to those of ordinary skill in the art. As an example, an antisense oligonucleotide can be synthesized to be between about 10 and about 100 nucleotides long. In some embodiments, the antisense oligonucleotide can be synthesized to be between about 1 and 30 nucleotides long. The synthesis of nucleic acid molecules is well know n in the art, as is the synthesis of modified antisense oligonucleotides to improve biological activity in comparison to unmodified antisense oligonucleotides (Tullis, 1991. U.S. Pat. No. 5,023,243).

[0080] Similarly, the expression of a gene encoding a histone H1.0 may be inhibited by the hybridization of an antisense molecule to a promoter or other regulatory' element of a gene, thereby affecting the transcription of the gene. Methods for the identification of a promoter or other regulatory element that interacts with a gene of interest are w ell known in the art, and include such methods as the yeast two hybrid system (Bartel and Fields, eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary, N.C.).

[0081] Alternatively, inhibition of histone Hl .0, can be accomplished through the use of an siRNA, shRNA, antisense oligonucleotide or ribozyme, or a combination thereof. Given the nucleotide sequence of the molecule, one of ordinary skill in the art could synthesize an antisense oligonucleotide or ribozy me withoutundue experimentation, provided with the disclosure and references incorporated herein.

[0082] In one embodiment, siRNA is used to decrease the level of histone H1.0. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See. for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391 (19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describe a chemical modification to siRNAs that aids in systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3' overhang. See. for instance, Schwartz et al.. 2003, Cell. 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the present invention also includes methods of decreasing levels of histone H1.0 at the protein level using RNAi technology7.

[0083] Exemplary siRNA that target histone H1.0 are provided in Table 1. In one embodiment, the siRNA for inhibiting histone H1.0 comprises the sequence of SEQ ID NO 35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO 65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68. In one embodiment, the siRNA for inhibiting histone H1.0 comprises the sequence of SEQ ID NO:59 or SEQ IDNO:60. In some embodiments at least one of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68 is used in combination with at least one additional siRNA that targets histone H1.0. For example, in some embodiments, the invention provides a combination of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68 for use for treating or preventing fibrosis or a fibrotic disease or disorder. In some embodiments, the invention provides a combination of SEQ ID NO:59 and SEQ ID NO:60 for use for treating or preventing fibrosis or a fibrotic disease or disorder.

[0084] In certain embodiments, the inhibitor described herein comprises a short hairpin RNA (shRNA) molecule. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzy mes (e.g., dicer) that cleaves the shRNA to form siRNA.

[0085] An exemplary shRNA that targets histone H1.0 is provided in Table 1. In one embodiment, the shRNA for inhibiting histone H1.0 comprises the sequence of SEQ ID NO:69. In some embodiments SEQ ID NO:69 is used in combination with at least one additional shRNA or siRNA that targets other histone Hl subtypes (e.g. Hl.1, Hl.2. Hl.3, Hl.4, and Hl.5).

[0086] In other related aspects, the invention includes an isolated nucleic acid encoding an inhibitor, wherein an inhibitor such as an siRNA, shRNA, GapmeR or antisense molecule, inhibits histone H1.0, a derivative thereof, a regulator thereof, or a downstream effector thereof.

[0087] In one embodiment, the antisense molecule is a GapmeR. GapmeR molecules typically have a central stretch of chemically modified DNA "gap” flanked by locked nucleic acids (LNA), which increase the binding affinity of GapmeR to the target RNA. In addition to specificity and highly efficient gene silencing, GapmeRs can easily internalize into target cells.

[0088] Antisense molecules of the invention may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30 nucleotides. In some embodiments, the antisense oligomers are about 15 to about 20 nucleotides. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0089] Ribozymes and their use for inhibiting gene expression are also well known in the art (see. e.g., Cech et al., 1992. J. Biol. Chem. 267: 17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933; Eckstein et al., International Publication No. WO 92 / 07065; Altman et al., U.S. Patent No. 5,168,053). Ribozymes are RNA molecules possessing the ability7to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences encoding these RNAs, molecules can be engineered to recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of this approach is the fact that ribozymes are sequence-specific.

[0090] There are two basic types of ribozymes, namely, tetrahymena-type (Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while hammerheadtype ribozymes recognize base sequences 11-18 bases in length. The longer the sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species.

[0091] In one embodiment of the invention, a ribozyme is used to inhibit histone H1.0. Ribozy mes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence of histone H1.0. Ribozymes targeting histone H 1.0 may be synthesized using commercially available reagents or they may be genetically expressed from DNA encoding them.

[0092] In one embodiment, the inhibitor of histone H1.0 may comprise at least one component of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding histone H1.0, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the inhibitor comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0093] When the inhibitor of the invention is a small molecule, a small molecule antagonist may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.

[0094] Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.

[0095] In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity' of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores.

[0096] In other related aspects, the invention includes an isolated peptide inhibitor, or combination thereof that inhibits histone H1.0. For example, in oneembodiment, the peptide inhibitor of the invention inhibits histone H1.0 directly by binding to histone H1.0 thereby preventing the normal functional activity' of histone H1.0. In another embodiment, the peptide inhibitor of the invention inhibits histone H1.0 by competing with endogenous histone H1.0. In yet another embodiment, the peptide inhibitor of the invention inhibits the activity of histone Hl .0 by acting as a transdominant negative mutant.Substrates

[0097] The present invention provides a scaffold or substrate composition comprising a modulator of the invention, an isolated nucleic acid of the invention, a cell expressing the modulator of the invention, or a combination thereof. For example, in one embodiment, a modulator of the invention, an isolated nucleic acid of the invention, a cell a cell expressing the modulator of the invention, or a combination thereof is incorporated within a scaffold. In another embodiment, a modulator of the invention, an isolated nucleic acid of the invention, a cell expressing the modulator of the invention, or a combination thereof is applied to the surface of a scaffold. The scaffold of the invention may be of any type known in the art. Non-limiting examples of such a scaffold includes a. hydrogel, electrospun scaffold, foam, mesh, sheet, patch, and sponge.Pharmaceutical Compositions

[0098] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or at least one other accessoryingredient, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0099] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable forethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary', if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to. humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0100] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for intrathecal, ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intratumoral, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

[0101] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0102] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0103] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise at least one additional pharmaceutically active agent, including, for example, chemotherapeutics, immunosuppressants, corticosteroids, analgesics, and the like.

[0104] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0105] As used herein, “parenteral administration"’ of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intrathecal, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intrastemal injection, intratumoral. and kidney dialytic infusion techniques. In one embodiment, the method of administration is through intrathecal injection.

[0106] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise at least one additional ingredient including, but not limited to, a suspending, stabilizing, or dispersing agent. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder orgranular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0107] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using anon-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0108] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. In some embodiments, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In someembodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. In some embodiments, dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0109] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0. 1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (e.g., having a particle size of the same order as particles comprising the active ingredient).

[0110] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise at least one additional ingredient including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0111] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as thedispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using anon-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to. Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0112] Additionally, the molecules may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. Various forms of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the molecules for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the chimeric molecules, additional strategies for molecule stabilization may be employed.

[0113] Nucleic acids may be included in any of the above-described formulations as the free acids or bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts that substantially retain the biologic activity of the free bases and which are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.

[0114] In addition to the formulations described previously, the molecules may also be formulated as a depot preparation. Thus, the molecules may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0115] Alternatively, other pharmaceutical delivery systems may be employed. Liposomes, lipid nanoparticles, and emulsions are well-known examples of delivery vehicles that may be used to deliver nucleic acids of the disclosure.Therapeutic Methods

[0116] In one embodiment, the invention provides methods of decreasing histone H1.0 activity, or expression, or decreasing the level of histone H1.0 RNA such that the modulation produces a therapeutic effect in a subject, or group of subjects. A therapeutic effect is one that results in an amelioration in the symptoms, or progression of a disease or disorder. In one embodiment, the disease or disorder is a fibrotic disease or disorder.

[0117] Exemplary fibrotic diseases that can be treated using the histone H1.0 inhibitor comprises include, but are not limited to. cardiac fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, lung fibrosis, asthma, COPD, Raynaud’s phenomenon, pulmonary fibrosis, cirrhosis, liver cirrhosis, atrial fibrosis, endomyocardial fibrosis, arthrofibrosis, Crohn’s Disease, mediastinal fibrosis, myelofibrosis, tubulointerstitial fibrosis, hepatic fibrosis, premacular fibrosis, retinal fibrosis, dermal fibrosis, wound-associated fibrosis, Peyronie’s disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, fibroma, scleroderma, systemic scleroderma, Sjogren syndrome and kidney fibrosis.

[0118] In one embodiment, the method comprises administering a composition described herein to a subject having, or having symptoms indicative of, a fibrotic disease or disorder. In some embodiments, the fibrotic disease or disorder is cardiac fibrosis.

[0119] In some embodiments, the methods comprise administration of a delivery vehicle comprising at least one histone H1.0 inhibitor. Any suitable format of delivery vehicle is contemplated. In some embodiments, the at least one delivery vehicle is a colloidal dispersion system, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, liposomes, and lipid nanoparticles. Exemplary colloidal systems for use as delivery7vehicles in vitro and in vivo include liposomes (e.g., an artificial membrane vesicle) and lipid nanoparticles.

[0120] The use of lipid formulations, as described above, is contemplated for the introduction of at least one histone Hl .0 inhibitor into a host cell (in vitro, ex vivo, or in vivo). In another aspect, at least one agent may be associated with a lipid. The at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, complexed with a lipid, contained or complexed with a micelle, or otherwise associated with a hpid. Lipid, lipid / nucleic acid or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed"’ structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.

[0121] In one embodiment, delivery of at least one agent comprises any suitable delivery method, including exemplary' delivery methods described elsewhere herein. In certain embodiments, delivery of at least one agent to a subject comprises mixing at least one agent with a transfection reagent prior to the step of contacting. In another embodiment, a method of the present invention further comprises administering at least one agent together with the transfection reagent. In another embodiment, the transfection reagent is a cationic hpid reagent.

[0122] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a proteinbased transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfectionreagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent know n in the art.

[0123] In some embodiments, delivery of at least one agent comprises the use of liposomes as delivery vehicles. “Liposome7’ is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al.. 1991 Glycobiology’ 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.

[0124] In one embodiment, the at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / nucleic acid or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed’’ structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.

[0125] In another embodiment, the transfection reagent forms a liposome. Liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity. In another embodiment, liposomes arehollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. In some embodiments, the liposomes comprise an internal aqueous space for entrapping water-soluble compounds. In another embodiment, liposomes can deliver at least one inhibitor to cells in an active form.

[0126] The present invention relates in part to methods of treating diseases or disorders in subjects in need thereof, the method comprising the administration of a composition comprising at least one deliver}’ vehicle comprising at least one histone H1.0 inhibitor. In some embodiments, the deliver}' vehicle is not targeted and the method includes administration of the deliver}’ vehicle to a target cell or tissue. In some embodiments, the delivery vehicle is targeted and the method includes administration of the delivery vehicle to allow targeted delivery to the target cell or tissue. In some embodiments, the target cell or tissue is a fibrotic cell or tissue. In some embodiments, the target cell or tissue is a cardiac cell or tissue.

[0127] It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the invention is not limited to treatment of a disease or disorder that is already established. Particularly, the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of a disease or disorder do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing a disease or disorder, in that a composition, as discussed previously elsewhere herein, can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the disease or disorder.

[0128] One of skill in the art, when armed with the disclosure herein, would appreciate that the prevention of a disease or disorder, encompasses administering to a subject a composition as a preventative measure against the development of, or progression of, a disease or disorder. As more fully discussed elsewhere herein,methods of modulating the level or activity of a gene, or gene product, encompass a wide plethora of techniques for modulating not only the level and activity' of polypeptide gene products, but also for modulating expression of a nucleic acid, including either transcription, translation, or both.

[0129] To practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate composition to a subject. The present invention is not limited to any particular method of administration or treatment regimen.

[0130] One of skill in the art will appreciate that the compositions of the invention can be administered singly or in any7combination. Further, the compositions of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and / or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that the compositions of the invention can be used to prevent or to treat a disease or disorder, and that a composition can be used alone or in any combination with another composition to affect a therapeutic result. In various embodiments, any of the compositions of the invention described herein can be administered alone or in combination with at least one modulator of an additional molecule associated with a disease or disorder.

[0131] Administration of the compositions of the invention to a human patient can be by any route, including but not limited to intravenous, intranodal, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via an aerosol, etc.), buccal (e.g., sub-lingual, etc.), topical (i.e., both skin and mucosal surfaces, including airway surfaces, etc.), intrathecal, intraarticular, intraplural, intracerebral, intra-arterial, intraperitoneal, oral, intraly mphatic, intranasal, rectal or vaginal administration, by perfusion through a regional catheter, or by direct intralesional injection. In one embodiment, the compositions of the invention are administered by intravenous push or intravenous infusion given over defined period (e.g., 0.5 to 2 hours). The compositions of the invention can be delivered by peristaltic means or in the form of adepot, although the most suitable route in any given case will depend, as is well known in the art, on such factors as the species, age, gender and overall condition of the subject, the nature and severity of the condition being treated and / or on the nature of the particular composition (i.e., dosage, formulation) that is being administered. In particular embodiments, the route of administration is via bolus or continuous infusion over a period of time, once or twice a week. In other particular embodiments, the route of administration is by subcutaneous injection given in at least one site (e.g. thigh, waist, buttocks, arm), optionally once or twice weekly. In one embodiment, the compositions, and / or methods of the invention are administered on an outpatient basis.

[0132] In one embodiment, the invention includes a method comprising administering a combination of compositions described herein (e.g., a combination of siRNA molecules or a combination of delivers’ vehicles encapsulating siRNA molecules). In certain embodiments, the method has an additive effect, wherein the overall effect of the administering a combination of compositions is approximately equal to the sum of the effects of administering each individual inhibitor. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering a combination of compositions is greater than the sum of the effects of administering each individual composition.

[0133] The method comprises administering a combination of composition in any suitable ratio. For example, in one embodiment, the method comprises administering two individual compositions (e.g., siRNA) at a 1: 1 ratio. In one embodiment, the method comprises administering for individual compositions (e.g., siRNA) at a 1 : 1 : 1 : 1 ratio. How ever, the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.Pharmaceutical Compositions

[0134] The formulations of the pharmaceutical compositions (e.g., comprising at least one histone H1.0 inhibitor) described herein may be prepared by any methodknown or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., at least one histone H1.0 inhibitor) into association with a carrier or at least one other accessory ingredient, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0135] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any. experimentation. Subjects to w hich administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0136] Pharmaceutical compositions (e.g., a composition comprising at least one histone Hl inhibitor) that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[0137] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0138] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0139] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise at least one additional pharmaceutically active agent.

[0140] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0141] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. Parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrastemal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

[0142] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or forcontinuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise at least one additional ingredient including, but not limited to, a suspending, stabilizing, or dispersing agent. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.

[0143] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using anon-toxic parenterally acceptable diluent or solvent, such as water or 1,3 butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0144] In various embodiments, the targeted delivery vehicles may be administered to a subject such that the delivery vehicle contacts the targeted cell in vivo. In other embodiments, the cell may be contacted with the delivery vehicles ex vivo and then transferred back to a subject in need with adoptive cell transfer. In thisembodiment, cells are removed from a patient and modified ex vivo by contacting them with the herein disclosed delivery' vehicles.

[0145] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry’ powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self propelling solvent / powder dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. At least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0146] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0. 1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent.

[0147] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise at least one additional ingredient including, but not limited to, a suspending, stabilizing, or dispersing agent. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.

[0148] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using anon-toxic parenterally acceptable diluent or solvent, such as water or 1,3 butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0149] As used herein, “additional ingredients” include, but are not limited to, at least one of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles andsolvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsify ing agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.EXPERIMENTAL EXAMPLES

[0150] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0151] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following w orking examples therefore are not to be construed as limiting in any way the remainder of the disclosure.Example 1: Histone HLO Couples Cellular Mechanical Behaviors to Chromatin Structure

[0152] Tuning of genome structure and function is accomplished by chromatin binding proteins, which determine the transcriptome and phenotype of the cell. This study focused on investigating how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors, and demonstrated that histone HLO, which compacts nucleosomes into higher-orderchromatin fibers, controls genome organization and cellular stress response. This study demonstrates that histone H1.0 has privileged expression in fibroblasts across tissue types and its expression is necessary and sufficient to induce myofibroblast activation. Depletion of histone H1.0 prevents cytokine-induced fibroblast contraction, proliferation and migration via inhibition of a transcriptome comprised of extracellular matrix, cytoskeletal and contractile genes through a process that involves locus-specific H3K27ac acetylation. Transient depletion of histone H1.0 in vivo prevents fibrosis in cardiac muscle. These findings identify an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling cellular force generation, nuclear organization and gene transcription.

[0153] This study focuses on understanding the molecular regulation of the linker histone Hl family of proteins, given their role in promoting chromatin folding (Fyodorov et al., 2018. Nature reviews. Molecular cell biology 19: 192-206). The mouse linker histone Hl family is comprised of five (Hl . 1-1.5) main isoforms, plus the oocyte specific Hloo, the testis specific Hit and the replacement variant H1.0. Linker histones bind the nucleosome, facilitating chromatin compaction and the formation of higher order structures comprised of multiple nucleosomes and associated DNA (Crane-Robinson, 2016, Biochim Biophys Acta 1859:431-435). Loss of function studies have shown that individual histone Hl isoforms are dispensable for normal mouse development (Fan et al., 2001, Mol Cell Biol 21 :7933-7943; Sirotkin et al., 1995, Proc Natl Acad Sci U S A 92:6434-6438), yet triple knockouts (deleting HL 3, H1.4 and H1.5) showed extensive developmental abnormalities (Fan et al., 2003, Mol Cell Biol 23:4559-4572), associated with an altered linker-core histone ratio, which was maintained when only one isoform w as deleted via compensatory upregulation of other isoforms. These findings highlight the central role of histone stoichiometry in controlling normal development and tissue homeostasis (Fyodorov et al., 2018, Nature reviews. Molecular cell biology 19: 192-206). In cancer cells, altering histone Hl levels substantially reorganized global chromatin structure, decompacting topologically associating domains (Sema-Pujol et al., 2022, NucleicAcids Res 50:3892-3910) and shifting the genome to a more relaxed state (Yusufova et al., 2021, Nature 589:299-305).

[0154] Fibroblast activation leads to expression of cytoskeletal and extracellular matrix genes through a process that requires the activity of histone modifying enzymes, including histone deacetylases, and chromatin readers, including BRD4 (Felisbino et al., 2018, JACC Basic Transl Sci 3:704-715). Furthermore, the ability- of chromatin remodeling enzymes to modulate gene expression can be influenced by the local topology of chromatin (Poleshko et al., 2017, Cell 171:573- 587), which may be regulated by the abundance of linker histone Hl. A fundamental unanswered question is how the cell processes stress signals at the nucleus to remodel chromatin for precise gene expression, integrating the nucleosome-targeted actions of chromatin remodeling machinery’ and the genome sculping behavior of chromatin structural proteins, to elicit different mechanical responses.

[0155] This study demonstrated that levels of histone H1.0 underpin a genome wide change in chromatin organization to facilitate transcriptional changes in cytoskeletal and extracellular matrix genes. Results showed that histone H1.0 is required for fibroblast activation in response to cytokine stimulation and overexpression of histone H1.0 is sufficient to activate fibroblasts in the absence of stimulation. Histone H1.0 acts locally to promote formation of more compact chromatin fibers and globally to condense the genome, in turn regulating cellular deformability. Histone H 1.0 is required for cytokine induced reprogramming of the activating chromatin modification histone H3 lysine 27 acetylation (H3K27Ac) and acts via modulation of HDACs and BRD4. Finally, this study demonstrated that these chromatin regulatory' actions of histone H1.0 affect a wide range of mechanical behaviors in the cell, including contractile force generation, cytoskeletal regulation, motility and extracellular matrix deposition.The methods and materials are now described.Animal care and use

[0156] Adult female and male C57BL / 6J (The Jackson Laboratory, cat. no. 000664) and C3H / HeJ (The Jackson Laboratory, cat. no. 000659) mice (8-12 weeks old) were obtained from Jackson Laboratory and used in the study. Male and female mice were used in this study but the groups were not powered to reveal sex differences. Animals were housed under normal light / dark cycles and with controlled ambient air temperature and humidity.Histone HLO knockdown in vivo in mice

[0157] HLO siRNA (Thermo Fisher Scientific, cat. no. 4404010) and scrambled negative control siRNA (Thermo Fisher Scientific, cat. no. 4457289) were purchased from Thermo Fisher Scientific. For each experimental group, siRNA (1.25 mg / kg body weight) was tail vein injected into either C57BL / 6J or C3H / HeJ mice on days 0, 7, and 14 using InvivoF ectamine 3.0 (cat. no. IVF3005; Thermo Fisher Scientific) according to manufacturer instructions and based on previous studies (Hu et al., 2019, Proc Natl Acad Sci U S A, 116:6172-6180). siRNA sequences are listed in Table 1.Table 1 : SequencesMurine models of fibrosis

[0158] Cardiac fibrosis was induced by daily intraperitoneal injection of isoproterenol (ISO) (Millipore Sigma, cat.no. 16504) for 14 days (C57BL / 6J: 80 mg / kg / day; C3H / HeJ: 20 mg / kg / day), beginning the day of the second siRNA injection (day 7 of the in vivo knockdown protocol described above). Mice in negative control groups were injected daily with vehicle (phosphate-buffered saline). Mice were euthanized and then hearts, kidneys, and lungs were harvested 14 days post-ISO treatment.Echocardiography

[0159] Heart function was measured by echocardiology before and after ISO treatments. Animals were anesthetized with 1.5% isoflurane and 95% 02 and chest hair removed. Continuous ECG monitoring was implemented, and heart rates were maintained between 400 and 500 beats per minute. Body temperature was set at 37°C using a heating pad. A Vevo 3100 imaging system was used to acquire M-mode images. LV systolic function was evaluated by calculating ejection fraction (EF%). LV diastolic function was measured by calculating the E / A ratio. All calculations were performed using the Vevo Lab 5.6. 1 system.Cell culture and TGF-B or Angiotensin II treatment

[0160] Adult female and male C57BL / 6J primary cardiac fibroblasts were isolated using enzymatic digestion (7 mg / ml collagenase type II: Worthington Biochemical Corporation, cat. no. LS004177) followed by centrifugation (644 x g for 8 minutes at 4°C) and cell plating in DMEM / F12 media supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (penicillin and streptomycin), and 0. 1% insulin- transferrin-selenium (ITS; Coming, cat. no. 354350). After 2 hours, cells were maintained in DMEM / F12 media supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (penicillin and streptomycin), human basic fibroblast growth factor (hbFGF, 1: 10000 concentration from 200X stock; Millipore Sigma, cat. no.11123149001) and 0.1% insulin-transferrin-selenium (ITS; Coming, cat. no. 354350). Media and floating cells were then removed, and fibroblasts were grown in DMEM / F12 media supplemented with 10% FBS, 1% antibiotics, hbFGF and 0.1% ITS until reaching 70-80% confluency.

[0161] Lung primary fibroblasts were isolated from adult female and male C57BL / 6J mice using a previously reported method involving collagenase digestion (Edelman et al., 2018Methods Mol Biol, 1809:59-67) and maintained in DMEM / F12 media supplemented with 20% FBS, 1% antibiotics (penicillin and streptomycin), hbFGF and 0.1% ITS until reaching 70-80% confluency. Mouse skin fibroblasts from passage 3 to 5 and human skin fibroblasts from passage 9 were maintained inDMEM / F12 media supplemented with 10% FBS, 1% antibiotics (penicillin and streptomycin). For all experiments involving fibroblasts, cell at 70-80% confluency was cultured in serum-free media (0.1% FBS) for 24h prior to TGF-J3 treatment (10 ng / mL; Novoprotein. cat. no. CA59) or Angiotensin II treatment (IpM. Sigma, cat. no. A9525).Immunoblotting and antibodies

[0162] Protein was extracted from primary cardiac fibroblasts and lung fibroblasts using homemade RIPA lysis buffer (150mM NaCL 5mM EDTA, 50mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing protease inhibitors (Roche, cat. no. 04693159001) and phosphatase inhibitors (Roche, cat. no. 04906837001). A homemade lysis buffer was used for protein extraction from whole heart, lung, or kidney tissue (50mM Tris pH 7.4. lOmM EDTA, 1% sodium dodecyl sulfate (SDS), lOmM sodium butyrate, 1.2mM phenylmethanesulfonyl fluoride, ImM sodium fluoride, ImM sodium orthovanadate) supplemented with protease inhibitor tablets (Roche, cat. no. 04693159001). Protein concentration was measured using a Pierce BCA Protein Assay (Thermo Fischer Scientific, cat. no. 23225). An equal amount of protein was loaded into an SDS- containing polyacrylamide gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad, cat. no. 1620115). Membranes were blocked with 5% BSA for one hour, incubated with appropriate primary’ and fluorescent secondary antibodies and developed using a ChemiDoc MP Imaging System (Bio-Rad). Unless otherwise stated, western blots were performed on whole cell lysate, which would include proteins bound to as well as within the cells.Immunofluorescence

[0163] Cells were fixed at either: 1 ) room temperature for 10 minutes using 4% paraformaldehyde (PF A) for Figs. 1 and 2; or 2) room temperature for 20 min using 1.6% PFA for Figure 6, Figure 12, and Figure 13. Fixed cells werepermeabilized and blocked for 1 hour using blocking buffer (5% BSA, 0.1% Triton X-100) and incubated overnight at 4°C with primary' antibody: anti-aSMA (1: 100, Abeam cat. no. ab7817), anti-Periostin (1 :50, R&D System, cat. no. AF2966), anti- FLAG (l: 100, Sigma, cat. no. B3111). anti-Hl.O (1: 100, Abeam, cat. no. abl34914). anti-Vimentin (1 :200, Abeam, cat. no. ab45939), or anti-Lamin A / C (1 :200, Abeam, cat. no. ab8984). Appropriate concentration of secondary antibodies was incubated at room temperature for Ih. Imaging was performed at a fluorescence microscope (Zeiss Axio Vert.Al) or confocal microscope (Nikon AIR. 60x). Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI). Secondary antibody staining alone was used as a negative control.Bulk RNA-seq and bioinformatics analysis

[0164] Pellets from 3 biological replicates of primary isolated cardiac fibroblasts transfected with H1.0 or scrambled siRNAs and treated with TGF-J3 or vehicle were processed for RNA isolation, library' preparation and sequencing. Ribosomal RNA was removed using KAPA RNA HyperPrep kit (Roche, cat. no. kk8561). Approximately 40 million paired-end reads per sample (2xl50bp) were generated during sequencing. Raw fastq.gz files were downloaded and processed as described (Chapski et al., 2021, J Mol Cell Cardiol, 160:73-86), with the following modifications. Salmon vl.4.0 (Patro et al., 2017, Nat Methods, 14:417-419) was used to pseudoalign reads to an mmlO index built from Ensembl build 102. DESeq2 (Love et al., 2014, Genome Biol, 15:550) was used to perform differential expression testing, specifically on genes that had at least 10 reads measured between the total samples, and significantly differentially expressed genes were defined as those with adjusted pvalue (padj) less than 0.01. Principal component analysis was performed using the plotPCAQ function in DESeq2 and visualized using ggplot2 (Wickham, H. in ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, 2016) in R. Heatmaps were visualized using gplots (https: / / cran.r- project.org / web / packages / gplots / index.html) in R, or Prism software v9.0 (GraphPadSoftware, San Diego, CA, USA). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using g:Profiler (Raudvere et al., 2019, Nucleic Acids Res 47:W191-W198) on the subset of genes upregulated by log2FoldChange of 1.5 with TGF-P (when compared to scrambled negative control) and then downregulated by log2FoldChange of 1 .5 in the H 1 .0 siRNA + TGF-P condition (when compared to TGF- alone).RT-qPCR

[0165] Total RNA was isolated from primary cardiac fibroblasts or pri maty- lung fibroblasts from C57BL / 6J mice using an RNA isolation kit (Zymo, cat. no. R1018) for RT-qPCR. Heart tissue from C57BL / 6J mice was homogenized and lysed with Trizol (ThermoFischer Scientific Cat#15596018). Total RNA was extracted following with manufacturer's instructions. cDNA was generated according to manufacturer instructions (Bio-Rad, cat. no. 1708891) and quantitative real-time PCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad) using SsoFast EvaGreen Supermix (Bio-Rad, cat. no. 1725201). All primer sequences used in this study are listed in Table 1.RNAi assay in vitro

[0166] Lipofectamine RNAiMAX (Thermo Fisher, cat. no. 13778150) transfection was performed following the manufacturer's protocols. Briefly, 500pL of Opti MEM Reduced Serum Media (Thermo Fischer Scientific, cat. no. 31985070) containing either Dharmacon’s siRNA targeting H1.0 (40nM, Horizon Discovery7cat. no. M-060325-01), Hl.l (80nM, Horizon Discovery7cat. no. M-049956-00), Hl.2 (80nM, Horizon Discovery cat. no. M-045246-00), Hl.3 (80nM, Horizon Discovery cat. no. M-051171-00). Hl.4 (80nM, Horizon Discovery cat. no. M-042536-01), Hl.5 (80nM, Horizon Discovery cat. no. M-049995-00), Thbs4 (40nM, Horizon Discovery cat. no. Cat.M-044016-01) or the appropriate concentration of siRNA scramble control (Horizon Discovery7cat. no. D-001206-14) were mixed with 500pL of OptiMEM Reduced Serum Media containing 20pL of Lipofectamine RNAiMAX (ThermoFisher Scientific cat.no. 13778075,). For the human skin fibroblast experiment, transfection was performed using Dharmacon’s siRNA targeting human H1.0 (40nM, Horizon Discovery cat. no. M-017209-01). After incubating the reagents for 10 minutes at 37°C, the solution was added to the cells and slightly agitated to mix. After 24 hours incubation at 37°C, the siRNA reagent solution was removed and replaced with appropriate media according to the downstream experiment. siRNA sequences are listed in Table 1.Viral infection in isolated cells

[0167] Isolated cardiac fibroblasts were infected with either mouse Adv-GFP- H1.0-FLAG (Vector Biolabs, custom generated for this study) or human Adv- HDACl(Vector Biolabs, cat.no. 1498) with Adv-GFP (Vector Biolabs, cat.no.1768) as a negative control, using a multiplicity of infection (MOI) of 200 plaque forming units per cell. After 24 hours incubation at 37°C, the solution was removed and replaced with DMEM / F12 media containing 10% FBS, 1% antibiotics (penicillin and streptomycin) and 0.1% ITS. For the Adv-GFP-Hl.O-FLAG experiments, cells were collected 48 hours after infection. In the case of the human Adv-HDACl experiments, an additional infection was performed 24 hours after initial infection, and cells were collected for downstream analyses 24 hours later, for a total of 48 hours of infection.AAV9 delivery in vivo

[0168] AAV9-Tcf21-GFP-shRNAmir encoding shRNA against histone Hl .0 was custom generated by Vector Biolabs for this study with sequences (shRNAmir) optimized to facilitate transcription of small non-coding RNAs, including shRNA. A separate vector, encoding a scrambled shRNA, was used as control. Transfection was validated by GFP fluorescence and histone Hl .0 abundance. AAV9 (3xl0Al Iparticles / mouse) were injected via tail vein into C3H / HeJ mice (8-12 weeks). Five weeks after injection. PBS or ISO (20 mg / kg / day) were injected for oneweek. Mice were euthanized and then hearts were harvested 7days post-ISO treatment. A similar strategy7was employed to target cardiac fibroblasts previously (Francisco, J. et al., 2021, Sci Rep 11, 10553). shRNA sequences are listed in Table 1.Collagen gel contraction assay

[0169] Primary cardiac fibroblasts were transfected with H1.0 or scrambled negative control siRNA for 48 hours. Fibroblasts suspended in 10% serum- supplemented DMEM / F-12 medium were seeded (0.5xl0A6 cells / mL) on collagen gels 24 hours prior to serum deprivation for 4 hours. At the beginning of contraction, gels were released from wells using a pipette tip and treated with TGF-P (lOng / mL; Novoprotein cat. no. CA59) for 24 hours. Primary cardiac fibroblasts transfected with Adv-GFP or Adv-GFP-Hl.O-FLAG for 48 hours were suspended in 10% serum- supplemented DMEM / F-12 medium, seeded (0.5xl0A6 cells / mL) on collagen gels for 8 hours and then released from w ells for 24 hours. Gel images w ere acquired by a ChemiDoc MP Imaging System (Bio-Rad). Gel area was calculated using ImageJ (Schneider et al., 2012. Nat Methods, 9:671-675) and Fiji (Schindelin et al., 2012, Nat Methods, 9:676-682) and contraction was reported as percentage of contraction.Traction force assay

[0170] Primary cardiac fibroblasts transfected with Cy3 labeled siRNA (Horizon Discovery, cat.no. D-001620-03) together with either scrambled or H 1.0 siRNA with or without TGF-P stimulation were seeded onto BSA conjugated to a 647-fluorophore micropattemed onto a flexible poly dimethylsiloxane (PDMS). Fibroblasts and patterned BSA dots were imaged and deformation of the dots quantified and converted into forces as described (Beussman et al., 2021. Acta Biomater).CCK-8 proliferation assay

[0171] Primary cardiac fibroblasts were seeded in a 48 well plate (1x104 cells / well) overnight, transfected with H1.0 or scrambled siRNA for 48 hours and then treated with TGF-P (lOng / mL) or vehicle for 24 hours. After 2 hours incubation with 20pL of CCK-8 solution to each well, absorbance at 450nm wavelength was recorded in a BioTek h 1 ,gy Hl Hybrid plate reader as a readout for cell proliferation.Wound healing assay

[0172] Wound healing experiments were performed on 1) primary mouse cardiac fibroblasts transfected with Adv-GFP-Hl.O-FLAG or Adv-GFP for 48 hours; 2) primary murine cardiac fibroblasts transfected with H1.0 or scrambled siRNA for 48 hours and then treated with TGF- (lOng / mL) or vehicle for 24 hours; or 3) human skin fibroblasts from passage 9 transfected with H 1.0 or scrambled siRNA for 48 hours. After transfection and / or TGF-P treatment, when cells were around 100% confluency, a scratch was made in the culture plate using a P200 pipette tip. Images were taken at 0 and 24 hours using a microscope (Zeiss Axio Vert. Al), and the percentage of w ound closure was calculated using ImageJ (Schneider et al.. 2012, Nat Methods 9:671-675) and Fiji (Schindelin et al., 2012, Nat Methods 9:676-682).Ingenuity Pathway Analysis (IP A)

[0173] Based on differential gene expression analysis of RNA-seq data detailed above, core analysis was applied in IPA to identify potential upstream regulators by comparing gene expression in Scramble +TGF-0 vs Scramble and H1.0 siRNA + TGF-P vs Scramble + TGF-P groups. The activation z-score (z > 2 indicates activation or z < -2 indicates inhibition) was applied to predict activation or inhibition state of upstream regulators. The related gene expression changes and pathways with upstream regulators were displayed in Figure 3 to illustrate a possible mechanistic network in TGF-P treated cardiac fibroblasts. The same genes and pathways were displayed for the H1.0 siRNA + TGF-P group. The Path Designer tool within IPA wasused to visualize these networks. For ease of visualization, some genes appear without lines (Ingenuity Systems).Immunohistology

[0174] Cardiac tissue samples were fixed in 10% formalin buffered solution (Sigma, cat. no. HT501128) overnight, dehydrated in 70% ethanol and sent to the UCLA Translational Pathology Core to generate paraffin blocks. Samples were cut into 4pm thick slices, put on slides, and stained with hematoxylin and eosin or Masson's trichrome stain (Sigma, cat. no. HT15-1KT) to detect fibrosis. Fibrotic area for each slide was quantified and expressed as the percentage of the area occupied by the whole heart on a given slide. For kidney and lung fibrosis quantification, where the whole organ was not able to be imaged at high resolution within the same field of view, five images were taken from each mouse using I Ox magnification (Zeiss Axio Vert.Al). ImageJ (Schneider et al., 2012, Nat Methods 9:671-675) and Fiji (Schindelin et al., 2012, Nat Methods 9:676-682) were used to calculate fibrotic area.Cardiac fibrosis quantification

[0175] For cardiac fibrosis quantification, whole mouse hearts were cut into 2- 3 pieces (~2mm in size). Subsequently, each piece was sectioned into 4pm thick slices, mounted onto slides, and subjected to staining with hematoxylin and eosin or Masson's trichrome stain (Sigma, cat. no. HT15-1KT) to detect fibrosis. The area of fibrosis was determined using color thresholding in Image J. The quantitative data presented are from 2-3 histological sections taken from 2 or more regions of ventricle, for a total of 5-6 images per heart.Kidney fibrosis quantification

[0176] For kidney fibrosis quantification, where the whole organ was not able to be imaged at high resolution within the same field of view, five images in TIFF format were randomly taken from each mouse using lOx magnification. ImageJ andFiji were used to calculate fibrotic area. Specifically, the color threshold option from Fiji was applied to set the appropriate threshold to distinguish Masson’s trichrome staining (deep blue) from background staining as follows, for each image: Fibrotic Area % = Deep Blue Area / Total Area.Targeted nuclease digestion and RT-qPCR

[0177] Chromatin accessibility in isolated cardiac fibroblasts or whole heart from C57BL / 6J mice was measured using the Chromatin Accessibility Assay Kit (Abeam, cat. no. abl 85901) according to the manufacturer's instructions. After chromatin digestion and DNA purification, a TapeStation 4200 (Agilent) was used to visualize DNA fragment size and intensity. Open chromatin is easily accessed bynucleases and digested more frequently, thereby showing a lower qPCR amplification signal relative to less accessible regions. To assess chromatin accessibility at specific loci within the periostin and aSMA promoters by RT-qPCR, three independent primer sets were designed for each promoter (sequences are provided in Table 1). Undigested DNA was used as a negative control.Co-immunoprecipitation (co-IP)

[0178] Primary- murine cardiac fibroblasts were transfected with Adv-GFP- H1.0-FLAG or Adv-GFP for 48 hours. The co-IP assay was performed using the FLAG Immunoprecipitation kit (Sigma, cat. no. FLAGIPT1-1KT) according to the manufacturer's instructions. Immunoprecipitated proteins were eluted using the SDS sample buffer included in the kit, and then subjected to immunoblotting.Hypo- and hypertonic treatment of cells

[0179] Isolated murine cardiac fibroblasts were exposed to: 1) a ( 1 : 1) mix of DMEM / F12 media supplemented with 10% FBS, 1% antibiotics (penicillin and streptomycin), and 0.1% ITS and water to reach a concentration of 140 mOsm (hypotonic treatment); or 2) a mix of DMEM / F12 media supplemented with 10%FBS, 1% antibiotics (penicillin and streptomycin), and 0. 1% ITS and 10X PBS (2mL media mixed with 213pL PBS) to reach a concentration of 560 mOsm (hypertonic treatment). One hour after treatment, cells were fixed using 1.6% formaldehyde (PF A) solution in phosphate buffered saline (PBS).Image analysis of chromatin condensation parameter (CCP)

[0180] Primary mouse cardiac fibroblasts were fixed with 1.6% PFA and stained with DAPI. Nuclear images were taken using a confocal microscope (Nikon, AIR, 60x). Images were converted to 8-bit format and each individual nucleus was cropped from the image field by the Fiji package within ImageJ (Schneider et al., 2012, Nat Methods 9:671-675; Schindehn et al., 2012, Nat Methods 9:676-682). Chromatin condensation parameter (CCP) was calculated using a previously published MATLAB script (Irianto et al., 2014, Med Eng Phys. 36:412-417; Irianto et al., 2013, Biophys J, 104:759-769 (2013). Briefly, the Sobel edge detection algorithm was applied to define edges within the nucleus. The density of edges within nucleus was then normalized to its cross-sectional area, giving the measured level of chromatin condensation.Cellular deformability assay

[0181] To measure the deformability of cardiac fibroblasts under different treatment conditions, suspended cells were filtered by air pressure through 10 pm porous membrane (Milhpore) on timescales of seconds using cellular microfiltration as previously described (Qi et al., 2015, Sci Rep 5: 17595). Cell deformability was determined by measuring the retention volume after 2-4 min of applied pressure. Large volume retained indicates cells are less deformable. Small volume retained indicates cells are more deformable. Prior to the filtration assay, cell viability (Trypan Blue staining) and cell size were measured by automated cell counter (TC20, BioRad). To perform the assay, 400 pL cell suspension (5xl0 5 / mL) were loaded into each well of a 96 w ell plate. The absorbance of retained cell volume was measured at562 nm by a plate reader (SpectraMax, M2). Retention was determined by the retained volume of cells divided by the initial volume.ChlP-seq and bioinformatics analysis

[0182] Hl .0 and FLAG ChlP-seq: Primary isolated murine cardiac fibroblasts were transfected with Adv-GFP-Hl.O-FLAG for 48 hours. FLAG and H1.0 chromatin immunoprecipitation was performed using anti-FLAG (Sigma, cat. no. F1804) and anti-Hl.O (Proteintech, cat. no. 17510-1-AP) antibodies. In a separate experiment, primary isolated mouse cardiac fibroblasts were transfected with H1.0 siRNA or scramble for 72 hours. Chromatin shearing was performed using the truChIP Chromatin Shearing kit (Covaris, cat. no. 520154) according to the manufacturer's instructions. DNA fragment size was assessed using a TapeStation 4200 (Agilent). Samples in the 300-500bp range were used for immunoprecipitation using the ChlP-IT High Sensitivity kit (Active Motif, cat. no. 53040). DNA was purified using a Zymo DNA Clean & Concentrator-5 kit (Zymo, cat. no. D4014). Library preparation and DNA sequencing were performed by the UCLA Technology Center for Genomics & Bioinformatics Core. Approximately 35 million paired-end reads per sample (2xl50bp) were generated and used for bioinformatic analyses. Alignment of paired-end reads to the mmlO genome was performed as described (Chapski et al., 2021, J Mol Cell Cardiol, 160:73-86). After using the bamCoverage function of deepTools (Ramirez et al., 2O16.Nucleic Acids Res 44:W160-165) with parameters — smoothLength 150 and — normalizeUsing RPGC to generate log2(IP / Input) bigWig tracks, the computeMatrix function was used with parameters - b 5000 -a 5000 —binSize 250 — nanAfterEnd —referencePoint TSS —skipZeros to calculate occupancy around transcription start sites of genes upregulated, downregulated. and unchanged with a given biological treatment. Visualization of occupancy as a ChlP-seq profile was performed using the plotProfile function of deepTools v3.0.2 with default parameters.

[0183] H3K27ac ChlP-seq: Three biological replicates of isolated cardiac fibroblasts from passage 1 were transfected with H1.0 or scrambled siRNAs for 48 hours and treated with TGF-0 (10 ng / mL) for 24 hours. H3K27ac immunoprecipitation was performed using the same experimental strategy as the H 1.0 ChlP-seq experiment, but using an anti-H3K27ac antibody (Abeam, cat. no. ab4729). For each biological condition, a combined input sample of sonicated genomic DNA was obtained from all three biological replicates. Library' preparation and paired-end sequencing were performed at the UCLA Technology Center for Genomics and Bioinformatics Core, resulting in -50-70 million read pairs (2xl50bp) per sample. Alignment was performed against the mmlO genome using bowtie2 (Langmead et al., 2012, Nat Methods, 9:357-359) followed by SAM-to-BAM conversion and sorting using samtools vl.7 (Li et al., 2009, Bioinformatics 25:2078-2079).

[0184] Peak calling for each sample was performed using MACS v2.2.7. 1 (Zhang et al., 2008, Genome Biol, 9:R137), using the callpeak function with the following layout and parameters: —treatment ChIP_replicate.sorted.bam —control Input_sorted.bam -f BAMPE -g mm. Differential occupancy of H3K27ac w as determined using the DiffBind package (Ross-Innes et al.. 2012, Nature 481:389-393) v3.6.1 in R, specifically on a set of consensus peaks measured in at least 3 samples across the experiment. Significantly differentially occupied regions w ere defined as those with FDR < 0.05. To visualize H3K27ac signal in differentially occupied (FDR < 0.05) regions with TGF-J3 that undergo an opposite change (no thresholding) in H3K27ac signal with H1.0 depletion, a heatmap visualization w as used. For each biological condition, read alignments from all three biological replicates were merged using the samtools vl.7 merge function, followed by sorting using the samtools vl.7 (Li et al., 2009. Bioinformatics, 25:2078-2079) sort function and then generated genome browser tracks (bigWig files) of the log2FoldChange in signal using the bamCompare function of deepTools v3.0.2 with the — smoothLength 150 parameter, the — outFileFormat bigwig -bl treatment_merged.bam and -b2 control_merged.bam parameters. These bigWigs were used as inputs for the computeMatrix function ofdeepTools (Ramirez et al., Nucleic Acids Res 44:W160-165) v3.0.2 with the following parameters: reference-point —referencePoint center -b 5000 -a 5000 — skipZeros. The matrix output of computeMatrix was used as input for the plotHeatmap function of deepTools v3.0.2 with -zMin -1.2 and — zMax 1.2, which generated the final heatmap visualization of the H3K27ac ChlP-seq data. The subset of closest genes to these regions of interest, whose expression increases (no thresholding) with TGF-P (compared to scrambled control) and decreases (no thresholding) in the H1.0 siRNA + TGF-P condition (compared to TGF-P alone) were examined by g:Profiler (Raudvere et al., 2019, Nucleic Acids Res, 47:W191- W198) using an adjusted p-value threshold of < 0.05.ChlP-qPCR

[0185] ChlP-qPCR against histone Hl .0 or BRD4 was performed using the chromatin immunoprecipitation method described above for ChlP-seq, but with qPCR as the endpoint. For ChlP-qPCR measurements at specific loci, eluted immunoprecipitated DNA was used to perform qPCR using primer sets designed to amplify specific regions of the periostin. aSMA or Sertad4 promoters. Primer sequences are listed in Table 1. Primers against the GAPDH promoter were used as a positive control.Single RNA-seq bioinformatics analysis

[0186] Figure 1 A-Figure IB were generated from the single-cell RNA-seq data website fibroXplorer.com (Buechler et al., Nature, 593:575-579), using 100 random cells per indicated condition. To generate Figure 1C, data w as downloaded from NCBI (GSE120064) (Ren et al., 2020, Circulation, 141 : 1704-1719). Reads aligning to predicted genes and mitochondrial transcripts (those beginning with "Gm" and "mt-," respectively) were removed from the UMI matrix. Seurat v4.0.1 (Hao et al., 2021, Cell, 184:3573-3587) was used to create a Seurat object and perform all downstream analyses. The Seurat object was split by sample, normalized, and the3000 most variable features identified in each dataset with SCTransform (Hafemeister et al., 2019, Genome Biol, 20:296). The Seurat objects corresponding to each sample were then integrated together with iterative pairwise integration (Stuart et al., 2019, Cell 177: 1888-1902). Principal Component Analysis (PC A) was performed for the first 50 principal components of the integrated object. An Elbow Plot was used to determine the dimensions to use (16) for identifying neighbors and clustering. The k- nearest neighbors and shared nearest neighbor graph for the dimensionality -reduced dataset was computed with FindNeighbors, with the default k.param of 20. Louvain clustering was performed with FindClusters, with a resolution of 1.2 and visualized by UMAP. With default assay set to "RNA," feature counts were normalized by cell (LogNormalize method of NormalizeData) and features centered and scaled bystandard deviation (ScaleData) for downstream differential gene expression analysis. Markers of each cluster were identified with the Wilcoxon Rank Sum test via FindAllMarkers, comparing each cluster to all other cells, only testing genes that are detected in at least 25% of cells in either the cluster of interest or the other cells, and only returning genes with p-value < 0.05. Cell type clusters were determined by gene expression levels of markers in Ren et al. Figure IE (Ren et al., 2020. Circulation 141 : 1704-1719). Hl isoform ("HlfO", "Histlhla", "Histlhlc", "Histlhld", "Histlhle", "Histlhlb", "Hlfx") expression levels in each cell type were calculated with AverageExpression.

[0187] For Figure 8B and Figure 8F. data was downloaded from NCBI (GSE109816 and GSE121893) (Wang et al., 2020, Nat Cell Biol, 22: 108-119). UMI and metadata tables from both sources were merged and intersected, respectively. Because there are cells in the UMI matrix for which metadata is not available, the UMI matrix was trimmed to include only those cells for which there is associated metadata. The UMI matrix and metadata was further filtered to include only the following: cells that express greater than 500 genes / cell, cells for which UMI count was within 2 standard deviations from the mean of loglO(UMI) of all cells, cells with mitochondrial gene expression ("MT-") less than 72%, and cardiomyocytes withsufficient UMIs (over 10000). Lastly, all mitochondrial genes ("MT-") were filtered from the UMI matrix. Seurat v4.0. 1 was used to create a Seurat object of the remaining 10,077 cells and perform all downstream analyses. The Seurat object was split by sample, and each of the 20 samples was normalized with Seurat's NormalizeData function, in which feature counts for each cell are divided by the total counts for that cell and multiplied by 10000 before being natural-log transformed using loglp. The most variable features in each dataset were identified by dividing features into 20 bins based on average expression and calculating z-scores for dispersion within each bin with FindVariableFeatures (selection.method = "mvp"). These variable features were then used for iterative pairwise integration (Stuart et al., 2019, Cell 177:1888-1902 el 821 ). PCA was performed for the first 50 principal components of the integrated object. The k-nearest neighbors and shared nearest neighbor graph for the dimensionality-reduced dataset was computed with FindNeighbors, with 10 dimensions used and the default k.param of 20. Louvain clustering was performed with FindClusters, with a resolution of 1 and visualized by UMAPPlot. To identify cardiac fibroblast cells, the default assay was changed back to "RNA" and feature counts were normalized by cell (LogNormalize method of NormalizeData) and features centered and scaled by standard deviation (ScaleData) for downstream differential gene expression analysis. For Figure 8B, Differential gene expression in each cluster was identified with the Wilcoxon Rank Sum test via Find AllMarkers, comparing each cluster to all other cells, only testing genes that are detected in at least 20% of cells in either the cluster of interest or the other cells, and only returning genes with p-value < 0.05. Cell type clusters were determined by gene expression levels of markers in (Wang et al., 2020, Nat Cell Biol, 22: 108-119). Hl isoform ("H1F0", "HIST1H1A", "HIST1H1C", "HIST1H1D", "HIST1H1E", "HIST1H1B", "H1FX") expression levels in each cell type were calculated with AverageExpression. For Figure 8F, the dataset was further subset to just those fibroblasts with nonzero expression of POSTN and H1F0, and the expression ofPOSTN and H1F0 in the resulting 51 cells was reported with FetchData. Scatterplots were plotted with ggpubr and Spearman correlation calculated with stat_cor().

[0188] For human data in Figure 8D, snRNA-seq data w as downloaded from the Chan Zuckerberg CELL x GENE Discover database (https: / / cellxgene.cziscience.com / collections / 8191c283-0 16-424b-9b61-c3el d6258a77). To convert the uploaded .h5ad file to a Seurat object, Scanpy was used to run sc.read_h5ad() and create a folder of matrix, features, and barcode files for import into Seurat v4.0. 1. Cardiac fibroblast nuclei, as identified by the associated metadata, were subset from the object. With default assay set to "RNA," feature counts were normalized by nucleus (LogNormalize method of NormalizeData) and features centered and scaled by standard deviation (ScaleData) for downstream differential gene expression analysis. The dataset was further subset for those fibroblasts with nonzero expression of POSTN and H1F0, and the expression of POSTN and H1F0 in the resulting 345 nuclei was reported with FetchData. Scatterplots were plotted with ggpubr and Spearman correlation calculated with stat_cor().

[0189] For human data in Figure 8E. the scRNA-seq data was downloaded as a Seurat object from NCBI (GSE183852). 23,549 cardiac fibroblast cells, as identified by the associated metadata, were subset from the object. With default assay set to "RNA," feature counts were normalized by cell (LogNormalize method of NormalizeData) and features centered and scaled by standard deviation (ScaleData) for downstream differential gene expression analysis. The dataset was further subset for those fibroblasts with nonzero expression of POSTN and H1F0, and the expression of POSTN and H1F0 in the resulting 1257 cells was reported with FetchData. Scatterplots w ere plotted with ggpubr and Spearman correlation calculated with stat_cor().Quantification and statistical analysis

[0190] Data are presented as the mean ± SD, unless otherwise indicated in the figure legends. Statistical analyses were performed using Prism software v9.0(GraphPad Software, San Diego, CA, USA) using Welch's t-test between two groups and one-way ANOVA with Tukey's multiple comparison analysis between three or more groups. A p-value less than 0.05 was considered statistically significant. Two sided tests were performed and all "replicates’ are biological replicates, meaning from different animals or cell isolations (depending on the type of experiment), unless otherwise noted.Data availability

[0191] Raw and processed RNA-seq and ChlP-seq data generated during this study were deposited in the National Center for Biotechnology Information Gene Expression Omnibus and are available for download using accession number: GSE215268.Code availability

[0192] All software used in this study was from public softw are packages described in the methods and detailed in the accompanying references.The experimental results are now described.Histone HEP enriched in fibroblasts and stress responsive

[0193] To investigate the role of linker histone isoforms in response to cellular stress, single cell RNA sequencing (scRNA-seq) data (Buechler et al., 2021, Nature 593:575-579) was examined to reveal the natural variation in these isoforms amongst cells. Because fibroblasts are ubiquitous cells present in nearly all organs of the body, a database of murine fibroblasts from various organs was examined and analysis showed that regardless of tissue of origin, linker histone Hl .0 is more highly expressed than other linker histone Hl variants (Figure 1 A). Fibroblasts play an important role in sensing extracellular tension and responding to organ level stress: therefore the linker histone variant transcripts in fibroblasts were further examinedfrom various injured murine tissues. In the mouse tissues surveyed, H1.0 is the predominant linker histone variant (Figure IB). Whether the same histone isoforms are operative in humans is unclear: histone H1.0 and Hl. 10 are expressed in fibroblasts from diseased human tissues (Figure 8A and Figure 8B) and analysis of three separate single cell RNA-seq datasets from human hearts revealed a positive correlation between histone H1.0 and periostin, a canonical marker of fibroblast activation, as did data from GTEx (Figure 8C through Figure 8F). Global transcriptome analyses in a genetically diverse population of mice administered the adrenergic agonist isoproterenol (Rau et al., 2015, Circulation Cardiovascular Genetics 8:40-49), which stiffens the muscle through increased fibrosis, showed a strong association of histone H1.0 levels with metrics of heart muscle pathology and dysfunction, including left ventricular mass and the echocardiography parameters E and A amplitude, measurements of the heart's ability to relax during diastole (Figure 9 A). Bulk RNA-seq analyses of mouse hearts showed that despite cardiomyocytes contributing the vast majority7of the heart mass, fibroblasts accounted for the greatest level of histone H1.0 transcript expression (Figure 9B), a finding also supported by analysis of single-cell RNA-seq data of murine cardiac cells (Figure 1C). Histone H 1.0 is known to be the principal, if not only, histone Hl isoform poly-adenylated in mammals. Figure 1 A and Figure IB depict data obtained from the poly-A capture of mRNAs (Buechler et al., 2021. Nature 593:575-579), revealing histone H1.0 and Hl.2 to be the most abundant at the transcript level. However, Figure 8B through Figure 8C show data that are not based on poly-A capture of mRNA (Wang et al., 2020, Nat Cell Biol 22: 108-119; Ren et al., 2020, Circulation 141 : 1704-1719), yvherein ribosomal RNA depletion vas employed, leading to the same observation. Thus, it can be reasoned that the greater abundance of histone H1.0 at the transcript level is not due to a bias of poly-A selection. Previous work also indicates this differential abundance is reflected at the protein level (Franklin et al., 2012, Mol Cell Proteomics 11 , Ml 1 1 014258).

[0194] To test this role of histone H1.0 in fibroblast activation, a primary adult mouse fibroblast model system was adopted treated with transforming grow th factor beta (TGF-P), a cytokine involved in stress response throughout the body, including in the heart (Bujak et al., 2007, Cardiovasc Res 74: 184-195). TGF-P treatment induced fibroblast activation as measured by periostin and alpha smooth muscle actin (aSMA) protein expression and demonstrated by actin stress fiber formation (Figure ID), concomitant with dynamic changes in histone H1.0 protein levels over time (Figure IE). Histone Hl.2, the second most abundant isoform in fibroblasts, was increased in abundance at 48h after TGF-p, whereas Hl.5 was decreased (Figure 9C). All other isoforms of histone Hl w ere unaffected by TGF-P treatment (Figure 9C).Histone HEP necessary and sufficient for fibroblast activation

[0195] Previous studies demonstrated compensatory upregulation of other isoforms following germline deletion of individual linker histones (Fan et al.. 2001, Mol Cell Biol 21 :7933-7943; Sirotkin et al., 1995, Proc Natl Acad Sci U S A 92:6434-6438). Therefore, an siRNA-mediated knockdown approach targeting the six main isoforms expressed in somatic cells was used. Knockdown of histone H1.0 prior to administration of the cytokine w as sufficient to prevent TGF-P-induced fibroblast activation as measured by periostin and aSMA transcript (Figure 9D) and protein abundance (Figure 2A). Depletion of histone H1.0 also prevented actin stress fiber formation (Figure 2B). Knockdown of histone H1.0 had modest effects on other Hl isoforms (Figure 9F), yet individual knockdown of the other five isoforms had no effect on fibroblast activation (Figure 9G through Figure 9K; note: knockdown of histone Hl.2, the next most abundant Hl isoform, does not affect TGF-p-induced gel contraction, Figure 9L), illustrating that even though genetic loss of linker histone Hl isoforms can be compensated developmentally (Fan et al., 2001, Mol Cell Biol 21:7933-7943; Fan et al., 2003, Mol Cell Biol 23:4559-4572), these individual isoforms play distinct roles in the somatic cell.

[0196] Depletion of histone H1.0 did not alter levels of core histones H3, H2A or H4, with only a modest change in the level of H2B (Figures 9E and Figure 10A through Figure 10C). thereby resulting in a decreased linker-core ratio. These observations are in contrast to germline knockouts of Hl isoforms, which result in compensatory alteration in other core histone levels and a maintenance of the linker to core nucleosome ratio — a key feature shown previously to regulate chromatin structure and nuclear architecture (Fyodorov et al., 2018, Nature reviews. Molecular cell biology 19: 192-206; Bujak et al., 2007, Cardiovasc Res 74: 184-195). Previous studies observed fewer linker histones per nucleosome in the setting of cardiac hypertrophy (Franklin et al., 2012, Mol Cell Proteomics 11, Mi l l 014258). These findings suggest that transient depletion of histone H1.0 alters the linker-core histone ratio, unmasking endogenous roles of linker histones that are compensated for in histone H 1.0 germline knockouts. If the fibroblasts are already activated, histone H1.0 knockdown does not reverse the effects of TGF-P (Figure 10D) whereas simultaneous knockdown at the time of TGF-P treatment was sufficient to block aSMA but not periostin expression (Figure 10E). It has been hypothesized that proper histone stoichiometry is necessary for stress response in fibroblasts and that once the transcriptional program is activated in response to agonist, the window for modulating chromatin architecture to prevent this stress response has closed.

[0197] To test whether changes in marker gene expression were indicative of phenotypic changes in activated fibroblasts, the effect of histone H1.0 depletion on distinct mechanical behaviors in primary cells was examined. A traction force assay was employed, in which cells were seeded onto fluorescently labeled bovine serum albumin (BSA) beads and the deformation of the beads was used to measure the force generated by individual fibroblasts (Beussman et al., 2021, Acta Biomater, 163:302- 311). Treatment with TGF-P induced robust traction force generation at the individual cell level and this response w as completely abrogated by depletion of histone H1.0 (Figure 2C Note: In traction force experiments, which measure single cells, all groups were co-transfected with an siRNA conjugated to Cy3 fluorescent tag alongwith either the scrambled siRNA or the siRNA against histone H1.0, and only cells expressing this tag were selected for measurement). When the contractile behavior of the whole population of cells on the culture dish was examined with a gel contraction assay, histone H1.0 was again found to be necessary for the TGF-P induced contractile phenotype (Figure 2D). Furthermore, overexpression of histone H1.0 demonstrated that increasing the levels of this protein in the nucleus (Figure 11 A) was sufficient to induce fibroblast activation in the absence of cytokine stimulation as measured by periostin and aSMA expression (Figure 2E) and gel contraction assay (Figure 2F).

[0198] To examine whether the relationship between histone H1.0 and fibroblast mechanical behaviors was more universal, a different activating stimuli was used to examine distinct mechanical properties of cells from various tissues and species. Histone H1.0 was necessary for activation of fibroblasts from mouse lung (Figure 10B), mouse skin (Figure 10C) or human skin (Figure 10G through Figure 10H). Activation of cardiac fibroblasts by angiotensin II was also dependent on histone H1.0 (Figure 10D). Depletion of histone H1.0 does not impair cell viability but w as sufficient to prevent cardiac fibroblast proliferation in response to TGF-P (Figure 2G) as measured by CCK-8 viability assay, as w ell as in the setting of a cell migration assay in which confluent cells are mechanically disrupted and allowed to close a pseudo wound (Figure 10E through Figure 10G). Overexpression of histone H1.0 promoted active wound closure in the absence of TGF-P stimulation (Figure 10H), demonstrating that histone H1.0 is sufficient to induce this proliferative response.Histone HEP coordinates chromatin remodeling machinery

[0199] To investigate molecular mechanisms whereby histone H 1.0 participates in fibroblast activation, RNA-seq was used to determine the transcriptome changes following TGF-P treatment that are dependent on histone H1.0. Depletion of histone H1.0 prevented a select subset of transcriptional changes induced by TGF-Ptreatment (Figure 3 A and Figure 12A). Ingenuity Pathway Analyses revealed that up- regulated genes whose expression was blocked by histone H1.0 knockdown are involved in key intracellular and extracellular processes (Figure 3B). KEGG analyses revealed an enrichment in pathways associated with ECM (Figure 3C and Figure 12C) and signaling via protein kinase B / Akt (activation of which is histone Hl .O-dependent (Figure 12C)), a protein associated with growth and proliferation. Among histone H1.0 target genes w as thrombospondin 4 (Thbs4), a secreted ECM protein known to positively regulate tissue healing and previously shown to be necessary for normal fibrotic deposition after cardiac muscle injury (Frolova et al., 2012, FASEB J 26:2363-2373). Knockdown of histone H1.0 blocked the TGF-P-induced increase in THBS4 at the transcript and protein level (Figure 12D). Thbs4 was required for TGF- P-induced activation of myofibroblast genes periostin and aSMA expression (Figure 12E through Figure 12F), demonstrating this to be a necessary downstream gene regulatory7target of histone H1.0.

[0200] It can be reasoned that histone Hl.O’s ability to directly bind chromatin and alter gene expression underpins these changes in gene expression and fibroblast phenotype. Therefore, the influence of histone Hl .0 levels on RNA polymerase II (RNAP II) expression and activation w as further examined. Histone H1.0 knockdown decreased the transcript levels of RNAP II subunit a and prevented the TGF-p- induced increase in subunit c, whereas subunit b was slightly increased and d was unchanged by histone H1.0 depletion (Figure 12G through Figure 12H). Histone Hl.0 depletion also decreased the proportion of RNAP II that is serine 2 phosphorylated in response to TGF-p stimulation (Figure 3D), indicating that histone H1.0 participates in TGF-P-induced activation of transcription in part by regulating RNAP II subunit levels as well as post-translational modification. One mechanism of chromatin remodeling is via histone acetylation, which alters local chromatin compaction and serves to recruit reader proteins, which in turn facilitate engagement of transcriptional machinery (Shahbazian et al., 2007, Annual review of biochemistry 76:75-100).Inhibition of histone deacetylases (HDACs), which remove acetyl groups from lysineson histones and other proteins, is sufficient to block fibroblast activation (Travers et al., 2021, Circulation 143: 1874-1890). Depletion ofhistone H1.0 led to an increase in total H3K27Ac (Figure 4A), a mark associated with transcriptionally active enhancers, suggesting a shift towards more active chromatin. To examine the role of histone Hl .0 in regulating chromatin accessibility via acetylation, it was concluded that histone H3K27Ac ChlP-seq in TGF-|3-treated cells in the presence and absence of histone H1.0. Remarkably, depletion ofhistone H1.0 blocks the locus specific changes in H3K27Ac induced by TGF-0 (Figure 4B). Genes whose increase in H3K27Ac occupancy was blocked by histone Hl.0 knockdown were enriched in pathways associated with cell migration, proliferation and ECM production (Figure 4C), demonstrating that histone H1.0 is necessary for the proper acetylation of chromatin around these genes.

[0201] Increased pressure in the heart, which leads to fibroblast activation, pathologic muscle grow th and heart failure, is associated with elevated HDAC activity (Zhang et al., 2002, Cell 110:479-488) and these data demonstrate that histone H1.0 is necessary for TGF-|3-induced upregulation of HDAC1 (Figure 4D). HDAC1 is preferentially expressed in fibroblasts versus muscle cells in the heart (Nural-Guvener et al., 2014, Fibrogenesis Tissue Repair 7: 10) and overexpression of HDAC1 is sufficient to diminish global levels of H3K27Ac (Figure 4E-Figure 4F; HDAC inhibition has been previously shown to block TGF-|3-induced production of ECM Barter et al., 2010, Matrix Biol 29, 602-612). Co-immunoprecipitation demonstrates that histone H1.0 can bind HDAC1 in fibroblasts (Figure 4G), as previously documented for other HDAC isoforms in human cell lines (Kalashnikova et al.. Nucleic Acids Res 41:4026-4035), implying this regulatory interaction can also occur at the protein level. Thus, one mechanism by which histone Hl .0 can regulate transcription is by preventing upregulation of HDAC and thereby reducing its gene silencing effect.

[0202] To further investigate the molecular basis for how altered histone acetylation levels may influence transcription, experiments examined expression ofthe chromatin reader protein BRD4, which binds acetylated lysines, facilitating recruitment of positive transcription elongation factor b (P-TEFb) thereby releasing transcriptional pausing. Inhibition of BRD4 has been previously show n to block cell growth (Maruyama et al., 2002, Mol Cell Biol 22:6509-6520) and fibroblast activation (Xiong et al., 2016, Oncotarget 7:69291-69308). Results showed that histone H1.0 depletion leads to a decrease in BRD4 transcript and protein levels (Figures 4H-I), suggesting that reduction in the abundance of this chromatin reader is part of the mechanism by which histone H1.0 inhibition prevents fibroblast activation. These observations are in agreement with histone H1.0 dependence of changes in phosphorylation of RNA Pol II at Ser2 (Figure 3D), given that BRD4 is known to recruit the essential RNA Pol II regulatory factor P-TEFb (Yang et al, 2005, Mol Cell 19:535-545) and promote Ser2 phosphorylation (Devaiah et al, 2012. Proc Natl Acad Sci U S A 109:6927-6932). Binding of BRD4 to known TGF-p target genes was examined using ChlP-qPCR: TGF-P induced robust recruitment of BRD4 to the transcription start sites of Sertad4 (Stratton et al, 2019, Circ Res 125:662-677), Acta2 and Postn, which was completely prevented in all cases by depletion of histone H1.0 (Figure 4J). These findings, together with previous studies, indicate that histone Hl .0 coordinates reorganization of H3K27Ac around TGF-P target genes in part by regulating histone acetylation via HDACs and transcription by BRD4.Histone HEP regulates global chromatin compaction

[0203] Unlike transcription factors or some modified core nucleosome histones, the distribution of linker histone Hl across the genome is fairly ubiquitous (Serna-Pujol et al., 2022. Nucleic Acids Res 5:3892-3910, Teif et al, 2020. Epigenetics & chromatin 13:26). ChlP-seq for histone Hl .0 (Figure 12B) was performed and regions of relative depletion were investigated as described (Ito-Ishida et al, 2018, Nature neuroscience 21:794-798). Histone H1.0 is depleted at genes undergoing altered expression following fibroblast activation, with a greater depletion observed in genes whose expression is increased (Figure 5A), suggesting that histoneH1.0 eviction is associated with chromatin relaxation (Yusufova et al., 2021, Nature 589:299-305). Results confirmed specific localization of histone H1.0 to several TGF- P target genes using ChlP-PCR (Figure 5B). indicating that while its genomic distribution is broad, it is not uniform. Immunoprecipitation of endogenous histone H1.0 (Figure 5A) or overexpressed, tagged histone H1.0 (Figure 121) gave similar results. Next the role of histone H1.0 in chromatin folding was directly tested by performing nuclease digestion of genomic DNA to reveal the relative ratio of compact (nuclease inaccessible) to relaxed (nuclease accessible) DNA. Overexpression of histone H1.0 increased the proportion of DNA that was compacted and thus nuclease inaccessible, whereas knockdown of histone H1.0 had the antithetical effect (Figure 5C). Interestingly, treatment with TGF- shifted the genome to a more compact state and knockdown of histone H 1.0 reversed this effect (Figure 5C), demonstrating that this behavior of histone H1.0 to modulate chromatin fiber accessibility is operative in the context of fibroblast activation. Targeted PCR was performed for regions of Acta2 and Postn, demonstrating that the presence of histone H1.0 tended to compact these regions of chromatin (Figure 5D). Histone H1.0 overexpression or TGF- treatment renders the Acta2 and Postn loci less accessible (thus less DNA was recovered by PCR), whereas knockdown of histone H1.0 has the opposite effect. These findings suggest that normal levels of histone H1.0 establish a microenvironment for expression or repression of genes, such that perturbing the balance of histone H1.0 levels prevents normal stress-activated transcription.

[0204] Nuclear deformability has been implicated in diseases such as cancer and fibrosis (Kalukula et al., 2022, Nature reviews. Molecular cell biology, 9:583- 602) and is a major contributor to whole cell rigidity’ (Chalut et al., 2012, Biophys J 103:2060-2070). Therefore, a role for histone H 1 .0 to control global genome and nuclear stiffness was investigated using a deformability assay (Qi et al., 2015, Sci Rep 5, 17595). Depletion of histone H1.0 had a robust effect to increase the deformability of cells under basal conditions and to a lesser degree following stimulation with TGF- P (Figure 6A). Neither cell viability nor cell size were significantly altered bymodulating histone H1.0 levels (Figure 13A through Figure 13B), whereas TGF-P treatment increased the size and stiffness of cells independent of the nucleus (Figure 13D through Figure 13F), likely contributing to the muted effect of histone H1.0 depletion on cellular deformability measurements following TGF-p. In contrast, increasing the abundance of nuclear histone H1.0 w as sufficient to increase cellular retention in the absence of TGF-p (Figure 6A), which is consistent with increased cell and nuclear stiffness. Up-regulation of numerous cytoskeletal genes by TGF-P was blocked by depletion of histone H1.0 (Figure 6B; myosins were also under control of histone H1.0, Figure 13C) providing a mechanistic explanation for the effect of histone H1.0 depletion to alter cell compliance changes following TGF-p. To directly evaluate the role of histone H1.0 in genome compaction, nuclei w ere imaged and chromatin condensation parameter w as quantified, a measurement of global chromatin architecture (Irianto et al. ,014, Med Eng Phys 36:412-417), following modulation of histone H1.0 levels or hypotonic or hypertonic treatments as positive controls (which respectively decompact or compact chromatin. Figure 6C). Depletion of histone H1.0 levels caused global chromatin decondensation (Figure 6D) whereas augmentation of histone H1.0 caused condensation (Figure 6E). Combined with Figure 5, these findings demonstrate that histone H1.0 modulates chromatin compaction on a genome-wide scale — directly controlling overall cell deformability — via a local mechanism in which more histone H1.0 leads to more restrictive topology (Figure 6F).Histone HEP controls fibrosis in vivo

[0205] To investigate a role for histone H1.0 to control responses to physical stress in vivo, a model of catecholamine stimulation w ith isoproterenol, a nonselective P-adrenergic receptor agonist that increases cardiac work and thus tension on the muscle fiber, w as employed in addition to inducing fibrosis (Judd et al., 1969, Circ Res 25:201-214). Notably, catecholamines can also directly increase cellular tension in non-muscle cells (Kim et al., 2019, FASEB J 33:3997-4006; Nguyen et al., 2016,Integr Biol (Camb) 8: 1232-1245). Previous studies had shown that the fibrotic effects of isoproterenol are a complex trait strongly influenced by the genetic background of the mouse (Rau et al., 2015, Circulation Cardiovascular Genetics 8:40-49) and the two strains of mice were examined: C57BL / 6J, which has a modest fibrotic response, and C3H / HeJ, which exhibits a more pronounced response (Figure 13G). These analyses of C3H / HeJ mice demonstrated a positive correlation between histone H1.0 abundance and fibrotic deposition after isoproterenol treatment (Figure 13H). Administration of isoproterenol induced cardiac muscle hypertrophy in both strains, which was attenuated by coadministration of siRNA against histone H1.0 (Figure 7A through Figure 7B). Furthermore, in vivo depletion of histone H1.0 blocked isoproterenol from inducing an increase in the ratio of early to late peak diastolic filling velocities (E / A ratio) (Figure 7A-Figure 7B), a measure of diastolic function, where an E / A ratio > 2 is consistent with a less compliant, stiffer ventricle. No effect on ejection fraction, a measurement of systolic function, was observed following histone H1.0 depletion (Figure 131 and Figure 15). siRNA treatment was sufficient to deplete H1.0 in both mouse strains (Figure 7Band Figure 14A) as well as to block activation of fibrotic genes, including periostin and collagen 1A1, as measured by protein / transcript abundance (Figure 7C through Figure 7D). Knockdown of histone H1.0 was sufficient to transiently decondense chromatin in heart muscle as measured by nuclease accessibility (Figure 7E), indicating that the in vivo mechanisms of protection work through actions of histone El 1.0 to globally remodel the genome through local actions at the chromatin fiber. Results showed a significant prevention of isoproterenol-induced fibrosis (Figure 7F), demonstrating that histone H1.0 is essential for the transcriptional program driving production of ECM in vivo. Results also showed fibrosis in the kidneys of these isoproterenol treated animals, which was partially attenuated by depletion of histone H1.0 (Figure 14B through Figure 14C). Importantly, in vivo depletion of histone H1.0 with an orthogonal technique (AAV9- mediated delivery' of shRNA against a different region of the histone H1.0 transcript) was sufficient to recapitulate the phenot pes observed with siRNA-mediateddepletion (Figure 16). Taken together, these findings demonstrate a powerful effect of histone H1.0 to regulate fibrosis in vivo through its actions to control chromatin packaging.

[0206] One manner in which cells alter their microenvironment in response to physical and chemical stressors is via fibrosis, or the deposition of extracellular matrix proteins, thereby altering parenchymal mechanics. Results showed that tuning of histone Hl levels and chromatin compaction is necessary for response to stress stimuli and that the linker histone H1.0 isoform has a privileged role in this process. Results also demonstrated that augmenting histone H1.0 levels can recapitulate the chromatin organization, gene expression and mechanical cell behaviors in the absence of changes to cellular tension or cytokine stimulation. These findings support a central role for histone H1.0 as a molecular regulator of fibroblast stress response, coupling chromatin organization with cellular mechanical properties.

[0207] Alteration of histone Hl levels in vivo has been shown to shift global chromatin architecture between a relaxed (less Hl) or more compact (more Hl) state, and depletion of Hl promotes the development of lymphoma (Yusufova et al., 2021, Nature, 589:299-305), indicating that remodeling genome packaging through histone Hl is a conserved mechanism across cell types. These findings indicate that the levels of histone H1.0 can control chromatin condensation and thereby expression of genes associated with the cytoskeleton, force generation, ECM, and cellular motility. These findings raise the intriguing possibility that histone H1.0 may work directly via changes in nuclear compliance — that is, to change nuclear stiffness as a mechanism to change cell stiffness — in parallel w ith effects of histone H1.0 to control transcription of genes associated with altering cellular rigidity. Results demonstrated that the abundance of histone H 1.0 on chromatin is directly associated with chromatin compaction: depleting histone Hl .0 led to fiber relaxation and global decondensation, whereas overexpression had the opposite effect. These changes in chromatin accessibility prime the actions of other chromatin remodelers such as HD AC 1 andBRD4, which in turn modulate transcription of stress responsive genes in a histone Hl.O-dependent manner. It can be speculated that the observed role of histone H 1.0 to influence TGF-P induced Pol2 phosphorylation may be dependent on the concomitant effects on BRD4 and H3K27 acetylation in the context of transcriptional regulation.

[0208] The enrichment of histone H1.0 results showed across fibroblast populations is in agreement with this isoform being the only polyadenylated version of the linker histone family (Doenecke et al., 1988, Adv Enzyme Regul, 27: 107-120) and thus, the only one likely to be strongly expressed in non-dividing cells. Deletion of histone H1.0 in vivo did not adversely affect mouse development (Sirotkin et al., 1995, Proc Natl Acad Sci U S A, 92:6434-6438), likely due to compensation by other linker histone family members (linker to core nucleosome ratio was unchanged in these animals (Sirotkin et al.. 1995, Proc Natl Acad Sci S A, 92:6434-6438)). Subsequent studies depleted other histone Hl isoforms by germline knockout: loss of individual isoforms Hl.3, Hl.4 or Hl.5 failed to influence mouse development — including when combined with simultaneous loss of histone H 1.0 in a double knockout model — again due to compensatory upregulation of other linker histone isoforms (Fan et al., 2001, Mol Cell Biol, 21:7933-7943). Triple knockouts for Hl.3, Hl.4 and Hl.5 were lethal, with no embryos surviving past El l.5 (Fan et al., 2003, Mol Cell Biol, 23:4559-4572 (2003). While the exact stoichiometry of linker histones to nucleosome core particles at individual loci is uncertain and likely varies across the genome (and between different cell types), in somatic cells the average ratio approximates 1. When the linker to core ratio is maintained around 1, by altered expression of other isoforms in the setting of genetic knockout, there is no overt phenotype, whereas in the setting of triple deletions, a decreased linker to core ratio is associated with widespread developmental defects (Fan et al.. 2003, Mol Cell Biol. 23:4559-4572 (2003). In the present study, transient knockdown of histone Hl .0 was associated with neither major alterations in other linker histones nor changes in the expression of the core nucleosome histones (H2A, H3 and H4, with minimal changein H2B): thus, this intervention induced a transient decrease in the linker to core histone ratio, concomitant with perturbations in chromatin structure and the responsiveness of the cells to growth stimulus. Analysis of single nucleus RNA-seq data from dilated and arrhythmogenic cardiomyopathies (Reichart et al., 2022, Science, 377, eabol984) revealed that histone H1.0 was the only variant increased in fibroblast subpopulations that also expressed periostin and other single cell RNA-seq datasets from human hearts all show a positive correlation between histone H1.0 and periostin (Figure 8). Previous work has shown that levels of histone Hl variants are dynamic during development and reprogramming in the mouse embryo (Izzo et al., 2017, J Cell Biol, 216:3017-3028) as well as in the adult mouse heart following pressure overload (Franklin et al., 2012, Mol Cell Proteomics, 11, Mi l l 014258), linking changes in global chromatin organization with the phenotypic shifts associated with maturation and disease.

[0209] These results support a model in which perturbation of normal chromatin architecture is an organizing feature to regulate cellular response to stress. This mechanism is centrally controlled by levels of histone H1.0 and is used by the cell to alter local chromatin compaction and to change the global stiffness of the cell to respond to altered mechanical or cytokine environment. Recent investigations have shown that linker histones participate in gene regulation through mechanisms beyond their ability to compact the chromatin fiber (Prendergast et al., 2021, Genes Dev, 35:40-58). The actions of histone H1.0 are not merely to turn genes on or off. Results showed that chromatin fibers are relaxed by histone H1.0 depletion, in agreement with previous studies of linker histones, yet histone Hl .0 levels alone are not predictive of transcription. For example, the stress-activated genes periostin and aSMA, whose increase in expression is blocked by histone H1.0 depletion, are bound by histone H1.0 and more compacted when it is present. Thus, during agonist stimulation, histone Hl .0 dependent changes in chromatin organization must facilitate the actions of other transcriptional machinery.

[0210] At the molecular level, the actions of histone Hl .0 in cardiac fibroblasts involve direct binding to chromatin and compacting local fibers. These data demonstrate that histone H1.0 levels influence global and gene-specific deposition of histone H3K27Ac. a mark of gene and enhancer activation. Depletion of histone Hl .0 completely blocked changes in H3K27Ac induced by TGF-|3, including in fibrosis associated genes, notwithstanding global changes in acetylation being increased following histone H1.0 knockdown, demonstrating the specificity of this regulation. This process also involved decreased expression of HD AC 1 and decreased expression of BRD4, a bromodomain containing histone reader necessary7for binding to acetylated histones to recruit transcriptional machinery. Small molecule based HD AC inhibition can prevent fibrosis in the heart (Kee et al., 2006, Circulation, 113:51-59; Lee et al., 2007, Am J Physiol Heart Circ Physiol, 293:H968-977; Williams et al.. 2014, J Mol Cell Cardiol, 67: 112-125). Similarly, small molecule based inhibition of BRD4 has independently been shown to block fibrosis across the same organs (lung (Tang et al., 2013, Am J Pathol, 183:470-479), liver (Ding et al., 2015, Proc Natl Acad Sci U S A, 112: 15713-15718) and heart (Stratton, et al., 2019, Circ Res. 125:662-677; Alexanian et al.. 2021, Nature, 595:438-443)). In addition, previous studies in fibroblasts have shown that HD AC inhibition blocks BRD4- dependent gene activation (Travers et al., 2021, Circulation, 143: 1874-1890). Depletion of histone H1.0 led to a decrease in HD AC levels and a global decondensation of chromatin, in agreement with previous work showing that HD AC inhibition caused global chromatin decondensation and deacetylation in living cells (Strickfaden et al., 2020, Cell, 183: 1772-1784) and plays a critical role in genome protection during the mechanical disruptions of mitosis (Schneider et al., 2022, Nature, 609:183-190). Histone Hl itself has been shown to be regulated by acetylation and HD AC inhibition — either by direct interaction with Hl or through chromatin decondensation — has been shown to increase histone Hl mobility on chromatin (Li et al., 2018, Nucleic Acids Res, 46:7716-7730). Both histone acetylation and BRD4 levels have been shown to drive chromatin phase separation(Gibson et al., 2019, Cell, 179:470-484), a behavior linked to the global condensation and local fiber compaction. These findings provide a molecular link between the actions of histone H1.0 to regulate chromatin condensation and the previous observations of these histone modifiers in the setting of pathologic fibroblast activation.

[0211] While histone H1.0 is dispensable for organismal development due to compensator^' upregulation of other isoforms, these findings reveal a necessary role for this protein to regulate fibroblast activation and cellular stiffness in the adult mouse. Notably, knockdown of histone H1.0 alone had neither discernable effects on cell physiology in culture nor on organ function or histology in vivo. These observations likely represent a distinct behavior of chromatin in non-proliferating adult cells: in response to stress, global chromatin changes are necessary for transcriptional activation and involve histone H1.0, mirroring distinct effects shown for other chromatin structural proteins such as CTCF in primordial versus terminally differentiated cells (Nora et al., 2017, Cell, 169:930-944 e922, Rosa-Garrido et al., 2017, Circulation, 136:1613-1625). These changes in chromatin architecture are responsive to cytokines like TGF- , and hormones like angiotensin II and isoproterenol, serving to directly alter cellular stiffness by changing nuclear deformability' and by ensuring proper expression of cytoskeletal and ECM proteins. In this model, histone H1.0 directly links chromatin structure with cellular stress response, providing a mechanism to ensure that the microenvironment of the cell is coupled to the necessary transcriptional program to elicit distinct mechanical behaviors of the cell.

[0212] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0213] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

CLAIMSWhat is claimed is:1 . A composition comprising at least one histone H 1 .0 inhibitor.

2. The composition of claim 1, wherein the composition comprises at least one antisense RNA molecule.

3. The composition of claim 2, wherein the antisense RNA molecule comprises an siRNA or shRNA molecule.

4. The composition of claim 3, wherein the composition comprises at least one siRNA selected from the group consisting of SEQ ID NO:35, SEQ ID NO:

36. SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68.

5. The composition of claim 3, wherein the composition comprises an shRNA comprising the sequence of SEQ ID NO: 69.

6. A method of modulating chromatin condensation in a cell, the method comprising administering a composition of any one of claims 1-5.

7. The method of claim 6, wherein the method comprises decreasing the level of chromatin compaction in the cell.

8. The method of claim 6, wherein the cell comprises a fibroblast.

9. A method of inhibiting stress-induced activation of fibroblasts, the method comprising administering a composition of any one of claims 1-5.

10. The method of claim 9, wherein the method comprises decreasing the level of chromatin compaction in the cell.

11. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering a composition of any one of claims 1-5 to the subject.

12. The method of claim 11, wherein the disease or disorder is a fibrotic disease or disorder.

13. The method of claim 11, wherein the disease or disorder is selected from the group consisting of cardiac fibrosis, interstitial lung disease, idiopathic pulmonary fibrosis, lung fibrosis, asthma, COPD, Raynaud's phenomenon, pulmonary fibrosis, cirrhosis, liver cirrhosis, atrial fibrosis, endomyocardial fibrosis, arthrofibrosis, Crohn’s Disease, mediastinal fibrosis, myelofibrosis, tubulointerstitial fibrosis, hepatic fibrosis, premacular fibrosis, retinal fibrosis, dermal fibrosis, wound- associated fibrosis, Peyronie’s disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, fibroma, scleroderma, systemic scleroderma, Sjogren syndrome and kidney fibrosis.

Images