In vitro reconstitution method for histone h3-h4 octasome

Abstract

To develop a method for reconstituting an H3-H4 octasome in vitro under physiological conditions.SOLUTION: Provided is a method for reconstituting an H3-H4 octasome, including: (a) incubating a mixture containing a wheat germ cell-free extract, mRNAs encoding histone H3 and H4 respectively, and template DNA; or (b) incubating a mixture containing a wheat germ cell-free extract and mRNAs encoding histone H3 and H4 respectively, and subsequently incubating it with template DNA.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for reconstituting histone H3-H4 octasomes in vitro, and to functional histone H3-H4 octasomes constructed by said method. [Background technology]

[0002] All genomic information (DNA) of eukaryotes is highly folded into a chromosomal structure called chromatin and stored in the cell nucleus. Chromatin is composed of repeating units of nucleosomes, which are the basic structure. Nucleosomes are composed of an octameric core protein consisting of four types of histone proteins (H2A, H2B, H3, and H4) wrapped around a double-stranded DNA of about 150 bp. It is believed that the spatiotemporal structural changes of chromosomes enable DNA replication, repair, and transcription during the cell cycle, and the factors that control these processes are DNA methylation and various chemical modifications of histone proteins. In addition, the existence of variant histones with functions homologous to the four canonical histones (H2A, H2B, H3, and H4) is also known, and they contribute to structural changes in chromosomes.

[0003] Nucleosomes are a typical histone-DNA complex in chromatin of cells, but the existence of other histone-DNA complexes has also been known. Among them, H3-H4 octasomes are nucleosome-like particles with a size comparable to that of nucleosomes, and unlike nucleosomes, they have an octameric core protein composed only of two types of histones, H3 and H4. In recent years, the existence of H3-H4 octasomes has been suggested in model yeasts (Non-Patent Document 1). In Non-Patent Document 1, H3-H4 octasomes were reconstructed by a conventional salt dialysis method, and it was revealed that H3-H4 octasomes have a three-dimensional structure in which 120-130 bp double-stranded DNA is wrapped around an octameric core protein composed of four copies of H3-H4 dimers.

[0004] The salt dialysis method is well known as a method for reconstructing chromatin in a test tube, and a mixture of double-stranded DNA and histones (recombinant histones) heterologously expressed in E. coli is subjected to salt dialysis to refold the protein complex. However, the salt dialysis method has problems such as 1) the structure of histones heterologously expressed in E. coli is broken and insoluble, so they need to be solubilized under non-physiological conditions, and 2) they are reconstructed in a salt concentration-dependent manner (i.e., under non-physiological conditions), so they cannot provide chromatin or octasomes reconstructed in a state close to the natural state. In addition, the inventors' group recently developed a technique for reconstructing chromatin using a wheat germ cell-free extract by co-expression of solubilized histones derived from human and Drosophila and chromatin reconstructing factors (Non-Patent Document 2, Non-Patent Document 3). However, it is unclear whether the H3-H4 octasome can be reconstructed using a wheat germ cell-free extract, and there have been no cases to date in which an expression system using a wheat germ cell-free extract has been applied to the reconstruction of the H3-H4 octasome. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nozawa et al., PNAS, 2022, Vol. 119, No. 45, e2206542119 [Non-Patent Document 2] Okimune et al., BMC Biotechnol. 20, 62 (2020) [Non-Patent Document 3] Endo et al., FEBS Open Bio, 11 (2021), 1552-1564 Summary of the Invention [Problem to be solved by the invention]

[0006] In the future, elucidation of the molecular mechanism of the H3-H4 octasome in the eukaryotic nucleus is considered to be essential for understanding the genome structure and function that are at the root of life phenomena, but for this purpose, a chromosome structure that can be evaluated in a state close to the native state is required. Under these circumstances, the present invention aimed to develop a method for reconstituting the H3-H4 octasome in vitro under physiological conditions. [Means for solving the problem]

[0007] As a result of intensive research, the inventors have surprisingly succeeded in reconstructing a functional H3-H4 octasome under physiological conditions in a wheat germ cell-free system by co-expressing mRNAs encoding animal or plant histone proteins H3 and H4, respectively, in the presence of template DNA. Furthermore, they have also succeeded in reconstructing a functional H3-H4 octasome containing a variant histone. Furthermore, they have found that the reconstruction of the H3-H4 octasome is promoted by adding and co-expressing mRNAs encoding chromatin remodeling factors. Thus, the present invention has been completed.

[0008] Therefore, the present invention provides the following aspects. [1] A method for reconstituting the H3-H4 octasome, comprising the steps of: (a) incubating a mixture containing a wheat germ cell-free extract, mRNA encoding each of histones H3 and H4, and template DNA; or (b) incubating a mixture containing a wheat germ cell-free extract and mRNA encoding histones H3 and H4, respectively, followed by incubation with template DNA; A method comprising: [2] The method described in [1], wherein the histones are derived from animals or plants. [3] The method described in [2], wherein the animal is a human. [4] The method according to any one of [1] to [3], wherein H3 is a variant histone. [5] The method according to any of [1] to [4], wherein the mixture further comprises an mRNA encoding a chromatin remodeling factor, or the mixture further comprises incubating with a chromatin remodeling factor. [6] Reconstituted H3-H4 octasome consisting of histones H3 and H4 expressed in wheat germ cell-free extract and template DNA. [7] The reconstituted H3-H4 octasome described in [6], wherein the histones are of animal or plant origin. [8] The method of [7], wherein the animal is a human. [9] A reconstituted H3-H4 octasome as described in [6] or [7], in which H3 is a variant histone.

[10] H3-H4 octasome reconstituted by the method described in [1] or [5].

[11] A method for screening a substance that promotes or inhibits assembly of the H3-H4 octasome, comprising: (1) (a) incubating a mixture containing a wheat germ cell-free extract, mRNA encoding each of histones H3 and H4, and template DNA in the presence of a candidate substance; (b) incubating a mixture containing a wheat germ cell-free extract and mRNA encoding each of histones H3 and H4 in the presence of a candidate substance, followed by incubation with template DNA; or (c) incubating a mixture containing a wheat germ cell-free extract and mRNA encoding histones H3 and H4, respectively, followed by incubation with template DNA in the presence of a candidate substance; and (2) To assess whether the H3-H4 octasome was reconstituted. A method comprising:

[12] The method according to

[11] , wherein in (a) or (b), the candidate substance is co-expressed in a wheat germ cell-free extract. Effect of the Invention

[0009] According to the present invention, H3-H4 octasomes can be easily reconstituted in vitro under physiological conditions. Furthermore, the H3-H4 octasome reconstitution process of the present invention can be used in a method for screening novel factors that promote or inhibit H3-H4 octasome reconstitution. Since the present invention provides H3-H4 octasomes reconstituted under physiological conditions, the present invention provides an important evaluation system for epigenetics research and can be applied to drug discovery screening, etc. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows the results of a supercoiling assay of human H3-H4 octasomes reconstituted in wheat germ cell-free extracts without or with chromatin remodeling factors. In the figure, RC indicates "relaxed DNA" and SC indicates "supercoiled DNA." The results without the chromatin remodeling factor Nap1L1 are shown in the "-" lane, and the results with the chromatin remodeling factor Nap1L1 are shown in the "+" lane. [Diagram 2] FIG. 2 shows the results of an experiment to determine the optimal amount of human chromatin remodeling factor Nap1L1 to be added for reconstitution of the human H3.X-H4 octasome in wheat germ cell-free extracts. [Diagram 3] Figure 3 shows the results of a supercoiling assay of human H3-H4 octasomes reconstituted in wheat germ cell-free extracts without or with chromatin remodeling factors. In the figure, RC indicates "relaxed DNA" and SC indicates "supercoiled DNA." The results without the chromatin remodeling factor AtNAP1;3 are shown in the "-" lane, and the results with the chromatin remodeling factor AtNAP1;3 are shown in the "+" lane. [Figure 4] Figure 4 shows the results of a supercoiling assay for plant H3-H4 octasome reconstitution using wheat germ cell-free extracts, in which RC indicates "relaxed DNA" and SC indicates "supercoiled DNA." [Diagram 5]FIG. 5 shows the results of MNase assays for plant H3-H4 octasome reconstitution with wheat germ cell-free extracts. [Figure 6] Figure 6 shows the results of a supercoiling assay for the reconstitution of plant H3-H4 octasomes using chromatin remodeling factors in wheat germ cell-free extracts. In the figure, RC indicates "relaxed DNA" and SC indicates "supercoiled DNA." DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Wheat germ cell-free extract is known as a cell-free protein synthesis system. The solution extracted from wheat germ contains the apparatus such as ribosomes and tRNA necessary for protein synthesis, so that the desired protein is synthesized by adding mRNA encoding the desired protein and reaction substrates for protein translation (various amino acids, energy sources such as ATP, etc.). The cell-free protein synthesis system can also express multiple proteins simultaneously. Such a wheat germ cell-free extract can be prepared by a method known in the art. Cell-free protein synthesis systems using wheat germ extract are well established, and various reagents and kits are commercially available. The wheat germ cell-free extract used in the present invention may be prepared by a method known in the art, or a commercially available wheat germ cell-free extract may be used.

[0012] Since the wheat germ cell-free extract may be contaminated with endogenous DNA, the extract may be pretreated with a non-specific endonuclease, such as, but not limited to, micrococcal nuclease, to remove the endogenous DNA.

[0013] In the present invention, histones H3 and H4 may be derived from any organism, for example, from animals, plants, or fungi such as yeast. Examples of animals include, but are not limited to, vertebrates such as mammals (e.g., humans, rats, mice, etc.), amphibians, reptiles, birds, and fish, and invertebrates such as insects. Preferably, human or plant H3 and H4 are used. As a plant, for example, but not limited to, histones from Arabidopsis thaliana, which is widely used as a model plant, are used in the examples described below.

[0014] Histone H3 may be a variant histone. Each histone has a main type called canonical histone and a number of subtypes called variant histones, but no variants of H4 have been reported. In the present invention, H3 and H4 may be a combination of a canonical histone and a variant histone.

[0015] It is known that there are many variant histones in H3, and their genes are also known. Examples of variants of human histone H3 include, but are not limited to, H3.2, H3.3, H3.X, and H3.Z. Examples of variants of plant histone H3 include, but are not limited to, H3.3, H3.6, H3.11, H3.12 (CENH3), H3.14, and H3.15. The canonical histone of H3 is designated as "H3.1".

[0016] Examples of combinations of H3 and H4 for reconstructing the human H3-H4 octasome include, but are not limited to, a combination of H4 with any one selected from the group consisting of H3.1, H3.2, H3.3, H3.X, and H3.Z, preferably a combination of H4 with any one selected from the group consisting of H3.2, H3.3, and H3.X, and more preferably a combination of H3.2 or H3.3 with H4. Examples of combinations of H3 and H4 for reconstructing the plant H3-H4 octasome include, but are not limited to, a combination of H4 with any one selected from the group consisting of H3.1, H3.3, H3.6, H3.11, H3.12 (CENH3), H3.14, and H3.15, preferably a combination of H4 with any one selected from the group consisting of H3.1, H3.3, H3.6, H3.12 (CENH3), H3.14, and H3.15, more preferably a combination of H4 with any one selected from the group consisting of H3.1, H3.3, H3.6, H3.12 (CENH3), and H3.15.

[0017] The mRNA encoding histones may be prepared by methods known in the art, or may be prepared using commercially available kits or reagents. The mRNA encoding histones may be prepared, for example, by preparing a cDNA library using total RNA extracted from cells, cloning a desired gene from the library, and transcribing it in vitro, or by transcribing chemically synthesized cDNA based on known genetic information in vitro. For example, the transcription solution from cDNA may be used as it is (unpurified) as the mRNA encoding histones, or mRNA may be isolated and purified from the transcription solution. Examples of mRNAs encoding human histones include, but are not limited to, mRNAs encoding H3.1 (e.g., RefSeq accession number NM_003520.1), mRNAs encoding H3.2 (e.g., RefSeq accession number NM_021059.2), mRNAs encoding H3.3 (e.g., RefSeq accession number NM_002107.7), mRNAs encoding H3.X (e.g., RefSeq accession number NM_001371919.1), and mRNAs encoding H4 (e.g., RefSeq accession number NM_175054.2). Human genome information can be obtained from public databases such as the NCBI (National Center for Biotechnology Information) database [RefSeq (NCBI Reference Sequences) and Database of genes from NCBI RefSeq genomes].Examples of mRNAs encoding Arabidopsis histones include, but are not limited to, mRNAs encoding H3.1 (locus number AT1G09200), mRNAs encoding H3.3 (locus number AT4G40030), mRNAs encoding H3.6 (locus number AT1G13370), mRNAs encoding H3.11 (locus number AT5G65350), mRNAs encoding H3.12 (CENH3) (locus number AT1G01370), mRNAs encoding H3.14 (locus number AT1G75600), mRNAs encoding H3.15 (locus number AT5G12910), and mRNAs encoding H4 (locus number AT2G28740). Genomic information of Arabidopsis can be obtained from public databases such as TAIR (The Arabidopsis Information Resource) and NCBI databases.

[0018] The amount of mRNA encoding each histone to be used is not particularly limited, but preferably each mRNA is used in an equal amount by weight.

[0019] In the present specification, the template DNA refers to any DNA that wraps around a histone molecule expressed in a wheat germ cell-free extract and reconstructs the H3-H4 octasome together with the histone. The template DNA is an exogenous DNA that is not contained in the wheat germ extract. The sequence of the template DNA is not limited and may have any sequence. The template DNA preferably has a non-translated sequence. The template DNA is preferably a double-stranded DNA. The template DNA may be circular or linear. The length of the template DNA is not particularly limited as long as it is 150 bp or more, but may be preferably 160 bp or more, for example, 2000 bp to 10,000 bp.

[0020] In the present invention, histone protein is expressed by incubating mRNA encoding histone in a wheat germ cell-free extract, which is a cell-free protein synthesis system, and H3-H4 octasome is reconstructed by the expressed histone and template DNA. In one aspect of the present invention, a one-step construction method (co-expression construction method) is provided, which includes incubating a mixture containing a wheat germ cell-free extract, mRNA encoding two histones, histones H3 and H4, and template DNA. In another aspect of the present invention, a two-step reconstruction method (post-translational construction method) is provided, which includes incubating a mixture containing a wheat germ cell-free extract and mRNA encoding two histones, histones H3 and H4, and then incubating with template DNA.

[0021] As described above, in the present invention, in order to express histone proteins from mRNA encoding histones in a wheat germ cell-free extract, the above mixture is incubated in the presence of a reaction substrate solution for protein translation. The reaction substrate solution for protein translation contains an amino acid mixture, creatine kinase for producing ATP (adenosine triphosphate) as an energy source, and the like. The amino acid mixture preferably contains at least 20 types of L-amino acids corresponding to all codons. The amino acid mixture may contain amino acid analogs or isomers, or amino acids labeled with isotopes or the like, depending on the purpose. The reaction substrate solution for protein translation preferably contains an amino acid mixture and ATP. The reaction substrate solution for protein translation may further contain various ions (e.g., potassium ions, magnesium ions, ammonium ions, etc.), nuclease inhibitors (e.g., ribonuclease inhibitors, nuclease inhibitors, etc.), reducing agents (e.g., dithiothreitol, etc.), antibacterial agents (e.g., sodium azide, ampicillin, etc.), buffers, polyethylene glycol, folate, and the like. The amounts of the above components to be used can be appropriately determined by those skilled in the art based on the ranges usually used in cell-free protein synthesis. Various reagents are commercially available as reaction substrate solutions for protein translation. These commercially available reagents may be used in the present invention.

[0022] The mixture can be incubated in a known manner, such as by layering, dialysis, or batch. The layering method is a method in which the mixture is injected under the upper layer of a reaction substrate solution, or the reaction substrate solution is placed on top of the mixture to form two layers, and then incubated. The dialysis method is a method in which the mixture is placed inside a dialysis membrane and the reaction substrate solution is placed on the outside to incubate.

[0023] The incubation time and temperature of the mixture are not particularly limited as long as they are suitable for the translation reaction of the protein, and can be appropriately determined by those skilled in the art. For example, but not limited to, it may be about 17 to 26°C for about 8 to 96 hours. Furthermore, in the post-translational construction method, the incubation time and temperature with the template DNA may also be appropriately determined by those skilled in the art, and may be, for example, about 17 to 26°C for about 8 to 96 hours.

[0024] In the post-translational assembly method, histone proteins expressed in wheat germ cell-free extract may be isolated and then incubated with template DNA, or a translation reaction solution containing histone proteins expressed in wheat germ cell-free extract may be used to incubate with template DNA.

[0025] Thus, according to the method of the present invention, the H3-H4 octasome is reconstituted in vitro under physiological conditions. The method of the present invention can reconstitute the H3-H4 octasome without using chromatin remodeling factors (particularly, without using exogenous chromatin remodeling factors).

[0026] As used herein, "under physiological conditions" refers to salt concentrations, pH, etc. similar to those in vivo. Therefore, the H3-H4 octasome reconstituted by the method of the present invention is constructed under conditions similar to those in vivo, and is considered to have a structure similar to that of natural H3-H4 octasomes occurring in vivo.

[0027] In the method of the present invention, a chromatin remodeling factor may be further used. Depending on the combination of two histones H3 and H4 used, the efficiency of H3-H4 octasome remodeling may be low or the remodeling of H3-H4 octasome may be difficult. In such a case, it is preferable to use a chromatin remodeling factor.

[0028] As used herein, the term "chromatin remodeling factor" refers to any substance that aids in the remodeling of chromatin, including chromatin-associated factors that bind to histones and aid in the association or detachment of histones from DNA, histone chaperone factors, and chromatin remodeling factors. Many types of chromatin remodeling factors are known, and any of these chromatin remodeling factors may be used in the present invention. Examples of chromatin remodeling factors include, but are not limited to, CAF-1 (Chromatin assembly factor-1), NAP1, Acf1, SWI, SNF, ISWI, NRP1, NRP2, and the like. One or more types of chromatin remodeling factors may be used.

[0029] The chromatin remodeling factor may be derived from the same source as the histone used, or may be derived from a source different from that of the histone used. For example, for the reconstruction of an animal histone H3-H4 octasome, a chromatin remodeling factor derived from an animal of the same species as the histone may be used, a chromatin remodeling factor derived from an animal different from that of the histone may be used, or a chromatin remodeling factor derived from a plant may be used. For example, for the reconstruction of a human histone H3-H4 octasome, a chromatin remodeling factor derived from a human may be used, a chromatin remodeling factor derived from an animal other than human, or a chromatin remodeling factor derived from a plant may be used. For example, for the reconstruction of a plant histone H3-H4 octasome, a chromatin remodeling factor derived from a plant may be used, or a chromatin remodeling factor derived from an animal, such as a chromatin remodeling factor derived from a human, may be used.

[0030] In the present invention, it was found that chromatin remodeling factors promote the formation of the H3-H4 octasome. Until now, factors that promote the formation of the H3-H4 octasome were unknown, and the present invention has revealed for the first time that chromatin remodeling factors have the activity of supporting the formation of the H3-H4 octasome.

[0031] The use of the chromatin remodeling factor may be carried out by adding an mRNA encoding the chromatin remodeling factor to the above mixture, or by incubating the above mixture with the chromatin remodeling factor. That is, a mixture containing a wheat germ cell-free extract, mRNA encoding two types of histones H3 and H4, mRNA encoding a chromatin remodeling factor, and template DNA, or a mixture containing a wheat germ cell-free extract, mRNA encoding two types of histones H3 and H4, and mRNA encoding a chromatin remodeling factor may be incubated as described above to co-express the chromatin remodeling factor with histones, or a mixture containing a wheat germ cell-free extract, mRNA encoding two types of histones H3 and H4, and template DNA may be incubated in the presence of a chromatin remodeling factor, or a mixture containing a wheat germ cell-free extract and mRNA encoding two types of histones H3 and H4 may be incubated to express histones, and then incubated with template DNA and a chromatin remodeling factor. The amount of the chromatin remodeling factor used is not particularly limited and may be appropriately determined by those skilled in the art.

[0032] The H3-H4 octasome reconstituted by the method of the present invention may be isolated and purified from the reaction solution by methods known in the art, such as precipitation using polyvalent cations and sucrose gradient ultracentrifugation.

[0033] In a further embodiment of the present invention, there is provided a reconstituted H3-H4 octasome, which is composed of two histones, H3 and H4, expressed in a wheat germ cell-free extract and a template DNA. The octasome may contain a variant histone. The reconstituted H3-H4 octasome is an H3-H4 octasome reconstituted by the method of the present invention.

[0034] Thus, the in vitro reconstituted H3-H4 octasome can be used, for example, in epigenetic studies, for example, to detect previously undescribed H3-H4 octasome-specific chemical modifications, such as, but not limited to, nucleosome acetylation, methylation, ubiquitination, and phosphorylation, by using the in vitro reconstituted H3-H4 octasome as a substrate for previously reported nucleosome-modifying enzymes.

[0035] In a further embodiment of the present invention, a method for screening a substance that promotes or inhibits the construction of H3-H4 octasome is provided, which comprises incubating the reaction solution for reconstruction with a candidate substance in the above-mentioned H3-H4 octasome reconstruction method of the present invention.Therefore, the screening method comprises: (1) (a) incubating a mixture containing wheat germ cell-free extract, mRNAs encoding two histones, H3 and H4, and template DNA in the presence of a candidate substance; (b) incubating a mixture containing wheat germ cell-free extract and mRNAs encoding two histones, H3 and H4, in the presence of a candidate substance, and then incubating with template DNA; or (c) incubating a mixture containing wheat germ cell-free extract and mRNAs encoding two histones, H3 and H4, and then incubating with template DNA in the presence of a candidate substance; and (2) evaluating whether H3-H4 octasome is reconstructed.

[0036] The candidate substance may be any substance known or suspected to have the function of remodeling the H3-H4 octasome, promoting the remodeling of the H3-H4 octasome, or inhibiting the remodeling of the H3-H4 octasome, including, but not limited to, substances known as chromatin remodeling factors, such as NAP1 and nucleoplasmin.

[0037] The candidate substance may be added to the reaction for reconstitution of the H3-H4 octasome as an isolated protein, or may be expressed as mRNA encoding the candidate substance in a mixture containing a wheat germ cell-free extract and together with the two histones H3 and H4.

[0038] Since the H3-H4 octasome has a structure similar to that of a nucleosome, in which double-stranded DNA is wrapped around a core histone octamer, a method known in the art for detecting chromatin can be used to evaluate whether the H3-H4 octasome has been reconstituted. For example, the presence or absence and level of H3-H4 octasome formation in the reaction solution after incubation can be evaluated by supercoiling assay, micrococcal nuclease (MNase) method, observation with an atomic force microscope (AFM) or a cryo-electron microscope, etc. The supercoiling assay is a method for evaluating the efficiency of supercoiling by analyzing DNA topology changes caused by chromatin formation, and the presence or absence and level of octasome formation can be measured by detecting supercoiled template DNA generated by removing histone molecules from the reconstituted H3-H4 octasome in electrophoresis. When the H3-H4 octasome is normally formed, supercoiled DNA is generated by decomposing histones. On the other hand, when an incomplete chromatin structure is formed, such as when the incorporation of octasomes is incomplete, or when the chromatin structure itself is not formed, decomposition of histones produces loose (relaxed) DNA or linear DNA that is not supercoiled. In electrophoresis, supercoiled DNA moves faster than relaxed DNA or linear DNA. The supercoiling method is particularly suitable when using a circular template DNA. The MNase method uses micrococcal nuclease to selectively digest the linker DNA between the octasomes that are linked together like beads, and the length of the DNA wrapped around the core histones is measured by electrophoresis, so that it can be confirmed whether the H3-H4 octasomes have been formed normally. If the H3-H4 octasomes are formed normally, mono-octasomes are produced by MNase digestion, and a DNA band of about 120 to 150 bp wrapped around the core histones can be confirmed by electrophoresis.

[0039] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. EXAMPLES

[0040] [material] In the examples, the following reagents were used unless otherwise specified. For protein expression, wheat germ cell-free extract (CellFree Sciences), SUB-AMIX translation buffer (CellFree Sciences), and creatine kinase (Roche Diagnostics, GmbH) were used. For octasome assembly reaction, pBluescript II SK plasmid DNA (Addgene) (hereinafter referred to as "pBSK plasmid"), SUB-AMIX solution (CellFree Sciences), creatine kinase (Roche Diagnostics, GmbH), and topoisomerase I (Takara Bio Inc.) were used. For gel analysis, 1 kbp DNA molecular weight marker (Toyobo) and protein size marker (Bio-Rad Laboratories) were used.

[0041] Preparation of histone mRNA and chromatin remodeling factor mRNA All genes encoding human proteins used in the examples were prepared by cloning a cDNA library of a human chronic myeloid leukemia-derived cell line (K562 strain) originally prepared by the inventors into a wheat germ cell-free protein expression vector (pEU vector, CellFree Sciences). All genes encoding Arabidopsis thaliana proteins used in the examples were prepared by cloning a cDNA library of Arabidopsis thaliana originally prepared by the inventors into a wheat germ cell-free protein expression vector (pEU vector, CellFree Sciences). In brief, total RNA of human chronic myeloid leukemia-derived cells and Arabidopsis thaliana was extracted by a conventional method, and a cDNA library was synthesized using reverse transcriptase. Genes encoding human histone H3 (variant histones H3.2, H3.3, and H3.X), H4, and chromatin remodeling factor (Nap1L1) were amplified from a cDNA library of a human chronic myeloid leukemia-derived cell line (K562 line) and inserted into the pEU vector. Genes encoding Arabidopsis histone H3 (canonical histone H3.1, variant histones H3.3, H3.6, H3.12, H3.14, and H3.15), H4, and chromatin remodeling factors (AtNap1;3 and NRP2) were amplified from a cDNA library of Arabidopsis thaliana and inserted into the pEU vector.

[0042] The human histones used were H3.2 (gene H3C14; RefSeq accession number NM_021059.2), H3.3 (gene H3F3A; RefSeq accession number NM_002107.7), H3.X (gene H3Y2; RefSeq accession number NM_001371919.1), and H4 (gene H4C16; RefSeq accession number NM_175054.2). The human chromatin remodeling factor used was Nap1L1 (gene NAP1L1; RefSeq accession number NM_001330231.2). Arabidopsis histones include H3.1 (gene HTR2; locus number AT1G09200; RefSeq accession number NM_100790), H3.3 (gene HTR4; locus number AT4G40030; RefSeq accession number NM_001342565.1), H3.6 (gene HTR6; locus number AT1G13370; RefSeq accession number NM_101207.3), and H3.12 (gene HTR12; locus number A). T1G01370; RefSeq accession number NM_100019.2), H3.14 (gene HTR14; locus number AT1G75600; RefSeq accession number NM_106212.3), H3.15 (gene HTR15; locus number AT5G12910; RefSeq accession number NM_121294.1), and H4 (gene HIS4; locus number AT2G28740; RefSeq accession number NM_128434.4) were used as chromatin remodeling factors of Arabidopsis. AtNAP1;3 (locus number AT5G56950; RefSeq accession number NM_125077.4), and NRP2 (locus number AT1G18800; RefSeq accession number NM_101738.3) were used as chromatin remodeling factors of Arabidopsis. The respective amino acid sequences are shown below.

[0043] [Table 1]

[0044] [Table 2]

[0045] [Table 3]

[0046] [Table 4]

[0047] The above plasmid construct (transcription template DNA) was subjected to in vitro transcription reaction at 37°C for 4 hours using a transcription kit in a WEPRO7240 protein synthesis kit (CellFree Sciences) according to the manufacturer's protocol to obtain an mRNA synthesis solution. 1 μL of DNA degrading enzyme (RNase-free DNase I, Nippon Gene) was added to the mRNA synthesis solution per 40 μL volume, and the solution was incubated at 37°C for 30 minutes to substantially completely digest the transcription template DNA. After repeated extraction twice with phenol (50 volumes) / chloroform (50 volumes) / isoamyl alcohol (1 volume) adjusted to pH 5.5, 1 / 10 volume of 3M NaOAc was added to the aqueous layer, and the mRNA was precipitated and recovered with 2.5 volumes of ethanol. The precipitate was washed once with 70% ethanol to remove free nucleotides, then dissolved in RNase-free water (Nippon Gene), and the RNA concentration was measured using NanoDrop1000 (Thermofisher Scientific). The mRNA concentration was adjusted to 1 μg / μL and used in the following examples.

[0048] Example 1: Reconstitution of the human H3-H4 octasome by wheat germ cell-free protein synthesis Reconstitution of human H3-H4 octasomes using wheat germ cell-free extract was performed by the bilayer method and confirmed by the supercoiling method. Briefly, 1.0 μL of an equal mixture of mRNAs encoding human histone H3 (H3.2, H3.3, or H3.X) and mRNAs encoding human histone H4 (containing ∼7 μg of each mRNA), 0.25 μg of pBSK plasmid (template DNA), 2 U of topoisomerase I, 5.0 μL of wheat germ cell-free extract (WEPRO7240), and 0.4 μg of creatine kinase were mixed and adjusted to a final volume of 10.4 μL with 1xSUB-AMIX solution to obtain a mixture (hereinafter referred to as the "co-expression and reconstitution mixture"). The mixture was gently placed in each well of a 96-well plate so that it was the lower layer of 103.0 μL / well of 1xSUB-AMIX solution to form a bilayer. The reaction was carried out at 26° C. for 16 hours to allow the octasome to be assembled.

[0049] 55.0 μL of the octasome assembly reaction solution obtained above was purified by phenol / chloroform / isoamyl alcohol (25:24:1, v / v) extraction (pH 8.0) followed by ethanol precipitation, and the purified DNA was resuspended in HD buffer (25 mM HEPES, 1 mM DTT, pH 7.6) containing a trace amount of ribonuclease A (Macherey-Nagel GmbH & Co.). Supercoiled plasmid DNA was separated by 0.8% TBE agarose gel electrophoresis in 0.5xTBE buffer, visualized with ethidium bromide (Nippon Gene), and analyzed by a gel documentation system (Bio-Rad Laboratories). As a control, the pBSK plasmid used as the template DNA was also electrophoresed. The results are shown in Figure 1.

[0050] In addition, it was confirmed by Western blotting of wheat germ cell-free extract that the H3-H4 octasome confirmed by the supercoiling method does not contain H2A and H2B derived from wheat germ extract.

[0051] As shown in Figure 1, human H3-H4 octasomes were reconstituted by incubating a mixture of wheat germ cell-free extract, mRNAs encoding two types of human histones, H3 and H4, and template DNA (lanes marked with "-"). Among the three combinations of H3-H4 octasomes, two (H3.2-H4 octasome and H3.3-H4 octasome) showed strong supercoiled DNA (SC) bands. However, for the combination of H3.X-H4 octasome, the SC band was faint, indicating that the efficiency of reconstitution of H3-H4 octasomes was very low.

[0052] Example 2: Reconstitution of the human H3-H4 octasome using chromatin remodeling factors 1 An experiment was carried out to see whether the combination (H3.X-H4 octasome) in which the reconstitution efficiency of the H3-H4 octasome was low in Example 1 could be reconstituted using a chromatin reconstitution factor. mRNA (5 μg / 1 μL of reaction solution) encoding a human chromatin reconstitution factor (Nap1L1) was added to a coexpression / reconstitution mixture prepared in the same manner as described in Example 1, and the coexpression / reconstitution reaction was carried out in the same manner as in Example 1, and the result was confirmed by the supercoiling method.

[0053] As a result, we found that co-expression of human chromatin remodeling factor Nap1L1 promoted H3.X-H4 octasome formation (Figure 1, lane "+"). Therefore, when H3-H4 octasome remodeling is difficult in wheat germ cell-free extract, it is possible to reconstruct the octasome by adding exogenous chromatin remodeling factors. Furthermore, we found that this H3-H4 octasome remodeling system can be used to screen candidate substances that promote or inhibit octasome remodeling.

[0054] Furthermore, the optimal amount of chromatin remodeling factor to be added was tested. 0, 5, 10, 15, or 20 μg of mRNA encoding a human chromatin remodeling factor (Nap1L1) was added to 113 μL of the above co-expression / remodeling mixture containing mRNA encoding H3.X and H4. The co-expression / remodeling reaction was carried out in the same manner as in Example 1, and confirmed by the supercoiling method. The results are shown in FIG. 2. As a result, it was found that an addition amount of about 5 μg to 10 μg was optimal.

[0055] Example 3: Reconstruction of human H3-H4 octasome using chromatin remodeling factors 2 An experiment was conducted to see whether the combination (H3.X-H4 octasome) that showed low reconstitution efficiency of H3-H4 octasome in Example 1 could be reconstituted using AtNAP1;3, a chromatin reconstitution factor from Arabidopsis thaliana. mRNA encoding the chromatin reconstitution factor from Arabidopsis thaliana (AtNAP1;3) was added to a coexpression / reconstitution mixture prepared in the same manner as described in Example 1, and a coexpression / reconstitution reaction was carried out in the same manner as in Example 1, and the result was confirmed by the supercoiling method.

[0056] As a result, we found that co-expression of Arabidopsis chromatin remodeling factor AtNAP1 promoted H3.X-H4 octasome formation (Figure 3, lane "+"). Therefore, when H3-H4 octasome reconstitution is difficult in wheat germ cell-free extract, it is possible to reconstitute the octasome by adding exogenous chromatin remodeling factors. Furthermore, although NAP1;3 is derived from plants, it was shown that it can promote the formation of heterologous H3-H4 octasome, indicating that H3-H4 octasome reconstitution is possible by using chromatin remodeling factors across species. Furthermore, we found that this H3-H4 octasome reconstitution system can be used to screen candidate substances that promote or inhibit octasome reconstitution.

[0057] Example 4: Reconstitution of plant H3-H4 octasomes by wheat germ cell-free protein synthesis Arabidopsis thaliana, a model plant, was used as the plant. Reconstruction of Arabidopsis thaliana H3-H4 octasomes using wheat germ cell-free extract was performed by the overlay method and confirmed by the supercoiling method. Briefly, 1.0 μL of an equal mixture of mRNAs encoding Arabidopsis thaliana histone H3 (H3.1, H3.3, H3.6, H3.12, H3.14, or H3.15) and mRNAs encoding Arabidopsis thaliana histone H4 (containing ~7 μg of each mRNA), 0.25 μg of pBSK plasmid (template DNA), 2 U of topoisomerase I, 5.0 μL of wheat germ cell-free extract (WEPRO7240), and 0.4 μg of creatine kinase were mixed and adjusted to a final volume of 10.4 μL using 1xSUB-AMIX solution to obtain a mixture (hereinafter referred to as the "mixture for co-expression and reconstruction"). The mixture was gently placed in each well of a 96-well plate so that 103.0 μL / well of 1×SUB-AMIX solution was placed under the mixture to form a bilayer. The mixture was allowed to react at 26° C. for 16 hours to form octasomes.

[0058] The octasome assembly reaction solution obtained above, 55.0 μL, was purified by phenol / chloroform / isoamyl alcohol (25:24:1, v / v) extraction (pH 8.0), followed by ethanol precipitation, and the purified DNA was resuspended in HD buffer (25 mM HEPES, 1 mM DTT, pH 7.6) containing a trace amount of ribonuclease A (Macherey-Nagel GmbH & Co.). Supercoiled plasmid DNA was separated by 0.8% TBE agarose gel electrophoresis in 0.5xTBE buffer, visualized with ethidium bromide (Nippon Gene), and analyzed by a gel documentation system (Bio-Rad Laboratories). As a control, the pBSK plasmid used as the template DNA was also electrophoresed. The results are shown in Figure 4.

[0059] As shown in Figure 4, the Arabidopsis H3-H4 octasome was reconstituted by incubating a mixture of wheat germ cell-free extract, mRNAs encoding two Arabidopsis histones H3 and H4, and template DNA. Among the six H3-H4 octasome combinations, five (H3.1-H4 octasome, H3.3-H4 octasome, H3.6-H4 octasome, H3.12-H4 octasome, and H3.15-H4 octasome) showed strong supercoiled DNA (SC) bands. However, for the H3.14-H4 octasome combination, the SC band was faint, indicating that the efficiency of reconstitution of the H3-H4 octasome was very low.

[0060] Furthermore, the octasome assembly reaction solution obtained above was evaluated by the MNase method. Briefly, 100 μL of the octasome assembly reaction solution was added with Micrococcal nuclease (Takara Bio) at a final concentration of 0.1 U / μL and calcium chloride at a final concentration of 2.5 mM, and incubated at 37 °C for 1 to 6 min. The reaction was stopped by adding 5 mM EGTA, and purified by phenol / chloroform / isoamyl alcohol extraction and then ethanol precipitation. The purified DNA was resuspended in HD buffer (25 mM HEPES, 1 mM DTT, pH 7.6) containing a trace amount of ribonuclease A (Macherey-Nagel GmbH & Co.), subjected to 2.0% TAE agarose gel electrophoresis in 1 × TAE buffer, and analyzed by ethidium bromide staining. Measurement of the average monooctasome DNA length was performed by a gel documentation system (Bio-Rad Laboratories). The results for H3.1-H4 octasome, H3.6-H4 octasome, and H3.12-H4 octasome are shown in Figure 5. As a result, it was found that the H3-H4 octasome was reconstructed by this example.

[0061] Example 5: Reconstitution of the plant H3-H4 octasome using chromatin remodeling factors An experiment was conducted to see whether the combination (H3.14-H4 octasome) in which the reconstitution efficiency of H3-H4 octasome was low in Example 4 could be reconstituted using a chromatin reconstitution factor. mRNA (5 μg) encoding an Arabidopsis chromatin reconstitution factor (Nap1;3 or NRP2) was added to a coexpression / reconstitution mixture prepared in the same manner as described in Example 4, and the coexpression / reconstitution reaction was carried out in the same manner as in Example 4, and confirmed by the supercoiling method. The results are shown in Figure 6.

[0062] As a result, it was found that co-expression of Arabidopsis chromatin remodeling factors Nap1;3 or NRP2 promoted H3.14-H4 octasome formation (Figure 6). Therefore, when H3-H4 octasome remodeling is difficult in wheat germ cell-free extract, it was found that octasomes can be reconstructed by adding exogenous chromatin remodeling factors. Furthermore, it was found that this H3-H4 octasome remodeling system can be used to screen candidate substances that promote or inhibit octasome remodeling. Furthermore, the remodeling efficiency differed between the two chromatin remodeling factors used in the examples, suggesting that these two types recognize different combinations of H3 and H4.

Claims

1. A method for reconstituting the H3-H4 octasome, comprising: (a) incubating a mixture containing a wheat germ cell-free extract, mRNA encoding each of histones H3 and H4, and template DNA; or (b) incubating a mixture containing a wheat germ cell-free extract and mRNA encoding histones H3 and H4, respectively, followed by incubation with template DNA; A method comprising:

2. The method of claim 1 , wherein the histones are of animal or plant origin.

3. The method of claim 2, wherein the animal is a human.

4. The method of any one of claims 1 to 3, wherein H3 is a variant histone.

5. The method of claim 1 , wherein the mixture further comprises an mRNA encoding a chromatin remodeling factor or further comprises incubating the mixture with a chromatin remodeling factor.

6. Reconstituted H3-H4 octasome consisting of histones H3 and H4 expressed in wheat germ cell-free extract and template DNA.

7. The reconstituted H3-H4 octasome of claim 6, wherein the histones are of animal or plant origin.

8. The reconstituted H3-H4 octasome of claim 7, wherein the animal is a human.

9. The reconstituted H3-H4 octasome of any one of claims 6 to 8, wherein H3 is a variant histone.

10. An H3-H4 octasome reconstituted by the method of claim 1 or 5.

11. A method for screening for a substance that promotes or inhibits assembly of the H3-H4 octasome, comprising: （１） (a) incubating a mixture containing a wheat germ cell-free extract, mRNA encoding each of histones H3 and H4, and template DNA in the presence of a candidate substance; (b) incubating a mixture containing a wheat germ cell-free extract and mRNA encoding histones H3 and H4, respectively, in the presence of a candidate substance, followed by incubation with template DNA; or (c) incubating a mixture containing a wheat germ cell-free extract and mRNA encoding histones H3 and H4, respectively, followed by incubation with template DNA in the presence of a candidate substance; and (2) To assess whether the H3-H4 octasome was reconstituted. A method comprising:

12. The method according to claim 11 , wherein in (a) or (b), the candidate substance is co-expressed in a wheat germ cell-free extract.

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