Cell cycle early G1 phase regulator

Abstract

The present invention addresses the problem of providing a cell cycle control agent based on a novel mechanism, by identifying a novel gene involved in a control mechanism of a cell cycle for canceration or differentiation of cells and using the gene and a translation product thereof.　The present invention provides a control agent for the initial stage of the G1 phase of the cell cycle, said control agent containing: C7orf26 gene having the activity of arresting the cell cycle at the initial stage of the G1 phase and causing a transition from the G1 phase to the G0 phase; and a translation product thereof.

Description

Agent for controlling the early stage of the G1 phase of the cell cycle

[0001] The present invention relates to the use of a novel factor having an activity of arresting the cell cycle at the early stage of the G1 phase and causing the cell to transition from the early stage of the G1 phase to the G0 phase.

[0002] The cell cycle refers to the cycle of cell division in eukaryotes and consists of four phases: the G1 phase, which is the preparatory period for DNA replication; the S phase, during which DNA replication occurs; the G2 phase, which is the preparatory period for mitosis; and the M phase, which is the mitotic phase. One cell cycle is formed by these four phases. Cells replicate themselves by accurately repeating the cycle of G1-S-G2-M-G1. In addition, differentiated cells such as nerve cells and diaphragm muscle cells, as well as many somatic cells, lose their ability to divide and move towards the quiescent phase (G0 phase). General cells assume their specific morphology and perform their original activities (differentiation of stem cells, hormone production by endocrine cells, information transmission by nerve fibers, muscle contraction, etc.) in the G0 phase. Cancer cells are abnormal cells that do not transition to the G0 phase and continue to proliferate without limit. The difference between cancer cells and normal cells is thought to lie in their ability to transition to the G0 phase.

[0003] The cell cycle includes checkpoints that monitor whether cells are progressing correctly, ensuring orderly proliferation and differentiation, and accurate replication of the genetic information they possess. These checkpoints include the G1 / S checkpoint, S phase checkpoint, G2 / M checkpoint, and M phase checkpoint. Their main roles are to check for DNA damage at the G1 / S checkpoint, normal replication at the S phase checkpoint, successful completion of DNA replication at the G2 / M checkpoint, and chromosome binding to the spindle at the M phase checkpoint. DNA damage checks are performed in the G1 phase, at the start of the S phase, during the S phase, and at the start of the M phase. If DNA is damaged or replication is insufficient, the checkpoint stops or slows down the cell cycle, preventing the next step from starting before the cells are ready. The cell cycle is driven by a complex of cyclin and cyclin-dependent kinase (CDK), which is activated when cyclin binds to it. The transition from G1 to S phase is triggered by the cyclin D / CDK4 (or CDK6) complex, which activates the cyclin E / CDK2 complex, causing the cell to move into the S phase. Subsequently, activation of cyclin A / CDK2 causes the cell to move from S phase to G2 phase, and activation of cyclin B / CDK1 causes the cell to proceed to M phase. Several proteins are known that block the function of the cyclin-CDK complex and halt the cell cycle when DNA damage is detected. p53 and its downstream counterpart p21 are representative examples, monitoring whether there are any problems in advancing the cell cycle at the four checkpoints mentioned above, and immediately halting the cell cycle if an abnormality is found. For example, at the G1 / S checkpoint, when DNA damage is detected, p53 is expressed, which in turn expresses the downstream p21, which binds to the cyclin-CDK complex, causing the CDK to lose its phosphorylation ability, and as a result, the cell cycle is stopped in the G1 phase.In the biological reactions of the cell cycle checkpoints, only p53 and p21 are transcribed and translated to create new proteins, and the production of p53 and p21 occurs in a very short reaction called "immediately early gene expression." The produced p21 binds to the cyclin-CDK complex and inhibits the phosphorylation and dephosphorylation function of CDK, resulting in the arrest of the cell cycle (Non-Patent Literature 1). If abnormalities occur in these factors involved in P53, p21, and the cyclin-CDK complex, checkpoint control cannot be achieved, the cell cycle does not arrest even if DNA abnormalities occur, damaged DNA is replicated as is, and it is thought that uncontrolled cell proliferation occurs, leading to cancer. Therefore, cancer is a representative disease caused by abnormalities in the cell cycle. On the other hand, the molecular mechanism of the transition from the G1 phase to the G0 phase has not been fully elucidated, but it is important in terms of promoting the differentiation of stem cells and progenitor cells, the growth and maintenance of organs and individuals, and the inherent roles of cells.

[0004] Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P. Molecular Biology of the Cell Sixth ed. Chapter 17. The cell cycle. Garland Science New York USA, 2014; pp.963-1020.

[0005] The present invention aims to identify novel genes and pathways involved in the regulatory mechanisms of the cell cycle for cell carcinogenesis and differentiation, and to provide cell cycle regulators based on novel mechanisms using these genes, their translation products, and conjugated substances.

[0006] The inventors discovered that a missense mutation (p.A425V) caused by a single nucleotide substitution in the C7orf26 gene exists in a large family with congenital eye abnormalities. Functional analysis of the C7orf26 gene, the causative gene for these eye abnormalities, revealed that the C7orf26 gene is a novel member of the integrator complex and is involved in mRNA maturation. Surprisingly, they also found that, as a further novel function, it is highly expressed during the M phase to the early G1 phase of the cell cycle and has the activity to arrest the cell cycle in the early G1 phase. This cell cycle arrest is achieved through the upregulation of the tumor suppressor gene p53. Furthermore, they found that the C7orf26 gene acts as a transcriptional cofactor for members of the E2F family of transcription factors (E2F4 and E2F1, etc.) in the promoter region of p53, increasing p53 expression, and that the integrator complex subunit (INTS) also acts in cooperation with C7orf26 as a transcriptional cofactor. Furthermore, it was shown that each member of INTS also possesses the cell cycle arrest effect of C7orf26 on its own. It was also confirmed that cells in which the C7orf26 gene was continuously expressed or highly expressed at physiological levels were subjected to cell death due to prolonged or high expression of p53. Moreover, when iPS cells into which the C7orf26 gene was introduced were differentiated into ectoderm, mesoderm, and endoderm cells, the number of differentiated cells increased compared to normal iPS cells during the early stages of development (ectoderm, mesoderm, and endoderm), and even further during the later stages of development (brain and retina). Specific genes were also highly expressed, demonstrating that the C7orf26 gene has the activity to arrest the cell cycle in the early G1 phase and then transition to the G0 phase, thus promoting cell differentiation. This invention is completed based on these findings.

[0007] In other words, the present invention encompasses the following: [1] A cell cycle early G1 phase regulator comprising any of the following proteins (A) to (C): (A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (B) A protein consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 2, and which has the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase; (C) A protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and which has the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase. [2] A cell cycle early G1 phase regulator comprising any of the following genes (D) to (F). (D) A gene comprising DNA consisting of the base sequence shown in Sequence ID No. 1; (E) A gene comprising DNA encoding a protein having a base sequence having 90% or more sequence identity with the base sequence shown in Sequence ID No. 1, and which has the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase; (F) A gene comprising DNA encoding a protein having the activity to hybridize under stringent conditions with DNA consisting of a base sequence complementary to the base sequence shown in Sequence ID No. 1, and which has the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase. [3] A cell cycle early G1 phase regulator according to [2], wherein the gene is inserted into a vector. [4] A cancer treatment drug comprising a cell cycle early G1 phase regulator according to any one of [1] to [3]. [5] A differentiation promoter for undifferentiated cells comprising a cell cycle early G1 phase regulator according to any one of [1] to [3]. [6] A differentiation promoter for undifferentiated cells according to [5], wherein the undifferentiated cells are stem cells or progenitor cells. [7] A method for promoting the differentiation of undifferentiated cells, comprising the step of expressing any of the following genes (D) to (F) in undifferentiated cells.(D) A gene comprising DNA consisting of the nucleotide sequence shown in Sequence ID No. 1; (E) A gene comprising DNA comprising a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 1, and encoding a protein that has the activity to arrest the cell cycle in the early G1 phase and transition from the early G1 phase to the G0 phase; (F) A gene comprising DNA comprising DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1, under stringent conditions, and encoding a protein that has the activity to arrest the cell cycle in the early G1 phase and transition from the early G1 phase to the G0 phase. This application claims priority to Japanese Patent Application No. 2020-020019, filed on 7 February 2020, and encompasses the contents described in the specification of said patent application.

[0008] In this invention, the C7orf26 gene, newly identified as having the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase, can arrest the cell cycle in the early G1 phase and induce cell death in uncontrolledly proliferating cells such as cancer cells. Therefore, the C7orf26 gene and its translation product are effective as pharmaceuticals for the prevention and / or treatment of diseases caused by or related to abnormal cell cycle regulation, such as cancer. Furthermore, when the C7orf26 gene is expressed in stem cells or progenitor cells of various cells such as muscle, nerve, and endocrine cells, it can stop cell division and proliferation in the stem cell and progenitor cell state, and promote differentiation by transitioning to the G0 phase. Moreover, while stem cells and progenitor cells that do not transition to the G0 phase are culled by cell death, it is expected that the expression of the C7orf26 gene will preserve such stem cells and progenitor cells that are destined to be culled in a quiescent state or direct them towards cells that can differentiate or function. Therefore, the present invention is effective for the efficient utilization of stem cells as raw materials for regenerative medicine materials, increasing the yield of regenerative medicine and cell therapy materials, increasing the yield of agricultural products, livestock products, and aquatic products, increasing the yield of immunotherapy and hormone preparations by improving the differentiation efficiency of the produced cells, treating infertility by enhancing the differentiation of germ cells, enhancing the reproduction and proliferation of beneficial organisms, and suppressing the reproduction and / or proliferation of harmful organisms (including pests, vermin, and harmful plants).

[0009] Figure 1A shows the flow cytometry analysis results at 0, 16, and 20 hours after administration of hydroxyurea (HU) to synchronize the cell cycle of HeLa cells. Figure 1B shows the relative expression levels of C7orf26 mRNA and Ki67 mRNA in HeLa cells at each phase of the cell cycle (error bars ± SD, n=3). Figure 1C shows the immunostaining of C7orf26 in HeLa cells at each phase of the cell cycle (scale bar: 5 μm). Figure 2 shows the changes in the relative expression levels of C7orf26, p53, p21, and Ki67 mRNA in HeLa / pPB-CuO-C7orf26 cells (HeLa cells in which the C7orf26 gene has been incorporated into the genome via a transposon using the PiggyBac Cumate Switch Inducible Vector, and C7orf26 is expressed in accordance with the concentration of added cumate) cultured in cumate-supplemented medium with gradually increasing concentrations. Figure 3A shows the flow cytometry results of HeLa / pPB-CuO-C7orf26 cells cultured in a medium continuously supplied with cumate (for 5, 6, and 8 days). These are HeLa cells in which the C7orf26 gene was incorporated into the genome via a transposon using the PiggyBac Cumate Switch Inducible Vector, and C7orf26 is expressed depending on the concentration of cumate added. Figure 3B shows the flow cytometry results of HeLa / pPB-CuO-C7orf26 cells synchronized with hydroxyurea (HU) or Ro3306. Figure 3C shows the relative expression levels of C7orf26, p53, p21, and Ki67 mRNA at each phase of the cell cycle in HeLa / pPB-CuO-C7orf26 cells synchronized with hydroxyurea (HU), Ro3306 treatment, or serum-free medium (serum (-)), as well as in HeLa / pPB-CuO-C7orf26 cells cultured in serum recovery medium after 24 hours of serum starvation. It is a schematic diagram showing the C7orf26 expression scheme in the cell cycle and the sites where transcriptional regulation by p53 is possible.Figures 5-1A and 5-1B show the results of a reporter assay to elucidate the transcriptional regulatory mechanism of p53 by C7orf26 and integrator complex subunits (INTS1, INTS2, INTS3, INTS4, INTS5, INTS6, INTS7, INTS8, INTS9, INTS10, INTS11, INTS12, INTS13, INTS14). Figure 5C shows the results of a reporter assay to elucidate the transcriptional regulatory mechanism of p53 by C7orf26 (in each figure, *: p<0.05, Mann-Whitney U-test, error bar: ±SD, n=3). Figure 5-2 shows the relative expression levels of C7orf26 and INTS1, INTS2, INTS3, INTS4, INTS5, INTS6, INTS7, INTS8, INTS9, INTS10, INTS11, INTS12, INTS13, and INTS14 during the cell cycle in hydroxyurea (HU)-synchronized HeLa cells. Figure 6-1A shows the relative expression levels of E2F1, E2F4, and E2F6 during the cell cycle in hydroxyurea (HU)-synchronized HeLa cells. Figure 6-1B shows the results of a reporter assay of p53 expression in HeLa cells in which E2F was knocked down using siRNA (*: p<0.05, Mann-Whitney U-test, error bars: ±SD, n=3). Figure 6-1C shows the results of Western blot analysis using an anti-E2F4 antibody (a-E2F4) on HeLa cell extracts transfected with pCMV-GFP or pCMV-C7orf26-Flag. Figure 6-2A shows the results of a reporter assay of C7orf26 expression in HeLa cells transfected with pCMV-E2F4 or pCMV-E2F6 (*: p<0.05, Mann-Whitney U-test, error bars: ±SD, n=3). Figure 6-2B shows the relative expression levels of C7orf26 during the cell cycle in HeLa cells synchronized with hydroxyurea (HU) and treated with E2F4 and E2F6 siRNA (*: p<0.05, Mann-Whitney U-test, error bars: ±SD, n=3). Figure 6-2C shows the changes in C7orf26 expression levels during the cell cycle in HeLa cells after administration of a CDK1 inhibitor.Figure 7A shows the flow cytometry results (decrease in live cells and increase in dead cells) of HeLa / pPB-CuO-C7orf26 cells (HeLa cells in which the C7orf26 gene was incorporated into the genome via a transposon using the PiggyBac Cumate Switch Inducible Vector, and C7orf26 is expressed depending on the concentration of added cumate) cultured in a medium supplied with cumate in succession (5, 6, and 8 days). Figure 7B shows the flow cytometry results (decrease in live cells and increase in dead cells) of HeLa / pPB-CuO-C7orf26 cells cultured in a medium supplied with cumate in succession (5 and 8 days). Figure 8 shows the results of cell death induction tests in HeLa cells (uterine cancer cell line) and glioma cells U-251MG that overexpressed C7orf26 [ad: HeLa cells, eg: glioma cells U-251MG, h: mitochondrial staining of HeLa cells without C7orf26 expression (control)]. Figure 9A shows the relative expression levels of C7orf26, p53, p21, and Ki67 mRNA in human iPS cells HiPSC / pPB-CuO-C7orf26 (human iPS cells into which C7orf26 has been incorporated into the genome using the PiggyBac Cumate Switch Inducible Vector, and C7orf26 is expressed depending on the concentration of cumate added) differentiated into ectoderm in cumate-added medium (Cum+) and in cumate-free medium (Cum-) (expression level in Cum- is set to 1). Figure 9B shows the flow cytometry analysis results for HiPSC / pPB-CuO-C7orf26 cells differentiated into ectoderm in cumate-supplemented medium (Cum+) and in cumate-free medium (Cum-).Figure 9C shows the relative expression levels of C7orf26, Pax6, Pax3, and Sox1 mRNA in HiPSC / pPB-CuO-C7orf26 cells differentiated into ectoderm in cumate-supplemented medium (Cum+) and in cells differentiated into ectoderm in cumate-free medium (Cum-) (expression level in Cum- is set to 1). Figure 10A shows the relative expression levels of C7orf26, p53, p21, and Ki67 mRNA in human iPS cells HiPSC / pPB-CuO-C7orf26 (human iPS cells into which C7orf26 has been incorporated into the genome using the PiggyBac Cumate Switch Inducible Vector, and which express C7orf26 depending on the concentration of cumate added) differentiated into retinal ganglion cells in cumate-added medium (Cum+) and in cumate-free medium (Cum-) (expression level in Cum- is set to 1). Figure 10B shows the relative expression levels of Brn3B, Math5, and C7orf26 mRNA in HiPSC / pPB-CuO-C7orf26 cells differentiated into retinal ganglion cells in cumate-supplemented medium (Cum+) and in cumate-free medium (Cum-) (expression levels in Cum- are set to 1). Figure 10C shows the relative expression levels of Otx2, En1, and C7orf26 mRNA in HiPSC / pPB-CuO-C7orf26 cells differentiated into brain cells in cumate-supplemented medium (Cum+) and in cumate-free medium (Cum-) (expression levels in Cum- are set to 1).Figure 11A shows the flow cytometry results for human iPS cells HiPSC / pPB-CuO-C7orf26 (human iPS cells into which C7orf26 has been incorporated into the genome using the PiggyBac Cumate Switch Inducible Vector, and which express C7orf26 depending on the concentration of cumate added) differentiated into mesoderm in cumate-added medium (Cum+) and in cumate-free medium (Cum-). Figure 11B shows the relative expression levels of C7orf26 and TBXT mRNA in HiPSC / pPB-CuO-C7orf26 cells differentiated into mesoderm in cumate-added medium (Cum+) and in cumate-free medium (Cum-) (expression level in Cum- is set to 1). Figure 12A shows the flow cytometry results for human iPS cells HiPSC / pPB-CuO-C7orf26 (human iPS cells into which C7orf26 has been incorporated into the genome using the PiggyBac Cumate Switch Inducible Vector, and which express C7orf26 depending on the concentration of cumate added) differentiated into endoderm in cumate-added medium (Cum+) and in cumate-free medium (Cum-). Figure 12B shows the relative expression levels of C7orf26 and Sox17 mRNA in HiPSC / pPB-CuO-C7orf26 cells differentiated into endoderm in cumate-added medium (Cum+) and in cumate-free medium (Cum-) (expression level in Cum- is set to 1).

[0010] The present invention will be described in detail below. 1. Early G1 Phase Regulator of the Cell Cycle In the present invention, the human C7orf26 gene, which has been newly identified as having the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase, codes for a protein consisting of 449 amino acids. The base sequence of human C7orf26 cDNA (SEQ ID NO: 1) and the amino acid sequence of the translation product (SEQ ID NO: 2) are registered in the National Center of Biotechnology Information (NCBI) database as NCBI accession numbers NM_024067 and NP_076972, respectively. Furthermore, the C7orf26 gene used in this invention may be a C7orf26 gene derived from an animal species other than humans. The C7orf26 gene or its translation product derived from another animal can be obtained by searching a database of genomes and / or cDNAs of non-human mammals or a protein database using BLAST or FASTA, with the base sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 as a query, thereby obtaining the base sequence and amino acid sequence of C7orf26 derived from the desired animal species.

[0011] Therefore, the early G1 phase regulator of the cell cycle of the present invention comprises one of the following proteins (which may be referred to as "C7orf26 protein" in this specification): (A) a protein consisting of the amino acid sequence shown in Sequence ID No. 2; (B) a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence shown in Sequence ID No. 2, and which has the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase; (C) a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 2, and which has the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase.

[0012] Regarding (B) above, the range of "one to several" in "an amino acid sequence in which one or several amino acids are added, deleted, or substituted" is not particularly limited to the number of amino acids, as long as the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 retains the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase. For example, it means approximately 1 to 10, preferably 1 to 7, more preferably 1 to 5, and even more preferably 1 to 3. Deletion of an amino acid means selecting and deleting any amino acid from the amino acid sequence shown in SEQ ID NO: 2. Addition of an amino acid means adding one to several amino acids to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO: 2. Examples of amino acid substitutions include conservative amino acid substitutions.

[0013] The above-mentioned conservative amino acid substitutions refer to substitutions between amino acids with similar properties, such as polarity, electrical properties, and structural properties, including hydrophobic amino acids, polar amino acids, acidic amino acids, basic amino acids, amino acids with branched side chains, and aromatic amino acids. Examples of hydrophobic (nonpolar) amino acids include glycine, alanine, valine, leucine, isoleucine, and proline; examples of polar amino acids include serine, threonine, cysteine, methionine, asparagine, and glutamine; examples of acidic amino acids include aspartic acid and glutamic acid; examples of basic amino acids include lysine, arginine, and histidine; examples of amino acids with branched side chains include valine, isoleucine, and leucine; and examples of aromatic amino acids include phenylalanine, tyrosine, tryptophan, and histidine. Preferred conservative amino acid substitutions include substitutions between amino acids selected from valine, leucine, and isoleucine, phenylalanine and tyrosine, lysine and arginine, alanine and valine, and asparagine and glutamine.

[0014] The addition, deletion, or substitution of amino acids can be carried out by modifying the gene encoding the protein using methods known in the art. Mutations can be introduced into the gene using known methods such as the Kunkel method or the Gapped duplex method, or similar methods.

[0015] With respect to (C) above, "amino acid sequence having 90% or more sequence identity" means that the sequence identity of the amino acid sequence is at least 90%, preferably 95% or more, more preferably 97% or more, and most preferably 98% or more. The sequence identity of amino acids can be determined using methods well known to those skilled in the art, sequence analysis software, etc. Examples include the blastp program of the BLAST algorithm and the fasta program of the FASTA algorithm. Here, the sequence identity of the amino acid sequence is a value expressed as a percentage of the frequency at which the same amino acid appears in the same location when the amino acid sequence to be evaluated is compared with the amino acid sequence shown in Sequence ID No. 2.

[0016] In this invention, "cells" include all cells of animals, including humans, such as germ cells like sperm and eggs, somatic cells that make up living organisms, stem cells, progenitor cells, cancer cells, cells (cell lines) that are isolated from living organisms, acquire immortalization ability, and are stably maintained outside the body, and cells that are isolated from living organisms and have been artificially genetically modified. "Somatic cells that make up living organisms" refers to cells collected from any tissue, organ, or body such as skin, muscle, bone, cartilage, connective tissue, blood vessels, blood (including umbilical cord blood), bone marrow, heart, eye, brain, nerve, thymus, spleen, adrenal gland, lung, pancreas, liver, stomach, small intestine, large intestine, bladder, prostate, testes, ovaries, and uterus. Furthermore, the cells of this invention also include cells other than animal cells, such as plant cells and microbial cells.

[0017] Examples of somatic cells that make up living organisms include fibroblasts, bone marrow cells, red blood cells, granulocytes (neutrophils, eosinophils, basophils), monocytes, lymphocytes (T cells, B cells, NK cells), platelets, macrophages, dendritic cells, osteocytes, osteoblasts, chondrocytes, epidermal cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, endothelial cells, hepatocytes, cumulus cells, dendritic cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, muscle cells, pancreatic beta cells, etc. Stem cells include pluripotent stem cells and somatic stem cells. "Pluripotent stem cells" are stem cells that have the ability to differentiate into all cells present in the living organism, derived from the ectoderm, mesoderm, and endoderm (multipotency), and also possess proliferative capacity. Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by transplantation (ntES cells), pluripotent germ cells (mGS cells), spermatogonial stem cells (GS cells), adult pluripotent stem cells (MAPC cells), and MUSE cells, but iPS cells and ES cells are preferred. "Somatic stem cells" refer to somatic stem cells present in bone marrow, blood, skin (epidermis, dermis, subcutaneous tissue), fat, hair follicles, brain, nerves, liver, pancreas, kidneys, muscles, and other tissues, such as hematopoietic stem cells, vascular endothelial stem cells, mesenchymal stem cells, hepatic stem cells, neural stem cells, pancreatic stem cells, intestinal stem cells, and germline stem cells. "Cancer cells" refer to cells that have derived from the aforementioned somatic cells and acquired unlimited proliferative capacity. "Progenitor cells" refer to cells that are in the intermediate stage of differentiation from the aforementioned stem cells into specific somatic cells or germ cells, and include hematopoietic progenitor cells, myeloid progenitor cells (erythroblasts, myeloblasts, promyelocytes, megakaryocytes, etc.), and lymphoid progenitor cells (monoblasts, lymphoblasts, etc.). "Cell lines" refer to cells that have acquired unlimited proliferative capacity through artificial manipulation outside the body.

[0018] In this invention, "early G1 phase control" refers to halting the cell cycle in the early G1 phase via p53 upregulation and transitioning from the early G1 phase to the G0 phase. "Early G1 phase" refers to the period after the M phase (mitosis) in which cells undergo division, until cyclin D-cyclin-dependent kinase (CDK) 4 / 6, which acts as an engine to propel cells into the next cell cycle phase (S phase), begins to function. The activity of Cyclin D-CDK 4 / 6 can be suppressed by p53 and p21, and halting the cell cycle during this period enables the transition to the G0 phase. The "activity that halts the cell cycle in the early G1 phase and transitions from the early G1 phase to the G0 phase" is achieved by introducing the C7orf26 gene into cells, overexpressing it, and enhancing the expression of p53 and p21, and can be confirmed by PCR and FACS analysis. Furthermore, the above halting includes not only complete halting but also deceleration. Furthermore, "having the activity to arrest the cell cycle in the early G1 phase and to transition from the early G1 phase to the G0 phase" means that it is substantially equivalent to the activity possessed by the protein consisting of the amino acid sequence described in Sequence ID No. 2.

[0019] The C7orf26 protein described above can be produced by genetic engineering methods using a polynucleotide containing the gene encoding it. For example, it can be produced by preparing RNA by in vitro transcription from a recombinant vector having a polynucleotide containing the gene encoding the C7orf26 protein, and then performing in vitro translation using this as a template, or by operably ligating a polynucleotide containing the gene encoding the C7orf26 protein into a suitable vector, introducing this into a host cell to create transformed cells, and expressing the target C7orf26 protein from these transformed cells. The vector can be appropriately selected and used if it is suitable for the host cell into which it will be introduced. The vector also contains a polynucleotide containing the gene encoding the C7orf26 protein operably ligated to a suitable promoter, and preferably includes a transcription termination signal, i.e., a terminator region, downstream of the polynucleotide. Furthermore, it may also include selection marker genes for selecting transformed cells (e.g., drug resistance genes, genes that complement nutrient requirement mutations). It may also include sequences encoding tag sequences useful for the isolation and purification of expressed proteins. The vector may also be integrated into the genome of the host cell. The introduction of the vector into host cells can be carried out using known transformation methods such as the competent cell method, the protoplast method, and the calcium phosphate coprecipitation method.

[0020] Any host cell capable of expressing the above-mentioned vector can be used to introduce the vector and express recombinant protein. Commonly used known microorganisms include bacteria, yeast, fungi, and mammalian cells. Examples of bacteria include Gram-negative bacteria such as Escherichia coli, and Gram-positive bacteria such as Bacillus or Streptomyces. Recombinant cells can be cultured using a known method suitable for the host cell.

[0021] The expressed protein can be purified by collecting the bacterial cells from the culture supernatant of host cells by centrifugation, grinding them with ultrasound or glass beads, removing solid matter such as cell fragments by centrifugation, and then preparing a crude enzyme solution. This crude solution can then be purified using one or more known methods used for purifying proteins and peptides, such as ammonium sulfate precipitation, precipitation separation with organic solvents (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectric focusing chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrates or antibodies, reverse-phase column chromatography, chromatography such as HPLC, and filtration such as microfiltration, ultrafiltration, and reverse osmosis filtration.

[0022] The C7orf26 protein can also be produced by chemical synthesis based on its amino acid sequence. When chemically synthesizing the C7orf26 protein, known chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method) can be used.

[0023] Furthermore, according to another aspect of the present invention, the cell cycle G1 phase early regulator of the present invention includes a gene encoding the C7orf26 protein. Specific examples of the gene include any of the following genes (D) to (F) (which may be referred to as the "C7orf26 gene" in this specification): (D) A gene containing DNA consisting of the nucleotide sequence shown in Sequence ID No. 1; (E) A gene containing DNA encoding a protein having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 1, and which has the activity to arrest the cell cycle in the early G1 phase and transition from the G1 phase to the G0 phase; (F) A gene containing DNA encoding a protein that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1, and which has the activity to arrest the cell cycle in the early G1 phase and transition from the G1 phase to the G0 phase.

[0024] With respect to (E) above, "a nucleotide sequence having 90% or more sequence identity" means that the sequence identity of the nucleotide sequence is at least 90%, preferably 95% or more, more preferably 97% or more, and most preferably 98% or more. The sequence identity of a nucleotide sequence can be determined using methods well known to those skilled in the art, sequence analysis software, etc. Examples include the blastn program of the BLAST algorithm and the fasta program of the FASTA algorithm. Here, the sequence identity of a nucleotide sequence is a value expressed as a percentage of the frequency at which the same nucleotide appears in the same location when the nucleotide sequence to be evaluated is compared with the nucleotide sequence described in Sequence ID No. 1.

[0025] With respect to (F) above, "stringent conditions" means conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed, and can be appropriately determined by referring to, for example, Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions can be set by the temperature and salt concentration in the solution during Southern hybridization, and the temperature and salt concentration in the solution during the washing step of Southern hybridization. More specifically, for example, conditions in which the reaction temperature of hybridization is in the range of 25 to 70°C, preferably 50 to 70°C, more preferably 55 to 68°C, and / or conditions in which the formamide concentration in the hybridization solution is in the range of 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%. Furthermore, the sodium salt concentration during the washing of the filter after hybridization is 15 to 750 mM, preferably 15 to 500 mM, more preferably 15 to 300 mM, 15 to 200 mM, or 15 to 100 mM.

[0026] The method for obtaining the C7orf26 gene is not particularly limited. For example, a human cDNA library (prepared according to a conventional method from suitable cells expressing the gene of the present invention) can be synthesized using a known method, and the target cDNA can be isolated using probe DNA prepared based on the nucleotide sequence of Sequence ID No. 1. The obtained cDNA can be amplified by conventional gene amplification methods such as PCR, NASBN, or TMA. Alternatively, cDNA can be obtained by RT-PCR using a primer set prepared based on the nucleotide sequence of Sequence ID No. 1 and mRNA isolated from human cells as a template. Homologous genes exist in other animals and can also be used.

[0027] To make the above-mentioned C7orf26 protein or C7orf26 gene available for introduction into cells, the following methods can be used, for example. Here, the cells can be either in vitro cells (cultured cells) or in vivo cells (cells in the body).

[0028] The C7orf26 protein can be formulated into a form that can be introduced into cells, for example, by mixing the protein molecule with a pharmacologically acceptable carrier solution without altering its structure or function. Such drugs can be introduced into in vitro cells, for example, by microinjection. Alternatively, intracellular introduction methods using lipids can also be employed.

[0029] Another embodiment involves creating a fusion protein in which a cell membrane transmissible peptide is linked to the N-terminus of the C7orf26 protein, thereby enabling the protein to be introduced into cells. By incorporating this cell membrane transmissible peptide, the C7orf26 protein can pass through the cell membrane and be taken into the cell. As the cell membrane transmissible peptide, the protein transduction domain (PTD) of HIV-1 / TAT or the PTD of the Drosophila homeobox protein Antennapedia can be used. A fusion DNA fragment can be created by linking a DNA fragment encoding the PTD region with the cDNA, and this fusion DNA fragment can be expressed in host cells such as E. coli to produce a fusion protein with the PTD peptide linked to the N-terminus. Alternatively, a fusion protein with the cell membrane transmissible peptide linked can also be produced by linking the C7orf26 protein and the PTD peptide via a divalent crosslinking agent (e.g., EDC or β-alanine).

[0030] On the other hand, the C7orf26 gene or C7orf26 mRNA can be made into a form that can be introduced into cells, for example, by incorporating it into an expression vector. A known eukaryotic cell expression vector having a promoter, splicing region, poly(A) addition site, etc., can be used as the expression vector, and a C7orf26 gene expression vector can be constructed by inserting the C7orf26 gene or C7orf26 mRNA into the cloning site of this expression vector.

[0031] This expression vector can be introduced into in vitro cells (cultured cells) by known methods such as electroporation, calcium phosphate method, liposome method, and DEAE dextran method.

[0032] Furthermore, for in vivo cells (cells in the body), the drug can be introduced into cells using means such as viral or nonviral gene transfer vectors to promote uptake into cells and enhance targeting of target cells. Drugs in this form can be introduced into the body for gene therapy. Examples of viral vectors include adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, lentivirus vectors, vaccinia virus vectors, and baculovirus vectors. Retrovirus vectors are particularly desirable because, after infecting cells, the viral genome is integrated into the host chromosome, allowing for stable and long-term expression of the foreign gene incorporated into the vector. Examples of nonviral vectors include liposomes, artificial lipid vesicles, hollow nanoparticles, and macromolecular compounds such as dendrimers.

[0033] 2. Pharmaceuticals for Cancer Treatment The early G1 phase control agent of the present invention, as specifically shown in the examples described later, arrests the cell cycle in cancer cells at the early G1 phase and induces cell death by increasing p53 expression. Therefore, it can be used as a pharmaceutical for the treatment and / or prevention of diseases caused by abnormal cell cycle regulation, or related diseases, particularly as a pharmaceutical for cancer treatment. Typical diseases caused by abnormal cell cycle regulation include malignant tumors (cancer), and the types are not particularly limited, but include gastric cancer, breast cancer, lung cancer, esophageal cancer, prostate cancer, liver cancer, colorectal cancer, kidney cancer, bladder cancer, skin cancer, uterine cancer, brain tumor, osteosarcoma, bone marrow tumor, leukemia, malignant lymphoma, etc. In particular, it is effective for intractable cancers such as glioma and undifferentiated cancers in combination with high differentiation by inducing the G0 phase. The presence and extent of induction of cell death can be detected by methods such as the TUNEL method, DNA ladder detection method, quantification of DNA fragmentation rate, and measurement of cell size distribution. Furthermore, it can suppress cell proliferation in diseases where cells proliferate excessively. These diseases are not particularly limited and include adenomas, such as pituitary adenoma, hyperthyroidism, pheochromocytoma, and polycystic ovary syndrome.

[0034] When using the early G1 phase control agent of the present invention for the treatment of cancer or hyperproliferative diseases of cells, it should be administered to mammals with cancer or hyperproliferative diseases in a therapeutically effective dose. Examples of mammals include humans, dogs, cats, sheep, goats, cattle, horses, and pigs. A "therapeutably effective dose for cancer or hyperproliferative diseases of cells" refers to the amount of this agent administered to proliferating cancer or hyperproliferative cells that results in the cessation of cancer or hyperproliferative cell proliferation, reduction of tumor size, or disappearance. The specific dose should be appropriately increased or decreased depending on the route of administration, the patient's age and weight, the type and malignancy of the cancer, the presence or absence of metastasis or recurrence, the type and proliferation rate of hyperproliferative cells, and the size of the tumor.

[0035] The methods of administration include intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, local, intratumoral, oral, perdermal, rectal, vaginal, nasal, and sublingual administration. Specifically, for example, solid tumors in various organs that can be easily accessed by surgery can be administered by local injection into or near the tumor using a stereotactic needle. Non-solid tumors such as leukemia, cancers in areas that are difficult to access by surgery such as brain tumors, and metastatic cancers can be administered by intravenous injection. In addition, the above administration methods can be appropriately selected and used depending on the type and location of the cancer.

[0036] When the early G1 phase control agent of the present invention is formulated as a cancer treatment drug, the C7orf26 protein or C7orf26 gene, or C7orf26 mRNA, is mixed with pharmacologically and pharmaceutically acceptable additives and can be formulated into various formulations such as tablets, powders, granules, fine granules, capsules, oral solutions (suspensions, syrups, emulsions, etc.), topical solutions (injections, sprays / aerosols, inhalants, ointments, etc.), injections, drips, and suppositories using methods known in the art. Pharmacologically and pharmaceutically acceptable additives may be appropriately selected depending on the dosage form and application, including formulation bases and carriers, excipients, diluents, binders, lubricants, coatings, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonic agents, pH adjusters, colorants, etc., and prepared in various known formulations that can be administered orally or parenterally, systemically or topically. The cancer treatment drug of the present invention, prepared in various formulations, can be administered orally or parenterally, systemically or topically. When administering the cancer treatment drug of the present invention orally, it may be formulated into tablets, capsules, granules, powders, pills, internal liquids, suspensions, emulsions, syrups, etc., or as a dried product that is redissolved at the time of use. Furthermore, when the cancer treatment drug of the present invention is administered parenterally, it is formulated into intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, intrathecal injection, subcutaneous injection, suppositories, etc., and in the case of an injectable formulation, it is provided in the form of a unit dose ampoule or a multi-dose container.

[0037] Gene therapy can be administered in either an ex vivo or in vivo manner, where target cells are extracted from the body and the gene is introduced. The cell cycle G1 phase early control agent of the present invention is applicable to both methods of treatment. In the ex vivo method, patient-derived cells are cultured outside the body, subjected to the gene introduction process, and then administered to the patient. In the in vivo method, the gene introduction vector is administered directly into the patient's body (organ tissue, skin, muscle, etc.). Furthermore, such cancer and hyperproliferative disease treatments may be used in combination with well-known cancer and hyperproliferative disease treatments, including surgery, chemotherapy, and radiation therapy.

[0038] 3. Differentiation-promoting agent for undifferentiated cells and method for promoting differentiation of undifferentiated cells The early G1 phase regulator of the cell cycle of the present invention can be used as a differentiation-promoting agent for undifferentiated cells because, as specifically shown in the examples described later, it can arrest stem cells in the early G1 phase of the cell cycle, then transition them to the G0 phase, and induce differentiation into target cells.

[0039] In the present invention, "undifferentiated cells" refer to undifferentiated cells that can differentiate into ectoderm cells, mesoderm cells, or endoderm cells, or that can promote their repair, and include stem cells, progenitor cells, and dedifferentiated (immature) mature cells. Stem cells are not particularly limited as long as they are in line with the purpose of the present invention, and include the aforementioned somatic stem cells and pluripotent stem cells (ES cells, iPS cells, etc.). Stem cells may be primary cultured cells, passaged cultured cells, or frozen cells. Here, ectoderm cells include sensory organ cells (retina and inner ear cells), nervous system cells (nerve cells (e.g., forebrain nerve cells, midbrain nerve cells, cerebellar nerve cells, hindbrain nerve cells, spinal nerve cells, etc.), neural tube cells, neural crest cells), and epidermal cells (epidermal cells, lens epithelial cells), while mesoderm cells include muscle cells (myoblasts, muscle satellite cells, etc.), skeletal cells (osteoblasts, osteocytes, chondrocytes, etc.), adipocytes, dermal cells, and circulatory system cells (heart Examples of endoderm cells include muscle cells, hematopoietic stem cells, red blood cells, platelets, macrophages, granulocytes, helper T cells, killer T cells, B lymphocytes, etc.), urogenital cells (tubular cells, mesangial cells, juxtaglomerular cells, testes, ovaries, etc.), and connective tissue. Endoderm cells include digestive system cells (hepatocytes, bile duct cells, pancreatic endocrine cells, acinar cells, pancreatic duct cells, absorptive cells, goblet cells, Paneth cells, enterocrine cells, etc.), and cells of tissues such as the lungs and thyroid.

[0040] The differentiation-promoting agent for undifferentiated cells of the present invention can be applied to all undifferentiated cells as long as they have equivalent characteristics in terms of differentiation direction and differentiation process. For example, the differentiation-promoting agent for undifferentiated cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cattle, sheep, goats, and pigs, as well as birds, reptiles, amphibians, fish, insects, crustaceans, and mollusks.

[0041] The application of the undifferentiated cell differentiation promoter according to the present invention to the cells may be in vitro or in vivo, and the effect can be exerted in either case. For example, when applying the undifferentiated cell differentiation promoter of the present invention in vitro, typically, an expression vector having the target C7orf26 gene downstream of an expression promoter is introduced into the target undifferentiated cells and cultured.

[0042] Examples of the expression promoter used here include the CMV promoter, SV40 promoter, etc. Examples of the expression vector include plasmid vectors and liposomes as non-viral vectors, and adenovirus vectors and retrovirus vectors as viral vectors. These expression vectors can also use commercially available products from each manufacturer. The expression vector may be integrated into the genome of the host cell. Further, as a method for introducing the expression vector into the cells, for example, the lipofection method, the electroporation method, the method of incorporating a gene into a viral vector and infecting, etc. can be used.

[0043] In the culture of undifferentiated cells into which the above gene has been introduced, the medium and additives used are not particularly limited. For example, media and additives generally used for culturing stem cells may be used. Specifically, a basal medium containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.), such as Dulbecco's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM / F-12), Glasgow Minimum Essential Medium (Glasgow MEM), Hank's balanced salt solution, etc. can be mentioned. In addition, depending on the target cells for differentiation induction, the medium may contain cell growth factors (e.g., fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin, etc.), cytokines (e.g., interleukins, chemokines, interferons, colony-stimulating factors, tumor necrosis factors, etc.), neurotrophic factors (nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), etc.), and other bioactive substances, such as amino acids (e.g., glycine, phenylalanine, lysine, aspartic acid, glutamic acid, etc.), vitamins (e.g., biotin, pantothenic acid, vitamin D, etc.), serum albumin, antibiotics, etc.

[0044] The incubator used for culturing undifferentiated cells is not particularly limited. For example, flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, roller bottles, etc. can be mentioned.

[0045] The culture vessel may be either non-adherent or adherent, and can be appropriately selected according to the purpose. Cell-adherent culture vessels may be treated with cell-supporting substrates such as extracellular matrix to improve cell adhesion. Examples of cell-supporting substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin.

[0046] The culture conditions for undifferentiated cells can follow the usual conditions used for culturing stem cells, etc., and no special control is required. For example, the culture temperature is not particularly limited, but is about 30-40°C, preferably 36-37°C. The CO2 gas concentration is, for example, about 1-10%, preferably about 2-5%.

[0047] The present invention will be described in more detail below with reference to examples. However, these examples are not intended to limit the present invention. The materials and test methods used in each example are shown below.

[0048] We purchased a human C7orf26 (NM_024067) ORF clone with a C-terminal Myc-DKK tag (Origene#:RC219786; pCMV C7orf26-Flag) and a negative control GFP expression clone (TaKaRa#Z2470N; pAcGFP1-C1). These expression vectors were prepared using the EndoFree Plasmid Maxi Kit (QIAGEN#:12362).

[0049] (Primers) The following is a list of primers used for gene expression analysis by quantitative PCR (real-time RT-PCR). C7orf26_F: 5'-CCGAGATTCTGCTGCCAGTTC-3' (SEQ ID NO: 3) C7orf26_R: 5'-GTGACAATCATTTCAAGCAAGTCCA-3' (SEQ ID NO: 4) p53_F: 5'-CAGCACATGACGGAGGTTGT-3' (SEQ ID NO: 5) p53_R: 5'-TCATCCAAATACTCCACACGC-3' (SEQ ID NO: 6) P21_F: 5'-TGTCCGTCAGAACCCATGC-3' (SEQ ID NO: 7) P21_R: 5'-AAAGTCGAAGTTCCATCGCTC-3' (SEQ ID NO: 8) Ki67_F: 5'-GCCTGCTCGACCCTACAGA-3' (SEQ ID NO: 9) Ki67_R: 5'-GCTTGTCAACTGCGGTTGC-3' (SEQ ID NO: 10) Pax6_F: 5'-TTTAAAGATCCTGGAGGTGGACATA-3' (SEQ ID NO: 11) Pax6_R: 5'-GCTCAGGTGCTCGGGTTCTA-3' (SEQ ID NO: 12) Brn3b_F: 5'-TGACACATGAGCGCTCTCACTTAC-3' (SEQ ID NO: 13) Brn3b_R: 5'-ACCAAGTGGCAAATGCACCTA-3' (SEQ ID NO: 14) Math5_F: 5'-CCCTAAATTTGGGCAAGTGAAGA-3' (SEQ ID NO: 15) Math5_R: 5'-CAAAGCAACTCACGTGCAATC-3' (SEQ ID NO: 16) Pax3_F: 5'-CCATACGTCCTGGTGCCATC-3' (SEQ ID NO: 17) Pax3_R: 5'-TGAACATGCCCGGGTTCTC-3' (SEQ ID NO: 18) Sox1_F: 5'-ATGCACAACTCGGAGATCA-3' (SEQ ID NO: 19) Sox1_R: 5'-GCGAGTACTTGTCCTTCTTG-3' (SEQ ID NO: 20) En1 F: 5'-GAGCGCAGGGCACCAAATA-3' (SEQ ID NO: 21) En1_R: 5'-CGAGTCAGTTTTGACCACGG-3' (SEQ ID NO: 22) Otx2_F: 5'-CAAAGTGAGACCTGCCAAAAAGA-3' (SEQ ID NO: 23) Otx2_R:5'-TGGACAAGGGATCTGACAGTG-3' (SEQ ID NO: 24) TBXT_F: 5'-TATGAGCCTCGAATCCACATAGT-3' (SEQ ID NO: 25) TBXT_R: 5'-CCTCGTTCTGATAAGCAGTCAC-3' (SEQ ID NO: 26) Sox17_F: 5'-AGCAGAATCCAGACCTGCACAA-3' (SEQ ID NO: 27) Sox17_R: 5'-TCAGCGCCTTCCACGACTT-3' (SEQ ID NO: 28)

[0050] (Cell Culture) HeLa cells were obtained from ATCC, 293T cells from Takara Bio, glioma cells U-251MG from DS BioPharma Medical, and human iPS cells 409B02 from RIKEN BRC. HeLa cells were grown in DMEM medium (Thermo Fisher Scientific CAT#:11965092) supplemented with 10% FBS and 1% Pen Strep. 293T cells were grown in 10% FBS and 1×GlutaMAX. TM Human iPS cells were grown in DMEM medium (Thermo Fisher Scientific CAT#:35050061) supplemented with the added supplement. Human iPS cells were grown on a feeder layer (inactivated MEFs) in Primate ES medium (ReproCELL) supplemented with 10 ng / mL of bFGF (Invitrogen).

[0051] (Cell cycle analysis) Cell cycle analysis was performed using the Click-iT Plus EdU flow cytometry assay kit (Thermo Fisher Scientific, #C10632) in combination with FxCycle PI / RNase staining solution (Thermo Fisher Scientific, #F10797), following the recommended staining protocol. HeLa cells were incubated with 10 μM eDU at 37°C for 1.5 hours and then harvested. The cells were fixed with Click-iT fixative for 15 minutes, followed by permeabilization with Click-iT saponin-based permeabilization and washing agent in the dark for 15 minutes. A Click-iT reaction with Alexa Fluor 488 pycolyl azaide was performed with a reaction volume of 0.6 mL for 30 minutes. After washing, the cell pellet was suspended in 0.2 mL of FxCycle PI / RNase staining solution and incubated in the dark at room temperature for 30 minutes. Subsequently, the cells were analyzed using an Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific). For EdU detection using Alexa Fluor 488 pycolyl azaide, 488nm excitation with a green emission filter (530 / 30nm) was used, and for PI detection, 488nm excitation with an orange emission filter (574 / 26nm) was used. Compensation analysis was performed using unstained HeLa cells and single-stained cells with either EdU or PI. Cell cycle population ratios were analyzed using Attune Cytometric Software v2.1.

[0052] (Cell cycle synchronization) For HeLa cells, the cell cycle was synchronized by culturing them for 24 hours in a medium containing 10 mM hydroxyurea (Sigma#H8627) or 10 μM Ro3306 (Abcam#ab141491), or in serum-free medium. Subsequently, the hydroxyurea-synchronized HeLa cells were cultured in normal medium and harvested after a predetermined period for expression analysis and cell cycle analysis.

[0053] (Luciferase Reporter Assay) The luciferase reporter assay was performed using the LightSwitch Luciferase Assay Kit (Active Motif #32031) according to the manufacturer's protocol. The reporter vector used was the LightSwitch promoter reporter GoClone (Active Motif #S717666, #S721662), which contains the c7orf26 and p53 promoter regions and a 3'-RenSP luciferase region downstream. This reporter vector, along with cDNA clones encoding c7orf26, p53, and INTS, was co-transfected into HeLa cells using FuGENE HD transfection reagent (Promega #E2311), and the cells in the 96-well plate were frozen 48 hours after transfection. After thawing, LightSwitch Luciferase Assay Reagent was added to the wells, and after a 30-minute reaction, the cells were transferred to a 1 / 2 AreaPlate-96 plate (PerkinElmer #6002350). Chemiluminescent signals were read using a 2030 ARVO X multi-label counter instrument (PerkinElmer). Signal ratios for each promoter activity were calculated in comparison to GFP-transfected cells.

[0054] (Cell Death Assay) HeLa cells were transfected with the pU6-shC7orf26-GFP vector and cultured in a 96-well plate or 8-well glass chamber after transfection. After 36 hours, the cells were fixed. Immunostaining with M30 CytoDEATH antibody (Roche # 12140322001) was performed on the fixed cells to detect cell death. After washing twice with PBS containing 0.1% Tween 20, the cells were incubated with M30 CytoDEATH antibody solution (1:50) at room temperature for 60 minutes. The cells were washed twice and incubated with Goat anti-Mouse IgG (H+L) secondary antibody and Alexa Fluor Plus 555 solution (1:500) (Thermo Fisher Scientific #A32727) at room temperature for 30 minutes. ProLong with DAPI After mounting with Gold Antifade reagent (Thermo Fisher Scientific #A32727), M30 CytoDEATH-positive cells were observed using DeltaVision ELITE (CORNES Technologies).

[0055] (Immunofluorescence) The specimen was fixed in 4% paraformaldehyde (pH 7.0) at room temperature for 20 minutes. After rinsing twice with PBS, the specimen was incubated with 0.1% Triton X-100 at room temperature for 15 minutes and rinsed twice with PBS. Next, the specimen was incubated with 3% BSA at room temperature for 60 minutes, followed by a primary antibody reaction at 4°C for 16 hours. After washing three times with PBS for 5 minutes each, a secondary antibody reaction was performed by incubation with the corresponding species-specific Alexa Fluor conjugate antibody at room temperature in the dark for 1 hour. After washing four times with PBS for 5 minutes each, the specimen was mounted on ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific #A32727) and observed with DeltaVision ELITE (CORNES Technologies).

[0056] (Knockdown of E2F by siRNA) E2F4-specific siRNA (Human E2F4 (1874) siRNA-SMARTpool L-003262-00-0005), E2F6-specific siRNA (Human E2F6 (1876) siRNA-SMARTpool L-003264-00-0005), E2F1-specific siRNA (E2F1 (1869) siRNA-SMARTpool, 5 nmol L-003259-00-0005), and a control siRNA containing a scrambled sequence (ON-TARGETplus Non-targeting Pool, #D-001810-10; NC siRNA) were purchased from Dharmacon. HeLa cells were seeded in 24-well plates two days before transfection. HeLa cells with a cell density of 50-70% are treated with 50 μL of Opti-MEMI medium containing 5 pmol of siRNA and Lipofectamine.TM Cells were transfected using RNAiMAX Transfection Reagent (Thermo Fisher Scientific #13778150) according to the manufacturer's instructions. For analysis, cells were harvested and fixed at different time points after transfection.

[0057] (Immunoprecipitation) 293T cells with a confluence of 70–80% in a 10 cm dish were transfected with 1.5 mL of Opti-MEM I medium containing 15 μg of pC7orf26-myc-Flag using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific #L3000001). After 72 hours of culture, proteins were extracted from the cells using CelLytic M Cell Lysis Reagent (Sigma #C2978) containing a protease inhibitor cocktail (Roche #04693116001) according to the manufacturer's instructions.

[0058] EZview TM Immunoprecipitation was performed using Red ANTI-FLAG M2 Affinity Gel (Sigma#: F2426) and FLAG Immunoprecipitation Kit (Sigma#: FLAGIPT1). The affinity gel was suspended in 1% BSA washing buffer and rotated at room temperature for 30 minutes. After washing four times with washing buffer, the affinity gel was suspended in cell lysate and rotated at 4°C for 16 hours. After washing four times with washing buffer, the C7orf26 Flag-fusion protein was isolated from the affinity gel by elution with 3x Flag peptide solution. The volume was concentrated using Amicon Ultra-0.5 (Millpore#: UFC5003) and then eluted using Qubit TM Protein content was measured using the R Protein Assay (Thermo Fisher Scientific#: Q33211).

[0059] (Immunoblot Analysis) Approximately 5 μg of extract and 1 μg of clarified eluate were boiled at 95°C for 5 minutes, separated on a 7.5% acrylamide SDS-PAGE gel, and transferred to a PVDF membrane (Bio-Rad #1620174) by wet transfer. The membrane was blocked with Blocking One (NAKARAI #03953), incubated at room temperature for 20 minutes, and washed with TBST. The membrane was incubated overnight with primary antibody (anti-E2F4 antibody) at 4°C, washed with TBST, incubated with HRP-conjugated secondary antibody at room temperature for 1 hour, and eluted with Blocking One. Immunoblastic bands were visualized using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientifi #34095).

[0060] (Real-time RT-PCR) Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). mRNA expression levels in each RNA sample were measured using the StepONE Sequence Detection System (Applied Biosystems). Reverse transcription polymerase chain reaction (RT-PCR) was performed using One Step SYBR TM PrimeScript TM The procedure was performed using the PLUS RT-PCR Kit (Takara Bio). Real-time PCR was performed using the aforementioned primers, and the expression levels of each gene were measured. The PCR conditions were: holding at 42°C for 5 minutes, followed by 95°C for 10 seconds, then 95°C for 5 seconds, and 60°C for 31 seconds, repeated for 40 cycles. mRNA expression levels were evaluated by the Ct (Threshold cycle) value.

[0061] (Example 1) Expression changes of C7orf26 during the cell cycle To analyze the expression changes of C7orf26 during the cell cycle, the cell cycle of HeLa cells was synchronized by adding hydroxyurea (HU) to the culture medium for 24 hours, and then the cell cycle was restarted by removing HU from the medium. After the restart of the cell cycle, HeLa cells were analyzed by flow cytometry after a certain period of time. At the time of cell cycle restart (hour 0), most HeLa cells were in the early S phase, progressing to the G2 / M phase at 16 hours, and an increase in cells in the early G1 phase was confirmed at 20 hours (Figure 1A). The expression of C7orf26 mRNA gradually increased with the progression of the cell cycle, peaking in the M phase 16 hours after HU removal, and high expression continued even in the early G1 phase 20 hours after HU removal. On the other hand, the expression of the cell proliferation marker Ki67 mRNA peaked in the G2 phase 12 hours after HU removal and gradually decreased in the M phase (Figure 1B). Immunostaining of HeLa cells at each phase of the cell cycle revealed that C7orf26 was present in the nucleus as expected (Figure 1C; upper left panel, white dots), and was prominently expressed in the cytoplasm during metaphase M (Figure 1C; upper right panel, white area). High expression of C7orf26 continued until telophase M (Figure 1C; lower left panel, white area), and staining was still observed until phase G1 (Figure 1C; lower right panel, white area). These results indicate that while housekeeping genes are stably expressed throughout the cell cycle, the expression of the C7orf26 gene fluctuates dramatically. High C7orf26 expression from phase G2 to M was expected because various alternative transcription mutants and their maturation are necessary for the dynamic cell behavior of mitosis. However, the fact that high expression continues until early phase G1, when cell division has already ended and numerous transcription mutants are no longer needed, highlights the noteworthy role of C7orf26 in the cell cycle.

[0062] (Example 2) Cell cycle regulation by C7orf26 To investigate the role of C7orf26 in the cell cycle, which is characterized by high expression even in the G1 phase, a gain-of-function experiment was conducted using a Cumate-Switch System that achieves physiological levels of C7orf26 increase. The activator cumate used was Water-soluble cumate solution (300 mg / mL, 10,000x) catalog number QM150A-1 from SBI (System Biosciences, LLC).

[0063] HeLa cells were transfected with the pPB-Cuo-C7orf26 vector containing the C7orf26 coding region using the PiggyBac Cumate Switch Inducible Vector (System Biosciences, LLC #PBQM812A-1) to clone a stable cell line, HeLa / pPB-Cuo-C7orf26 (this cell line expresses C7orf26 depending on the concentration of cumate added). This cell line was seeded in culture medium, and on day 2 after seeding, 1 / 1000 of the culture medium volume (final concentration 0.3 mg / ml) of cumate was added to the culture medium to 10x, and the cells were cultured to express C7orf26 mRNA. As shown in Figure 2, C7orf26 was expressed in the cells in a concentration-dependent manner depending on the cumate added to the culture medium. Furthermore, p53 expression increased in response to C7orf26, and the expression level of p21, which is downstream of p53, also increased in response to p53 expression. On the other hand, contrary to the upregulation of p53 and p21 expression by C7orf26, the expression level of Ki67 mRNA, a cell proliferation marker, was significantly decreased. Thus, it was confirmed that C7orf26 increases the expression of p53 and p21, which are transcription factors that suppress the cell cycle, and arrests the cell cycle as seen in the decrease in Ki67.

[0064] Cells continuously expressing C7orf26 in HeLa / pPB-Cuo-C7orf26 via continuous cumate supply were analyzed by flow cytometry. As shown in Figure 3A, compared to a control without cumate supply, continuous expression of C7orf26 for 5 days via continuous cumate supply increased the relative number of M-phase cells. With 6 days of continuous expression, the number of G1-phase cells increased, while the relative numbers of S-phase and G2 / M-phase cells decreased. With 8 days of continuous expression, the cell number decreased in all phases (G1, S, and G2 / M), but almost all cells were in the G1 phase, and the cell cycle stopped in the G1 phase without progressing.

[0065] The expression profiles of C7orf26, p53, p21, and Ki67 were analyzed at each phase of the cell cycle. To arrest the cell cycle at each phase, HeLa cells were cultured in medium supplemented with a cell cycle arrester or in serum-free medium, synchronized, washed, and then the cell cycle was restarted. Hydroxyurea (HU) was used to arrest the cell cycle in the S phase, and Ro3306 was used to arrest the cell cycle in the M phase. Flow cytometry analysis of HeLa cells showed that, compared to the control (Figure 3B, upper panel), the cell cycle of HeLa cells was arrested in the early S phase with HU (Figure 3B, middle panel) and arrested in the M phase with Ro3306 (Figure 3B, lower panel).

[0066] As shown in Figure 3C, compared to the control, C7orf26 expression was minimized in early S phase in cells whose cell cycle was arrested in S phase by HU and then restarted, accompanied by decreased expression of p53 and p21. In cells whose cell cycle was arrested in M ​​phase by Ro3306 and then restarted, C7orf26 expression was maximized in M ​​phase, and p53 and p21 were upregulated along with the increase in C7orf26. Furthermore, 24 hours of serum starvation (corresponding to cell cycle arrest in early G1 phase), which is a condition for inducing forced arrest in G0 phase in human fibroblasts, also induced C7orf26 expression, and in response to the increase in C7orf26 expression, the expression of p53 and p21 also increased. In late G1 phase, 4 hours after restarting the cell cycle from serum starvation, C7orf26 expression decreased, and the expression of p53 and p21 also decreased accordingly. Similarly, Ki67 expression decreased in early S phase in HU-induced cell cycle arrest cells, showed only a slight increase even during M phase cell division in Ro3306-induced cell cycle arrest cells, decreased in early G1 phase in serum-starvated cell cycle arrest cells, and was almost the same as the control in late G1 phase after serum restoration.

[0067] (Example 3) Mechanism of cell arrest induction in the G1 phase via the p53 pathway by C7orf26 The cell cycle has four cell cycle checkpoints, and it is thought that p53 controls the progression of the cell cycle at each checkpoint, and that the p53 pathway arrests the cell cycle. C7orf26 was highly expressed from the M phase to the early G1 phase, Ki67 decreased, and the G1 phase was arrested by C7orf26. Therefore, the main points at which C7orf26 acts on p53 were considered to be the initiation of cyclin D-CDK4 / 6 and cyclin E-CDK2 (limiting point). Of the two points, the initiation of cyclin D-CDK4 / 6 showed significant expression of the C7orf26 protein in the early G1 phase in immunohistochemistry, so it is most likely that C7orf26 specifically and strongly influences p53 in the early G1 phase (Figure 4).

[0068] The dose-dependent upregulation of p53 by C7orf26 observed in Example 2 was thought to be due to direct transcriptional regulation. Therefore, a reporter assay was performed in HeLa cells co-transfected with a reporter vector containing the p53 promoter region and pCMV-C7orf26-Flag. Compared to transfection with the control vector pCMV-GFP, the p53-Luc reporter signal was significantly increased by C7orf26 expression (Figure 5-1, A).

[0069] Furthermore, HeLa cells were co-transfected with a reporter vector containing the p53 promoter region and pCMV-INTSX-FLAG (X=1–14). Compared to transfection with the control vector pCMV-GFP, the p53-Luc reporter signal was also significantly increased by the expression of integrator complex subunits (INTS1, INTS2, INTS3, INTS4, INTS5, INTS6, INTS7, INTS8, INTS9, INTS10, INTS11, INTS12, INTS13, INTS14) (Figure 5-1, B).

[0070] These results indicate that C7orf26, in cooperation with the integrator complex subunit, upregulates p53. Since neither the integrator complex subunit nor C7orf26 contains a known promoter-binding domain, these findings suggest that they may be transcriptional coactivators of an unknown transcription factor that regulates p53 transcription.

[0071] Furthermore, surprisingly, the C7orf26-Luc reporter signal was also significantly increased by transfection with pCMV-p53-HA, indicating a positive feedback loop between C7orf26 and p53 (Figure 5-1, C).

[0072] Furthermore, the expression changes of the integrator complex subunits (INTS1, INTS2, INTS3, INTS4, INTS5, INTS6, INTS7, INTS8, INTS9, INTS10, INTS11, INTS12, INTS13, INTS14) during the cell cycle were analyzed in the same manner as described in Example 1. The mRNA expression of each member of the integrator complex subunits gradually increased with the progression of the cell cycle, similar to C7orf26, with most peaking in the M phase 16 hours after HU removal, and high expression continued even in the early G1 phase 20 hours after HU removal (Figure 5-2).

[0073] Next, we searched the web database JASPAR using the promoter region of p53, the target of C7orf26, and found members of the E2F family (E2F1, E2F4, E2F6) among the candidate transcription factors. Therefore, we analyzed the expression profiles of the E2F family transcription factors at each phase of the cell cycle. To halt the cell cycle at each phase, HeLa cells were cultured in medium supplemented with a cell cycle arrester or in serum-free medium, synchronized, and then washed to restart the cell cycle. Hydroxyurea (HU), which halts the cell cycle at the S phase, was used as the cell cycle arrester. After restarting the cell cycle by removing HU, E2F1 expression gradually decreased from the S phase to the G2 phase (0 to 8 hours), while E2F4 expression gradually increased in the G2 / M phase. This suggests that the increased E2F4 may contribute to the upregulation of C7orf26 during the G2 / M phase. E2F6 expression was relatively stable (Figure 6-1, A).

[0074] We investigated the changes in p53 reporter activity mediated by C7orf26 by knockdown of E2F cells (E2F1, E2F4, E2F6) using siRNA. Upregulation of p53 by C7orf26 was significantly reduced by siRNA of transcription factors E2F1 and E2F4. Therefore, E2F1 and E2F4 were considered candidate transcription factors that work in conjunction with C7orf26 and INTS to activate p53 transcription (Figure 6-1, B). Western blot analysis was performed on flag affinity eluates from HeLa cells transfected with pCMV-GFP or pCMV-C7orf26-Flag using an antibody against human E2F4. As a result, binding of C7orf26 to E2F4 was confirmed, supporting the idea that C7orf26 is a transcriptional cofactor of E2F4 in Figure 6-1, B (Figure 6-1, C).

[0075] Regarding E2F cells that express the C7orf26 gene, in HeLa cells overexpressing E2F4 and E2F6, E2F4 significantly increased the reporter activity of C7orf26-Luc more than E2F6 (Figure 6-2, A). Furthermore, when E2F4 and E2F6 siRNAs were applied to HeLa cells with synchronized cell cycles, C7orf26 expression was suppressed in the G2 and M phases (Figure 6-2, B). Therefore, it is presumed that E2F4 / 6 are the upstream genes that express the C7orf26 gene.

[0076] In HeLa cells with synchronized cell cycles, C7orf26 mRNA showed a cell cycle-dependent expression pattern, with peak expression of C7orf26 mRNA and protein occurring during the M phase (Figures 1B and 1C). Therefore, it was hypothesized that CDK1 is an upstream transcriptional repressor of C7orf26. HeLa cells were cultured in media with and without a CDK1 inhibitor, synchronized to the S phase with HU, and then the cell cycle was restarted by removing HU after 24 hours. In cells cultured in the CDK1 inhibitor-containing medium, C7orf26 mRNA levels clearly increased at 12 and 16 hours after HU removal (Figure 6-2, C). This result indicates that CDK1 is an upstream transcriptional repressor of C7orf26. As a result, it is hypothesized that at the early M phase checkpoint, C7orf26 is suppressed, preventing an increase in p53 expression, thus avoiding cell cycle arrest. After cell division, the cell enters the G1 phase where CDK1 is degraded, and only then does the G1 phase cell cycle arrest function of C7orf26 become active.

[0077] The series of experiments described above revealed that the C7orf26 protein acts as a transcriptional coactivator of p53 from M phase to G1 phase, working in conjunction with INTS and E2F1 / E2F4. It rapidly upregulates p53, causing abrupt cell cycle arrest in G1 phase and transition to G0 phase. The function of C7orf26 is suppressed by CDK1 until M phase, when its expression is released in G1 phase and rapidly increases due to positive feedback from p53. In addition, the integrator complex subunit protein, which works in conjunction with the transcription factor E2F4 / 1 protein, is rapidly formed during M phase (approximately 30 minutes) and works with C7orf26 in the early G1 phase to arrest the cell cycle at that point. These are thought to be the mechanisms by which C7orf26 specifically arrests the cell cycle at the checkpoint in the early G1 phase.

[0078] (Example 4) Confirmation of cell death induction (1) Induction of cell death by continuous expression of C7orf26 Figure 7 shows the flow cytometry results of HeLa / pPB-Cuo-C7orf26 in which C7orf26 was continuously expressed by cumate supplementation according to the same method as in Example 2. The staining reagent used was LIVE / DEAD from Thermo Fisher Scientific. TM After staining for 30 minutes using the Fixable Red Dead Cell Stain Kit (Catalog No. L34971), positive cells (488 nm) were analyzed. Weak fluorescence (low values) indicates cells containing dead cells or cell debris. After 8 days of continuous expression of C7orf26, the number of G1 phase cells decreased, accompanied by an increase in dead cells and cell debris (Figure 7A, arrowhead).

[0079] Furthermore, in HeLa / pPB-CuO-C7orf26 cells that continuously expressed C7orf26 for 5 days by supplementing with cumate, most cells survived except for a small number of dead cells, similar to the control (unsupplemented with cumate) HeLa / pPB-CuO-C7orf26 cells (Figure 7B, upper panel). In HeLa / pPB-CuO-C7orf26 cells that continuously expressed C7orf26 for 8 days by supplementing with cumate, while some living cells were present compared to the control (unsupplemented with cumate) HeLa / pPB-CuO-C7orf26 cells, the majority were dead (Figure 7B, lower panel).

[0080] These results indicate that C7orf26 induces G1 arrest in HeLa / pPB-CuO-C7orf26 cells, which ultimately leads to cell death.

[0081] (2) Induction of cancer cell death by forced expression of C7orf26 The C7orf26 gene was introduced into HeLa cells (uterine cancer cell line) and glioma cell line 251 (malignant brain tumor cell line) and forced expression was induced. When C7orf26 was forced into HeLa cells, after 24 hours (Figure 8a, b; a is C7orf26 staining alone, b is a merge with nuclear staining with DAPI), C7orf26 (white area in the figure) accumulated in the nucleus, and after 36 hours (Figure 8c, d), it was confirmed that the nucleus underwent cell death by autophagy and atrophied. Similarly, in glioma cell line 251, after 24 hours (Figure 8e, f; e is C7orf26-only staining, f is a merge with nuclear staining using DAPI), C7orf26 (white areas in the figure) was highly concentrated in the nucleus, and after 36 hours (Figure 8g), the nucleus was lysed by autophagy. Simultaneously, organelles such as mitochondria in the cytoplasm were also lysed by autophagy, leading to cell death (areas enclosed by dashed lines in the figure). In contrast, when the GFP gene was forced to be expressed instead of the C7orf26 gene, mitochondrial staining of HeLa cells (control) showed that in viable cells, mitochondria were neatly arranged as granules (Figure 8h, white dots).

[0082] (Example 5) Confirmation of stem cell differentiation promotion (1) Differentiation induction from human iPS cells to ectoderm The pPB-Cuo-C7orf26 vector containing the C7orf26 coding region was introduced into the human iPS 409B02 cell line using the PiggyBac Cumate Switch Inducible Vector (System Biosciences, LLC #PBQM812A-1), and a cumate-induced c7orf26-expressing iPS cell line was obtained by selecting the buromycin drug. Cumate-induced c7orf26-expressing iPS cell lines, grown by feeder-free culture using Stem Fit medium (Ajinomoto #AK02N), were seeded at a rate of 40,000 cells in 6 cm adhesive culture dishes (BD Falcon) coated with iMatrix-511 solution (0.5 mg / mL PBS Nippi #892012). After 24 hours of incubation, 10,000x Cumate Solution (System Biosciences, LLC #PBQM100A-1) was administered to a final concentration of 2x. The following day, the cells were transferred to ectoderm induction medium from the STEMdiff Trilineage differentiation kit (STEMCELL Technologies #ST-05230) to initiate induction (Day 0). The medium was changed daily thereafter, and the cells were harvested and fixed on day 7.

[0083] On day 5 after cumate administration (day 4 after the start of differentiation induction), cultured cells were harvested, and the expression levels of the C7orf26, p53, and p21 genes were analyzed by quantitative PCR. The results show the relative expression levels of each gene in cells differentiated in cumate-free medium (Cum-) as a baseline of 1, and in cells differentiated in cumate-supplemented medium (Cum+). Corresponding to the increase in C7orf26 expression due to cumate administration, the expression levels of p53 and p21 also increased, but the expression level of Ki67 decreased (Figure 9A). These results were consistent with the gene expression profile in HeLa cells described above.

[0084] The differentiation state was evaluated by flow cytometry. Sample preparation was performed using the Human Pluripotent Stem Cell Transcription Factor Analysis Kit (BD #560589) according to the included protocol. Separately, Alexa Fluor® 647 Mouse Anti-Human Pax6 (BD Biosciences # 562249) was purchased and used for staining for ectoderm marker analysis. An Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific) was used for measurement, and data analysis was performed using the included analysis software.

[0085] Compared to cells differentiated in a medium without cumate (Cum-), cells differentiated in a medium with cumate (Cum+) showed a peak in cell number at a higher fluorescence intensity, indicating an increase in the number of cells differentiated into the ectoderm. This result suggests that increased expression of C7orf26 enhances the transition of iPS cells, which had stopped at the G0 phase of the cell cycle, to the G1 phase and promotes differentiation into the ectoderm. Furthermore, when cultured cells were harvested on day 7 after the start of differentiation induction and the expression levels of C7orf26 and the Pax6, Pax3, and Sox1 genes were analyzed by quantitative PCR, the expression levels of Pax6, Pax3, and Sox1 increased in correspondence with the increase in C7orf26 expression due to cumate administration (Figure 9C).

[0086] (2) Differentiation induction of human iPS cells into retinal ganglion cells and brain cells The pPB-Cuo-C7orf26 vector containing the C7orf26 coding region was introduced into the human iPS cell line 409B02 using the PiggyBac Cumate Switch Inducible Vector (System Biosciences, LLC#PBQM812A-1), and a cumate-induced C7orf26-expressing iPS cell line was obtained by buromycin drug selection. Using the cumate-induced C7orf26-expressing iPS cell line grown by feeder-free culture using Stem Fit medium (Ajinomoto#AK02N), differentiation induction of retinal ganglion cells was performed as follows, according to the method described in the literature (Tanaka T et al., Sci Rep. 2015 Feb 10;5:834410.103). In a non-adhesion treated 96-well plate, 9000 iPS cells per well were seeded in serum-free medium (G-MEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid additive, 1 mM sodium pyruvate, 20 μM Y-27636, 3 μM IWR-1e) supplemented with 20% commercially available KSR. On day 2 after seeding, Matrigel was added to a final concentration of 0.5%, and on day 12, the medium was replaced with the same composition as above, but with IWR-1e removed and FBS (final concentration 1%) added. On day 15, CHIR99021 (final concentration 3 μM) and SAG (final concentration 100 nM) were added, and the cells were cultured for 3 days. On day 18, the culture medium was changed to serum-free retinal culture medium (composition: D-MEM / F12 with GlutaMAX, N2 supplement), and cultured for 6 days. On day 24, the culture medium was switched to serum retinal maturation medium (composition: D-MEM / F12 with GlutaMAX, N2 supplement, 1% FBS, 0.5 μM retinoic acid), and culture was continued for 3 days. On day 27, the cells were transferred to plates coated with poly-D-lysine and laminin to initiate differentiation into retinal ganglion cells, and at the same time, 10,000x Cumate Solution (System Biosciences, LLC #PBQM100A-1) was administered to a final concentration of 2x, and the cells were cultured in the presence of cumate. Colonies were harvested 7 days after the start of differentiation induction and RNA was extracted.

[0087] Using this RNA as a template, the expression levels of retinal ganglion markers (Brn3b, Math5) were evaluated by quantitative PCR. The results show the relative expression levels of each gene in cells differentiated in cumate-free medium (Cum-) as a baseline of 1, and in cells differentiated in cumate-supplemented medium (Cum+). Corresponding to the increase in C7orf26 expression due to cumate administration, the expression levels of p53 and p21 also increased, but the expression level of Ki67 decreased (Figure 10A). Furthermore, compared to cells differentiated in cumate-free medium (Cum-), in cells differentiated in cumate-supplemented medium (Cum+), the expression levels of Brn3b and Math5, particularly Math5, which are specific to retinal ganglion cell differentiation, increased with the rise in C7orf26, confirming enhanced differentiation into retinal ganglion cells (Figure 10B).

[0088] The centers of the differentiated colonies described above were collected, and RNA was recovered in the same manner. Using this RNA as a template, the expression levels of brain markers (Otx2, En1) were evaluated by quantitative PCR. Compared to cells differentiated in cumate-free medium (Cum-), cells differentiated in cumate-supplemented medium (Cum+) showed increased C7orf26 levels, along with increased expression levels of Otx2 and En1, which are specific to brain differentiation, confirming enhanced brain differentiation (Figure 10C).

[0089] (3) Differentiation induction from human iPS cells to mesoderm Cumate-induced C7orf26-expressing iPS cells, grown by feeder-free culture using Stem Fit medium (Ajinomoto #AK02N), were seeded at a rate of 15,000 cells in a 24-well multiplate (BD Falcon) coated with iMatrix-511 solution (0.5 mg / mL PBS Nippi #892012). After 3 days of culture, the medium was changed to mesoderm induction medium from the STEMdiff Trilineage differentiation kit (STEMCELL Technologies #ST-05230) to initiate differentiation (Day 0). Simultaneously, 10,000x Cumate Solution (System Biosciences, LLC #PBQM100A-1) was administered to a final concentration of 5x. The medium was changed daily thereafter, and on day 5 (Day 5), the cells were harvested and RNA extraction was performed. The expression of the mesoderm marker TBXT was analyzed by quantitative PCR to confirm the progress of differentiation. Similarly, FACS was performed, and the number of cells differentiated into mesoderm was measured using the mesoderm marker TBXT. Compared to the cell group differentiated in cumate-free medium (Cum-), the cell group differentiated in cumate-supplemented medium (Cum+) showed a peak in cell number at a high fluorescence intensity, indicating an increase in the number of cells differentiated into mesoderm (Figure 11A). Furthermore, compared to cells differentiated in cumate-free medium (Cum-), the expression level of TBXT, which is specific to mesoderm differentiation, increased in cumate-supplemented medium (Cum+) along with an increase in C7orf26, confirming enhanced mesoderm differentiation (Figure 11B).

[0090] (4) Differentiation induction from human iPS cells to endoderm Cumate-induced C7orf26-expressing iPS cells, grown by feeder-free culture using Stem Fit medium (Ajinomoto #AK02N), were seeded at a rate of 15,000 cells in a 24-well multiplate (BD Falcon) coated with iMatrix-511 solution (0.5 mg / mL PBS Nippi #892012). After 3 days of culture, the medium was changed to endoderm induction medium from the STEMdiff Trilineage differentiation kit (STEMCELL Technologies #ST-05230) to initiate differentiation (Day 0). Simultaneously, 10,000x Cumate Solution (System Biosciences, LLC #PBQM100A-1) was administered to a final concentration of 5x. The medium was changed daily thereafter, and on day 5 (Day 5), the cells were harvested and RNA extraction was performed. The expression of the endoderm marker Sox17 was analyzed by quantitative PCR to confirm the progress of differentiation. Similarly, FACS was performed, and the number of cells differentiated into endoderm was measured using the endodermal marker Sox17. Compared to the cell group differentiated in cumate-free medium (Cum-), the cell group differentiated in cumate-supplemented medium (Cum+) showed a peak in cell number at a high fluorescence intensity, indicating an increase in the number of cells differentiated into endoderm (Figure 12A). Furthermore, compared to cells differentiated in cumate-free medium (Cum-), the expression level of Sox17, which is specific to endodermal differentiation, increased in cumate-supplemented medium (Cum+) along with an increase in C7orf26, confirming enhanced endodermal differentiation (Figure 12B).

[0091] The present invention can be used in the fields of manufacturing pharmaceuticals for cancer treatment through cell cycle control, and in regenerative medicine and cell therapy materials. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. An agent for controlling the early G1 phase of the cell cycle, comprising any of the following proteins (A) to (C): (A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (B) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 with one or more amino acids added, deleted, or substituted, and having the activity of arresting the cell cycle at the early G1 phase and inducing a transition from the early G1 phase to the G0 phase; (C) a protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, and having the activity of arresting the cell cycle at the early G1 phase and inducing a transition from the early G1 phase to the G0 phase.

2. An agent for controlling the early G1 phase of the cell cycle, comprising any of the following genes (D) to (F): (D) a gene comprising DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1; (E) a gene comprising DNA consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, and encoding a protein having the activity of arresting the cell cycle at the early G1 phase and inducing a transition from the early G1 phase to the G0 phase; (F) a gene comprising DNA that hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1, and encoding a protein having the activity of arresting the cell cycle at the early G1 phase and inducing a transition from the early G1 phase to the G0 phase.

3. The agent for controlling the early G1 phase of the cell cycle according to claim 2, wherein the gene is inserted into a vector.

4. A pharmaceutical for treating cancer, comprising the agent for controlling the early G1 phase of the cell cycle according to any one of claims 1 to 3.

5. An agent for promoting differentiation of undifferentiated cells, comprising the agent for controlling the early G1 phase of the cell cycle according to any one of claims 1 to 3.

6. The differentiation promoter for undifferentiated cells according to claim 5, wherein the undifferentiated cells are stem cells or progenitor cells.

7. A method for promoting differentiation of undifferentiated cells, comprising the step of expressing any of the following genes (D) to (F) in undifferentiated cells: (D) a gene comprising DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1; (E) a gene comprising DNA consisting of a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, and encoding a protein having the activity of arresting the cell cycle at the early G1 phase and inducing a transition from the early G1 phase to the G0 phase; (F) a gene comprising DNA that hybridizes under stringent conditions to DNA consisting of a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1, and encoding a protein having the activity of arresting the cell cycle at the early G1 phase and inducing a transition from the early G1 phase to the G0 phase.

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