JP7501937B2 - Screening method for sperm chromatin condensation inhibitors targeting PHF7-mediated histone 3 ubiquitination - Google Patents

Description

This application is in the field of drug screening technology based on the molecular mechanism that clarified the regulation of sperm nuclear condensation by the regulation of histone 3 ubiquitination by PHF7.

Spermatogenesis is a complex process that produces spermatozoa that have half of the paternal genes. After meiosis, in which diploid spermatids derived from spermatogonia divide into four haploid spermatids, the round spermatids undergo a sperm maturation process to become spermatozoa, which involves cytoplasmic removal, nuclear condensation, acrosome formation, and tail growth.

Somatic chromatin is composed of histones, whereas sperm chromatin is mainly composed of arginine-rich protamines. The more condensed chromatin of sperm protects the genes from DNA damage and allows the paternal genes to be transmitted well to the offspring. During the sperm maturation process, histones in elongated sperm cells are removed from chromatin and exchanged for transfer protein (TNP) and finally for protamine (Wang et al. (2019a) Front Genet 10, 962.). Sperm with problems in histone-protamine exchange show abnormal morphology and reduced motility due to failure of nuclear condensation, which ultimately leads to problems in fertility (Wang et al., (2016). Journal of cellular biochemistry 117, 1429-1438).

Histone H4 hyperacetylation, which occurs in elongating spermatids before histone-protamine exchange, plays a role in histone removal and transcriptional regulation of genes required for sperm maturation (Goudarzi et al., 2014). Through numerous studies, many types of regulatory proteins involved in H4 hyperacetylation have been identified. PIWI regulates the subcellular location of RNF8, which promotes ubiquitination of H2A and H2B, which are required for H4 hyperacetylation in elongating spermatids, but the involvement of RNF8 in H4 hyperacetylation is still controversial (Gou et al., (2017) Cell 169, 1090-1104.e1013.; Lu et al. (2010) Nature 466, 508-512.; Sin et al. (2012). Genes Dev 26, 2737-2748.). CHD5, a chromatin remodeler, plays a role in sperm head development through H4 hyperacetylation and transcriptional regulation. After H4 hyperacetylation, BRDT recognizes H4K5/K8 acetylation at the hyperacetylated H4 end via the BD1 domain and participates in histone removal and transcriptional regulation together with chromatin remodelers. The recognition of acetylated chromatin via BD1 is strengthened by the interaction of the bromodomain with DNA. Furthermore, PA200, one of the units of sperm proteasome, is required for acetylation-mediated degradation of histones during sperm maturation (Qian et al. (2013) Cell 153, 1012-1024).

However, there is insufficient research into the molecular mechanisms of histone removal during sperm maturation, and there is a need to clarify this and develop drug technologies for the male reproductive system based on this.

This application aims to provide a method for screening for sperm nuclear condensation inhibitors based on the mechanism of regulation of PHF7 E3 ubiquitin ligase activity against histone H3K14 during spermatogenesis.

In one embodiment, the present application provides a method for screening a sperm chromatin condensation inhibitor, comprising the steps of: providing a eukaryotic cell that (over)expresses PHF7 (PHD finger protein 7) and BRDT (Bromdomain testis-specific) protein; contacting the cell with a test substance expected to inhibit the ubiquitination activity of PHF7 on the 14th lysine residue of histone 3 (H3); measuring the degree of ubiquitination of the H3 protein in the cell after the contact; and, if the ubiquitination of the H3 protein is increased in the cells contacted with the test substance compared to the cells not contacted with the test substance in the measuring step, selecting the test substance as a histone-protamine exchange inhibitor required for sperm chromatin condensation.

In one embodiment, the cells used in the method of the present application may be any cell that (over)expresses PHF7. In one embodiment, the cells are HEK (Human Embryonic Kidney) 293T cells that overexpress PHF7.

In one embodiment of the method of the present application, the cells are provided as a non-human animal model, and the measuring step further includes measuring the ubiquitination of the BRDT protein or the concentration of the BRDT protein in condensed sperm cells (spermatids) of the animal model, and if the ubiquitination of the BRDT protein increases compared to cells not treated with the test substance, or the concentration of the BRDT protein decreases compared to cells not treated with the test substance, the test substance is selected as a histone-chromatin exchange inhibitor required for sperm nuclear condensation.

Candidate substances for sperm nuclear condensation inhibitors screened using the method of the present application can be effectively developed as male contraceptives.

The screening method based on the mechanism identified in the present application and the substances identified thereby can inhibit PHF7 E3 ubiquitin ligase activity against histone H3K14, and while not affecting the transcriptional regulatory function of BRDT, they can effectively inhibit its function in histone removal by preventing the stabilization of BRDT and reducing its concentration, thereby inhibiting spermatogenesis. Specific inhibition of PHF7 using the substances identified through such a mechanism can be effectively used as a male contraceptive.

In particular, thienotriazolodiazepine JQ1 (CAS Number: 1268524-70-4), an inhibitor of the BRD family, which includes BRDT, BRD2, 3, and 4, has been proposed as a male contraceptive by targeting BRDT in mice. However, JQ1 has the risk of side effects as it inhibits BRD2, 3, and 4, which are expressed throughout the body. While BRDT plays a role from the early stages of spermatogenesis, PHF7, the mechanism of which has been elucidated in this application, is predominantly expressed in post-meiotic sperm cells and acts at the later stages of spermatogenesis, which may result in fewer side effects than when BRDT is targeted. In addition, since the E3 ubiquitin ligase activity of PHF7 is necessary for the sperm maturation process, PHF7 can be used as a diagnostic marker for male infertility testing.

The results show that Phf7 ^tKO male mice show spermatogenesis defects. (A) Schematic of mouse spermatogenesis. (B) Relative Phf mRNA levels in the testes of mice at different ages. (C) PHF7 protein levels in the testes of mice at different ages. (D) PHF7 protein levels in testicular cells of 8-week-old control and Phf7 ^tKO mice. (E) Fertility of control and Phf7 ^tKO male mice mated with wild-type female mice. (F) Testicular tissue analysis of control and Phf7 ^tKO mice using hematoxylin-PAS staining. (G) Epididymal tissue analysis of control and Phf7 ^tKO mice using hematoxylin-eosin staining. (H) Phase contrast microscope images of sperm from control and Phf7 ^tKO mice. (I) Motility of sperm from control and Phf7 ^tKO mice. (J) Test tube fertilization assay. Relative fertilization ability of control and Phf7 ^tKO sperm. (K) Confocal microscopy images of control and Phf7 ^tKO sperm. (L) Confocal microscopy images of control and Phf7 ^tKO sperm using histone H2A, TH2B, H3, and H4 antibodies. (M) Histone and protamine protein levels in control and Phf7 ^tKO sperm extracts. (N) Immunostaining of control and Phf7 ^tKO testis sections using H3 and PRM2 antibodies. Arrows indicate condensed sperm cells co-expressing H3 and PRM2. (O) Immunoblot analysis performed on control and Phf7 ^tKO sperm cell extracts. Figure 1 shows that PHF7 is an E3 ubiquitin ligase for lysine 14 of histone H3. (A) Immunoblot analysis performed on control and Phf7 ^tKO mouse testis cell extracts. Long exposure data of H3 immunoblot includes H3 band migrating below 25 kDa. (B) Immunoprecipitation analysis performed on control and Phf7 ^tKO mouse testis cell extracts. (C) Immunoblot analysis after transfection of Flag-PHF7 and Hismax-Ub into HEK293T cells. (D) In vitro ubiquitination assay for E2 screening using E. coli purified PHF7 and recombinant nucleosomes. Relative levels of histone ubiquitination are indicated below each immunoblot. (E) In vitro ubiquitination assay of nucleosomes proceeded for different incubation times. Relative levels of H3 ubiquitination are indicated below the H3 immunoblot. (F) In vitro ubiquitination assay using GST-H3 deletion mutants in the presence or absence of Ub. H3 ubiquitination ratios are indicated below the GST immunoblot. Asterisks (*) indicate polyubiquitination of H3. (G) Screening for advanced H3 ubiquitin residues after transfection of HEK293T cells with HA-PHF7 and H3-Flag KR mutant. (H) In vitro ubiquitination assay using histone extracts from HEK293T cells overexpressing H3-Flag WT and K14R mutant. We found that C160A knock-in mice lacking Phf7 enzyme activity exhibit defects in spermatogenesis and H3 ubiquitination. (A) Schematic of PHF7 WT, ΔPHD, and ΔRING mutants. Comparison of E3 ubiquitin ligase activity of Flag-PHF7 WT, ΔPHD, and ΔRING in HEK293T cells. (B) and (C) Defects in E3 ubiquitin ligase activity of PHF7 PHD domain mutant (B) and RING domain mutant (C) in HEK293T cells. (D) In vitro ubiquitination assay using GST-PHF7 RING domain mutants. (E) Schematic of Phf7 C160A KI mice. The KI allele has the restriction sites BamH I (GGATTC) and Afe I (AGCGCT). (F) Fertility of control and Phf7 ^CA/CA male mice mated with wild-type female mice. (G) Motility of control and Phf7 ^CA/CA male sperm. (H) IVF assay. Fertility of control and Phf7 ^CA/CA male sperm. (I) Confocal microscope images of control and Phf7 ^CA/CA male sperm. (J) Confocal microscope images of immunostaining of histones H2A, TH2B, H3, and H4 in control and Phf7 CA/CA male sperm. (K) Confocal microscope images of immunostaining of testis sections of control and Phf7 CA ^{/ CA} male mice using H3 and PRM2 antibodies. Arrows indicate condensed spermatids in which H3 and PRM2 are co-expressed. (L) Immunoblot analysis performed on control and Phf7 ^CA/CA male mouse sperm cell extracts. (M) Immunoblot analysis performed on control and Phf7 ^CA/CA male mouse testis cell extracts. Long exposure data of H3 immunoblot includes H3ub band below 25 kDa. We found that the E3 ubiquitin ligase activity of PHF7 is required for histone removal following H4 hyperacetylation. (A and B) t-SNE analysis of testicular cells from two mouse genotypes, with the designated cell types colored according to single-cell RNA sequencing (A) and the corrected expression levels of highly divergent genes in all cells (B). All genes and cells in the heatmap were sorted using Euclidean distance. Metadata for each cell is labeled on the heatmap (genotype, Phf7 ^f/f , gray; Phf7 ^tKO , red and cell type used in t-SNE analysis). (C) Relative mRNA levels of Tnp1/2 and Prm1/2 in testicular cells from control and Phf7 ^tKO mice. (D) Immunoblot analysis for TNPs and PRMs performed on sperm cell extracts from control and Phf7 ^tKO mice. (E) Immunoblot analysis for TNPs and PRMs performed on sperm cell extracts from control and Phf7 ^CA/CA mice. (F) Immunoblot analysis for H4ac and H4 performed on sperm cell extracts from control and Phf7 ^tKO mice. (G) Immunoblot analysis for H4ac and H4 performed on sperm cell extracts from control and Phf7 ^CA/CA mice. (H) Confocal microscopy images of immunostaining with H4ac and PRM2 antibodies on testis sections from mice of each genotype. (I) Schematic model of the function of PHF7 in H4 hyperacetylation followed by histone removal. The results indicate that PHF7 plays a role in maintaining the level of BRDT in early condensed spermatids. (A and B) Protein and relative mRNA levels of BRDT in spermatids from Phf7 ^f/f and Phf7 ^tKO mice (A), Phf7 ^+/CA and Phf7 CA ^/CA mice (B). (C and D) Confocal microscopy images of immunostaining with H4ac and BRDT antibodies in testis sections from Phf7 ^f/f and Phf7 ^tKO mice (C), Phf7 ^+/CA and Phf7 ^CA/CA mice (D). Roman numbers indicate the stages of spermatogenesis in the lavage duct. (E) Quantification of the percentage of H4ac and BRDT staining in the nuclei of elongated or condensed spermatids. We determined that the E3 ubiquitin ligase activity of PHF7 regulates the stability of BRDT. (A) Confocal microscopy images of immunostaining with BRDT and Flag antibodies in testis sections from Phf7 ^Flag/+ mice. Roman numerals indicate the stages of spermatogenesis in the lavage duct. Arrows indicate the nuclei of spermatids in which BRDT and Flag-PHF7 are co-expressed. (B) Conserved SPOP degron (D) sequence in the BET family. (C) Immunoblot analysis of HEK293T cells transfected with Flag-BRDT (1-555 amino acids) wild type and ΔD mutant in the presence or absence of MG132 treatment. (D) Immunoblot analysis of HEK293T cells transfected with Flag-BRDT, SPOP, and PHF7 wild type and C160A mutant. (E) Protein levels of SPOP, Cul3, BRD4, and PTEN in spermatids from Phf7 ^f/f , Phf7 ^tKO , Phf7 ^+/CA , and Phf7 ^CA/CA mice. (F) Ubiquitination assays performed after transfection of Flag-BRDT, SPOP, PHF7 wild-type and C160A mutant into HEK293T cells and treatment with MG132. (G) Confocal microscopy images of transfected HEK293T cells immunostained with HA or PHF7 antibodies and Flag antibodies. (H) In vitro ubiquitination assays using BRDT, SPOP-Cul3-Rbx1, and PHF7. We found that PHF7-mediated histone ubiquitination attenuates BRDT ubiquitination. (A) Immunoprecipitation of BRDT and PHF7 in HEK293T cells. (B) In vitro pull-down assay using E. coli-derived GST-BRDT and biotinylated nucleosomes containing H3ub or H4ac and streptavidin beads. The relative percentage of pulled-down GST-BRDT is indicated below the immunoblot. (C) In vitro ubiquitination assay using BRDT, SPOP-Cul3-Rbx1, and modified nucleosomes. (D) A model for regulation of BRDT stability via PHF7-mediated H3ub in early condensed spermatids (step 12). The results of generating PHF7-deficient mice are shown. (A) Schematic of the method for targeting the PHF7 gene. The targeting vector contains Phf7 exon 4, 5, 6, and 7 alleles (gray box) and the lacZ gene (purple box), loxP (green arrow), FRT (blue box), neomycin selection marker (light green box), and diphtheria toxin gene (DT, light blue box). (B) Images of testes and the ratio of testes weight to body weight of Phf7 ^{f/ f} and Phf7 ^tKO mice. Error bars indicate mean ± SD. (C and D) Confocal ^microscopy images of immunostaining for H3 (green), PRM1 (red, C), TNP2 (red, D), and DAPI (blue) in testicular sections of Phf7 f/f and Phf7 tKO mice. Arrows indicate sperm cells expressing PRM1 and TNP2 co-expressed with H3. LC-MS/MS analysis results for H3K14ub are shown. (A) LC-MS/MS analysis using in vitro modified histone H3 proteins. Peptide sequences and MS/MS analysis spectra corresponding to the modifications are indicated. (B) Difference in the number of peptide spectral matches (PSMs) for peptide sequences named in LC-MS/MS analysis. PHF7 overexpression and control samples from two repeated experiments are indicated. (C) LC-MS/MS analysis of ubiquitinated H2A and H2B proteins in the testes of Phf7 ^f/f and Phf7 ^tKO mice. Analysis of Phf7 ^CA/CA mice. (A) Hematoxylin-PAS staining of Phf7 ^+/CA and Phf7 ^CA/CA mouse testis sections. (B) Hematoxylin-eosin staining of Phf7 ^+/CA and Phf7 ^CA/CA mouse epididymis sections. (C) Phase contrast microscopy images of Phf7 ^+/CA and Phf7 ^CA/ CA mouse sperm. (D and E) Confocal microscopy images of immunostaining for H3 (green), PRM1 (red, D), TNP2 (red, E), and DAPI (blue) in Phf7 ^+/CA and Phf7 ^CA/CA mouse testis sections. Arrows indicate sperm cells in which PRM1 or TNP2 is co-expressed with H3. Single-cell RNA sequencing shows that PHF7-deficient mice have a similar transcript profile to wild-type mice. (A) UMAP plots with graph-based cluster coloring. (B) Mouse cells distributed on the UMAP plots. Red dots indicate cells derived from each sample in each plot. (C) Corrected expression levels of marker genes on the UMAP plots. (D) Distribution of the total number of cellular Unique Molecular Identifiers (UMIs). Phf7-disrupted mice show abnormal residual H4 acetylation in condensed spermatids. (A) Schematic of sperm maturation during the spermatogenesis cycle. Arabic numbers indicate steps (1-16) of sperm maturation: round spermatids (steps 1-8), elongating spermatids (steps 9-11), condensed spermatids (steps 12-13), and postcondensed spermatids (steps 14-16). (B and C) Confocal microscopy images of staining for H4ac (green), PNA (red), and DAPI (blue) on testis sections from Phf7 ^f/f , Phf7 ^tKO (B), Phf7 ^+/CA , and Phf7 ^CA/CA (C) mice. Roman numbers indicate spermatogenesis steps (I-XII) in the wash tube. Co-immunostaining of PRM1 or TNP2 with H4ac in testis sections, results related to Fig. 4. (A) Confocal microscopy images of immunostaining for H4ac (red), PRM1 (green), and DAPI (blue) on testis sections from Phf7 f/ ^f , Phf7 ^tKO , Phf7 ^+/CA , and Phf7 CA ^/CA mice. (B) Confocal microscopy images of immunostaining for H4ac (red), TNP2 (green), and DAPI (blue) on testis sections from Phf7 ^f/f , Phf7 ^tKO , Phf7 ^+/CA , and Phf7 ^CA/CA mice. Generation of Flag-PHF7 knock-in mice. (A) Schematic for Flag-PHF7 knock-in mice. The knock-in sequence includes PHF7 exon 2 allele (gray box), 3xFlag (red), 3xFlag (red), spacer (orange), and silent mutation (light blue). (B) Protein levels of Flag-PHF7 in testicular cell extracts from Phf7 ^+/+ (wild type) and Phf7 ^Flag/+ mice. Asterisks (*) indicate nonspecific bands. (C) Confocal microscopy images of immunostaining for Flag (green), PNA (red), and DAPI (blue) in Phf7 ^Flag/+ mouse testis sections. Roman numerals indicate spermatogenic stages (I-XII) in the lavage tube. (D) Confocal microscopy images of Phf7 ^Flag/+ mouse testis sections immunostained for Flag (green) and DAPI (blue). (E) Confocal microscopy images of Phf7 ^Flag/+ mouse testis sections immunostained for Flag (green), H4ac (red), and DAPI (blue). Roman numerals indicate spermatogenic stages (I-XII) in the lavage duct. Arrows indicate spermatids co-expressing Flag-PHF7 and H4ac.

This application is based on the discovery that PHF7 (PHD finger protein 7) is important for maintaining the stability of BRDT (bromodoma testis-specific), a histone removal factor, during sperm maturation. Spermatogenesis is a complex process that produces sperm, including somatic cell division, meiosis, and sperm maturation. During sperm maturation, histones are removed from the chromatin of post-meiotic spermatids and replaced with protamine. Although histone-protamine exchange during spermatogenesis is important for sperm nuclear condensation, it has not been sufficiently studied, and the regulatory mechanism behind this is needed to be elucidated.

Specifically, this application is based on the discovery that the PHF7 protein acts as an E3 ubiquitin ligase for the 14th lysine (K14) residue of histone H3 in post-meiotic sperm cells, and that such histone ubiquitination by PHF7 increases the amount of BRDT, a histone removal factor, and thus the PHF7 protein is an important epigenetic factor for chromatin condensation that regulates histone-protamine exchange.

Specifically, this application created PHF7-deficient mice and Phf7 C160A mutant mice with disrupted E3 ubiquitin ligase activity, and revealed that these mice exhibited deficiencies in histone-protamine exchange and abnormalities in the sperm maturation process due to dysregulation of the histone removal factor BRDT in early condensed sperm cells. In addition, the protein amount of the histone removal factor BRDT was reduced in mouse sperm cells, and differences in the expression level of BRDT at specific developmental stages were confirmed through immunoblotting and tissue immunostaining. This application also revealed that the activity of PHF7's E3 ubiquitin ligase for histone ubiquitination contributes to the stabilization of BRDT, i.e., increased concentration, by suppressing BRDT ubiquitination.

Therefore, deficiency or inhibition of expression or activity of PHF7 induces a deficiency of histone 3 with ubiquitinated lysine residue 14 (H3K14ub), which in turn reduces the amount of BRDT protein in early condensed spermatids, resulting in the development of abnormal spermatozoa in which histones are not replaced by protamine and remain.

Therefore, in one aspect, the present application provides a method for screening a sperm chromatin condensation inhibitor, which includes a step of selecting PHF7 as a histone-protamine exchange inhibitor required for sperm chromatin condensation.

While somatic chromatin is composed of histones, sperm chromatin is mainly composed of arginine-rich protamines. The more condensed chromatin of sperm protects the genes from DNA damage and allows the paternal genes to be transmitted well to the offspring. During the sperm maturation process, histones in elongated sperm cells are removed from the chromatin and replaced by transfer protein (TNP), and finally by protamine (Wang et al. (2019a) Front Genet 10, 962.). Sperm with problems in this histone-protamine exchange show abnormal morphology and reduced motility due to failure of nuclear condensation, which ultimately leads to problems in fertility (Wang et al., (2016). Journal of cellular biochemistry 117, 1429-1438).

In one embodiment, the method includes the steps of providing a eukaryotic cell expressing PHF7 and BRDT; contacting or treating the cell with a test substance expected to inhibit the ubiquitination activity of PHF7 at the 14th lysine residue of histone 3 (H3); measuring the degree of ubiquitination of the BRDT protein in the cells treated with the test substance; and, if the measuring step shows increased ubiquitination of the BRDT in the cells treated with the test substance compared to the cells not treated with the test substance, selecting the test substance as a candidate male contraceptive substance.

PHF7, the target of this application, is a member of the PHF family that has a PHD domain known as a histone methyl reader, and its human and mouse protein and gene sequences are publicly known by NCBI as follows: human protein sequence of PHF7: NP_057567.3, gene sequence: NC_000003.12, mouse protein sequence: NP_082225.1, gene sequence: NC_000080.6.

Previous research has shown that disruption of the PHF7 gene leads to defects in spermatogenesis and reduces H2A ubiquitination (Wang et al., 2019b). However, this study is the first to show that PHF7 acts as an E3 ubiquitin ligase for H3K14ub in post-meiotic sperm cells to maintain the stability of BRDT protein for histone removal during spermatogenesis.

H3, the substrate of the PHF7 E3 ubiquitin ligase identified in the present application, is a histone protein. Human and mouse H3 proteins and gene sequences are publicly known from NCBI as follows: human protein sequence of H3: NP_003520.1, gene sequence: NC_000006.12, mouse protein sequence: NP_038578.2, gene sequence: NC_000079.6. In numbering the amino acid residues, since the first amino acid in a protein is usually Met, the histone residue numbers in the present application exclude Met and place the next amino acid thereafter as number 1.

In general, histone proteins are basic proteins that pack DNA in eukaryotic cells to form structural units called nucleosomes. There are five histone families: H1/H5, H2A, H2B, H3, and H4. H2A, H2B, H3, and H4 constitute the core of the nucleosome and are H1/H5 linker histones. Core histones exist as dimers. The PHF7 E3 ubiquitin ligase of the present application ubiquitinates the 14th lysine residue of H3. Ubiquitin is a protein consisting of 76 amino acids, and the ubiquitin of the ubiquitinated protein regulates its own activity and the activity of other proteins by promoting protein degradation and regulating interactions with other proteins.

BRDT is first expressed in early spermatocytes and has various functions depending on the developmental stage. It is known that transcription begins to be repressed in round spermatids from the early stage of sperm maturation, and all transcription stops in sperm. However, the relationship with PHF7 has not been clarified. In this application, we have determined through experiments such as single-cell RNA sequencing that PHF7 deletion does not affect BRDT transcription, and that BRDT regulation by PHF7 acts at a stage after transcription repression, and that PHF7 acts as an important epigenetic factor in chromatin condensation that regulates histone-protamine exchange. The sequence of BRDT is publicly known by NCBI as follows: human protein sequence: NP_001717.3, gene sequence: NC_000001.11, mouse protein sequence: NP_473395.2, gene sequence: NC_000071.6.

The method of the present application includes a step of measuring the degree of ubiquitination of the histone protein in cells treated with a test substance to select a candidate substance. Methods for measuring the ubiquitination of histone 3 are known, and can be found, for example, in the Examples of the present application (Zhang et al. Nat. Commun 8:14799 (2017)). If the ubiquitination of histone 3 increases in cells treated with the test substance compared to untreated cells in the measuring step, the test substance is selected as a histone-protamine exchange inhibitor required for sperm chromatin condensation.

In one embodiment, the cells used in the method of the present application are eukaryotic cells derived from mammals, including humans or mice, that overexpress PHF7, for example, HEK (Human Embryonic Kidney) 293T, an established cell line that overexpresses PHF7. The established cell line can be purchased commercially. Methods for overexpressing a specific gene in an established cell line have been published, for example, a plasmid encoding the human PHF7 gene can be transfected into the desired cell line. However, the cell line is not limited thereto, and various cells that can achieve the objectives of the present application can be used.

In another embodiment, the cells used in the methods of the present application are part of an animal model, such as a mouse. A mouse is used in the methods of the present application.

The method of the present application includes a step of selecting the test substance as a histone-protamine exchange inhibitor required for sperm chromatin condensation if the ubiquitination of H3 is increased in cells contacted with the test substance compared to cells not contacted with the test substance during the measurement step.

In one embodiment, the cells in the method of the present application are provided as a non-human animal model, in which case the measuring step of the method of the present application includes measuring BRDT protein ubiquitination or the concentration of the BRDT protein in testicular cells of the animal, for example, a wild-type mouse, instead of or in addition to histone ubiquitination, and if the BRDT protein ubiquitination increases compared to cells not treated with the test substance, or the concentration of the BRDT protein decreases compared to cells not treated with the test substance, the test substance is selected as a histone-chromatin exchange inhibitor required for sperm nuclear condensation. As described in FIG. 7 and Example 6 of the present application, ubiquitination of BRDT promotes its degradation and reduces its concentration. Therefore, in the method of the present application, an increase in ubiquitination by the test substance reduces the concentration of the BRDT protein.

Therefore, a candidate substance that can inhibit the ubiquitination activity of PHF7 on the 14th lysine residue of histone 3 (H3) using the method of the present application ultimately increases the ubiquitination of BRDT, which leads to a decrease in the concentration of BRDT, and as a result, induces defects in chromatin condensation during spermatogenesis, inhibits spermatogenesis, and can be effectively used as a male contraceptive.

The cells used in the method of the present application may be provided as organoids or non-human animal models containing such cells. Such animal models may be produced using the examples of the present application or methods known in the art. In the present application, organoids are mini-organs produced in vitro in a three-dimensional environment that exhibits the microstructure of an actual organ. These are cells that have the ability to self-repair and differentiate and are produced in a three-dimensional culture environment. The method of producing organoids may refer to known methods (Organoid Culture Handbook, Ambiso).

The test substances used in the method of the present application include, but are not limited to, substances that are expected to regulate or change the function of PHF7 ubiquitin ligase as an epigenetic factor that regulates chromatin condensation during sperm maturation by H3 ubiquitination as identified in the present application, particularly to inhibit the ubiquitination of H3 K14, such as low molecular weight compounds, high molecular weight compounds, mixtures of compounds (e.g., natural extracts or cell or tissue cultures), or biopharmaceuticals (e.g., proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzymes and DNAzymes), or sugars and lipids.

In one embodiment of the present application, a small molecule compound can be used as the test substance. The test substance can be obtained from a library of synthetic or natural compounds, and methods for obtaining such compound libraries are known in the art. Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and natural compound libraries are available from Pan Laboratories (USA) and MycoSearch (USA). Test substances can be obtained by various combinatorial library methods known in the art, such as biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, "one-bead one-compound" library methods, and synthetic library methods utilizing affinity chromatography selection. Methods for the synthesis of molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90,6909,1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 90,6909,1993; 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, etc. For example, for drug screening purposes, compounds having low molecular weight and therapeutic effects can be used. For example, compounds with weights of about 1000 Da, such as 400 Da, 600 Da, or 800 Da, can be used. Depending on the purpose, such compounds can form part of a compound library, and the number of compounds constituting the library can vary from tens to millions. Such compound libraries can include, by way of example only, peptides, peptoids, and other cyclic or linear oligomeric compounds, and template-based small molecule compounds such as benzodiazepines, hydantoins, biaryls, carbocycles and polycycles (e.g., naphthalenes, phenothiazines, acridines, steroids, etc.), carbohydrates and amino acid derivatives, dihydropyridines, benzhydryls, and heterocycles (e.g., triazines, indoles, thiazolidines, etc.).

Also, for example, biologics can be used in screening. Biologics refers to cells or biomolecules, where biomolecules refer to proteins, nucleic acids, carbohydrates, lipids, or substances produced using cellular systems in vivo and in vitro. Biomolecules can be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, polynucleotides, peptides, antibodies, or other proteins or biological organic substances found in plasma.

In one embodiment, the increase or decrease by which a candidate substance can be selected using the method of the present application can be appropriately selected by a person skilled in the art in consideration of the results disclosed in the present application and the common sense of the person skilled in the art. For example, but not limited to, a test substance can be selected as a candidate substance by a decrease of about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 80% or more, about 100% or more, or a range therebetween, in the presence of the test substance compared to a control group not in contact with the test substance, but not excluding a range exceeding the above.

The following examples are presented to aid in understanding the present invention. However, the following examples are provided merely to facilitate understanding of the present invention, and the present invention is not limited to the following examples.

EXAMPLES Experimental Materials and Methods Generation of PHF7 conditional knockout mice: Three embryonic stem cells (ES cells) carrying the first allele deletion (tm1a) of PHF7 were obtained from the International Mouse Phenotyping Consortium (IMPC) and microinjected into C57BL/6 blastocysts. To remove the lacZ and neomycin cassettes, heterozygous F1s were crossed with FLPeR mice. The offspring mice were crossed with Stra8-Cre mice to obtain testis-specific PHF7 deletion mice. Phf7 ^f/f and Phf7 ^tKO male mice were used for experiments after 8 weeks of age. Genotyping primers are as follows:
Forward 5'-ATCAGTGTGTCCAGACTTCCATC-3'
Reverse 5'-TTATCGAGTGGAGGGACAGATGTG-3'.

All animal studies and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University.

Generation of Phf7 C160A knock-in mice and Flag-PHF7 knock-in mice: Phf7 C160A KI mice were generated from ES cells transfected with sgRNA/Cas9 as described previously. In particular, EGR-G01 [129S2×C57BL/6NCr-Tg(CAG/Acr-Egfp) ES cells were used for somatic insemination. The target sequence of sgRNA is 5'-GAACATCCACCAGGGGAGTT-3. To introduce a point mutation (TGT→GCT, C160A) into the 7th exon of PHF7, a HDR-conferring plasmid (pBluescript II SK(+) vector) was designed. Chimeric offspring carrying the C160A allele were bred with wild-type B6D2F1 mice to obtain heterozygous (Phf7 ^+/CA ) mice. Phf7 ^+/CA mice were then bred with C57BL/6J mice. Phf7 ^CA/CA mice were obtained by mating between Phf7 ^+/CA mice. Phf7 ^+/CA and Phf7 ^CA/CA mice were used for experiments after 8 weeks of age. Genotyping primers for the C160A allele are as follows:
Forward; 5'-TCACTGAGCACCAAGCAAATAGAC-3'
Reverse; 5'-TCTGAATCTTACACATACTTGAGCA-3'.

The KI allele has an AfeI restriction site (FIG. 3E) and genotypes are classified by AfeI reaction. Flag-PHF7 KI mice were generated by injecting gRNA/Cas9 mixture and reference DNA into eggs. The gRNA sequence is 5'-AAAACAAACATCCAAGATTG-3'. Genotyping primers for Flag-PHF7 allele are as follows:
Forward 5'-GGGTGGGACGACAAGAAGAG-3'
Reverse 5'-CTTGTCATCGTCATCCTTGT-3'.

All animal studies and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University and the Animal Experimentation Committee of the Institute for Microbial Diseases, Osaka University. Frozen sperm from these KI mice (STOCK PHF7<em2(C160A)Osb>Tg(CAG/Acr-EGFP)C3-N01-FJ002Osb, RBRC#11032, CARD#2939; and C57BL/6J-PHF7<em3(FLAG/PHF7)Osb>, RBRC#11056, CARD#2963) will be available at RIKEN BRC (https://mus.brc.riken.jp/en/) and Kumamoto University CARD (http://card.medic.kumamoto-u.ac.jp/card/english/). STOCK PHF7<em2(C160A)Osb> and C57BL/6J PHF7<em3(3xFLAG/PHF7)Osb> were obtained from Professor Masato Ikawa (Research Institute for Microbial Diseases, Osaka University).

Cell culture: HEK293T cells (ATCC) were cultured in DMEM media containing 10% FBS and antibiotics in a humidified incubator with 5% _CO2 . All cells used in this study were routinely tested for mycoplasma contamination.

Sperm motility: Sperm obtained from the epididymis were spread on a TYH drop. After 10 min of incubation, sperm motility was analyzed by a Computer-assisted sperm analysis (CASA) system (CEROS II).

In vitro fertilization assay: PMSG (pregnant mare serum gonadotropin) was injected into the abdominal cavity of B6D2F1 female mice, and 48 hours later, hCG (human chorionic gonadotropin) was injected. 14 hours after hCG injection, eggs were extracted from the trumpeter of each oviduct and cultured in TYH drops. Epididymal sperm were precultured in TYH drops for 2 hours. Sperm were added to the TYH drops containing the eggs. After 6 hours, the oocysts surrounding the eggs were removed by treating them with hyaluronidase for 5 minutes, and the pronuclei were observed under a phase contrast microscope.

Histology: Testicular tissue analysis was performed as previously described. Epididymis was fixed overnight at 4°C in 10% formalin. After fixation, tissue was dehydrated in increasing concentrations of ethanol from 50% to 100%. Dehydrated specimens were then inserted into paraffin wax via 100% xylene. For hematoxylin-eosin (H&E) staining, tissue was cut at 7 μm thickness, deparaffinized, rehydrated, and stained with hematoxylin for 3 min and eosin for 1 min. Images were obtained with a digital microscope.

Isolation of mouse testicular cells: Mouse testicular tissue was digested with collagenase A, DNase I, and trypsin in DMEM-F12 media. Trypsin was blocked with FBS, and cells were removed using 70 μm and 40 μm strainers. After centrifugation, red blood cells were removed with RBC lysis buffer. Cells were washed with HBSS. Sperm cells were isolated using STA-PUT, a gravity sedimentation method, modified from the previous protocol. A column was placed in 4% BSA in DMEM-F12 media, slowly piled with 2% BSA, and then testicular cells dissolved in 0.5% BSA were loaded on top. Sedimentation was carried out at room temperature for 150 minutes. Each fraction was classified according to cell morphology using a phase contrast microscope.

Histoimmunostaining: Sperm from the epididymis were dried on a slide, and sperm cells on the PLL-treated copper slip were incubated in PBS containing 1M DTT for 30 minutes and fixed in PBS containing 4% PFA for 10 minutes at room temperature. The fixed samples were permeabilized with PBS containing 0.1% Triton X-100 (PBS-T) for 10 minutes at room temperature. For staining, cells were incubated with antibodies at room temperature for 2 hours and then incubated with fluorescently labeled secondary antibodies for 1 hour. Histoimmunostaining using testis sections was performed in the same manner from blocking to mounting. Samples were observed under a confocal microscope. For double staining using two antibodies obtained from the same host, testis sections were first incubated with BRDT antibody overnight at 4°C, and then incubated with the next antibody in the order for 1 hour at room temperature. Alexa 488 AffiniPure Fab Fragment Donkey Anti-Rabbit IgG (H+L), H4ac antibody, Alexa 594 Donkey Anti-Rabbit IgG (H+L). The mounted samples were observed under a confocal microscope.

Transfection and cell lysis: Transfection was performed using PEI. All cells were washed with cold PBS, collected and lysed in Laemmli sample buffer. All extracts were analyzed by SDS-PAGE.

In vitro ubiquitination assays: Recombinant proteins PHF7, UBE1, GST-H3, GST-BRDT, and SPOP were purified using E. coli strain Rosetta. Histone ubiquitination assays using recombinant nucleosome, GST-H3, and histone extracts were performed at 30°C for 1 h using ubiquitin, UBE1, UbcH5b, UbcH5c, and PHF7 in assay buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl ₂ , 2 mM ATP, and 2 mM DTT). Flag-Cul3 and HA-Rbx1 were purified using Flag-M2 agarose beads and 3xFlag peptide from transfected HEK293T cells. GST-BRDT, His-SPOP, Flag-Cul3, HA-Rbx1, and PHF7 were incubated in the same buffer at 30°C for 30 minutes, and ubiquitin, UBE1, and UbcH5a were added to perform the BRDT ubiquitination assay at 30°C for 1 hour. After performing the ubiquitination assay with PHF7 and recombinant nucleosomes, the modified nucleosomes were removed with an Amicon Ultra Centrifugal Filters 50K membrane to remove PHF7, ubiquitin, UbcH5b, and 5c. The BRDT ubiquitination assay using the modified nucleosomes was performed in the same manner.

In vivo ubiquitination assay: Testicular cells were lysed in denaturing buffer (50 mM Tris-HCl [pH 7.5], 1% SDS, 70 mM β-mercaptoethanol) and boiled at 95°C for 5 min. All extracts were diluted with denaturing buffer (20 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, protease inhibitors) and immunoprecipitated overnight at 4°C using H3 antibody. HEK293T cells were transfected with Hismax-Ub and treated with MG132 for 6 h, followed by cell disruption in the same manner. Immunoprecipitation was performed at 4°C for 2 h using Flag antibody.

Immunoprecipitation: Transfected HEK293T cells were disrupted in EBC200 buffer (50 mM Tris-HCl [pH 8.0], 200 mM NaCl, 0.5% NP-40, protease inhibitors) and centrifuged. The supernatant was incubated with Flag antibody at 4°C for 2 hours. Protein A/G Sepharose beads were then added and incubated at 4°C for 1 hour. The beads were washed with EBC200 buffer and boiled in sample buffer for 10 minutes.

In vitro biotin-streptavidin pull-down assay: Recombinant protein GST-BRDT was purified using E. coli strain Rosetta. Recombinant biotinylated nucleosomes were used in an in vitro ubiquitination assay using PHF7 and incubated with streptavidin agarose resin in pull-down buffer (50 mM Tris-HCl [pH 7.4], 125 mM KCl, 5 mM MgCl ₂ , 0.5 mM EDTA, 0.5% Triton X-100, 5 mM DTT) for 1 h at 4°C. Nucleosomes attached to beads were washed with pull-down buffer and incubated with GST-BRDT protein for 2 h at 4°C. Beads were washed and boiled for 10 min.

LC-MS/MS analysis: LC-MS/MS analysis of peptide sample preps in the cell and in vitro modified histone samples was performed as previously described. Peptide sample preps and immunoaffinity purification of HEK293T cells overexpressing PHF7 and Phf7 ^f/f and Phf7 ^tKO mouse testis cells were performed with the PTMSCan Ubiquitin Remnant Motif (K-ε-GG) kit. Peptide samples were separated on an in-house packed long capillary column (100 cm x 75 μm id) and a trap column (3 cm x 150 μm id) using 3 μm Jupiter C18 particles. A flow rate of 300 nl/min and a linear gradient ranging from solvent A (water with 0.1% formic acid) to 40% solvent B (acetonitrile with 0.1% formic acid) for 60 min was used in conjunction with a nanoliquid UPLC (water) Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) operated with the following parameters: Data sets acquired at 60k or 7.5k resolution with precursor scan range m/z 300-1800, precursor isolation window 1.4Th, high level collision energy (NCE) 30%-energy collisional dissociation (HCD), dynamic exclusion period 30 seconds, and m/z 200 for full MS or MS/MS scans, respectively, were searched by MS-GF+ algorithm or MaxQuant software with precursor ion mass resistivity of 10 ppm based on SwissProt Homo sapiens proteome database. The discovery rate was set to 1% at protein and peptide level. The number of peptide spectrum matches was tabulated for each revealed peptide sequence. The intensity of peptide sequences of interest was determined by manual annotation at XIC (extracted ion chromatogram) level obtained from MaxQuant search results or using Qual Browser software.

Quantitative RT-PCR: RNA was extracted using Trizol and reverse transcription was performed using 2.5 mg of RNA using the M-MLV cDNA synthesis kit. The amount of mRNA was measured using SYBR TOPreal qPCR 2x PreMix and BioRad CFX384. The amount of mRNA was calculated using the ddCt method and all reactions were performed in triplicate. Expression was normalized using 18S rRNA and Hprt.

Single-cell RNA sequencing experiments: Single-cell RNA sequencing libraries were generated by isolating several thousand testicular cells into nano-liter droplets using Chromium Single Cell 3' Library & Gel Bead Kit v2. cDNA was generated in each droplet by reverse transcription. Barcode sequences and UMIs (Unique Molecular Identifiers) were subsequently added to each cDNA molecule. cDNA was pooled and sequencing libraries were prepped as described in the manual. Libraries were sequenced on the Nextseq 550 platform and Novaseq 6000 platform.

Single-cell RNA sequencing data analysis: Sequencing data were demultiplexed using bcl2fastq to generate fastq files. After demultiplexing, reads were aligned to the mouse reference genome and feature barcode matrices were generated using cellranger count with trimming and aggregation with cellranger aggr with default parameters. Subsequent analysis was performed using the Seurat program. After generating the feature barcode matrix, genes expressed in less than 3 cells and cells showing expression of less than 200 genes were removed. Additionally, cells showing more than 20% of mitochondrial gene expression and cells showing expression of less than 2,000 molecules were removed. Data were corrected by dimension reduction method. To visualize high dimension data, the 'mean.var. PCA (principle component analysis) was performed on highly diverse genes selected by FindVariableFeature with 'plot'. Cells were allocated to clusters divided into 10PC and 0.5 resolution parameter using FindCluster. Cells based on 10PC were inserted using UMAP to visualize cell cluster information.

Quantification and statistical analysis: Quantification of immunoblots was performed using Image J software. Statistical analysis was performed using GraphPad Prism software with Mann-Whitney U-test and Student's t-test to show group differences.

Example 1. Phf7 ^tKO male mice are used to investigate the involvement of PHF7 in spermatogenesis Based on the high expression of PHF7 in the testis, the function of PHF7 in spermatogenesis was analyzed. First, to investigate when PHF7 is expressed during spermatogenesis, we analyzed testis extracts from mice at different developmental stages. The following developmental stages are observed according to age: spermatogonia at 7 dpp, spermatocytes at 14 dpp, round spermatids at 21 dpp, elongated spermatids at 28 dpp, condensed spermatids at 30 dpp, and spermatozoa at 35 dpp (Figure 1A). Phf mRNA levels increased at 21 dpp (Figure 1B) and PHF7 protein levels increased at 28 dpp (Figure 1C), indicating that PHF7 is expressed in round and elongated spermatids.

To investigate the role of PHF7 in spermatogenesis, we generated testis-specific PHF7 knockout (KO) mice (hereafter Phf7 ^tKO ) ( FIG. 8A ). It can be seen that PHF7 protein expression is absent in testicular cells of Phf7 ^tKO mice compared to control (Phf7 ^f/f ) mice ( FIG. 1D ). We crossed control and Phf7 ^tKO mice with wild-type females, and Phf7 ^tKO males were infertile compared to control males ( FIG. 1E ). There was no significant difference in the ratio of testis weight to body weight between control and Phf7 ^f/f males ( FIG. 8B ). Furthermore, we did not observe any obvious defects in the lavage ducts of Phf7 ^tKO males when stained with hematoxylin-PAS ( FIG. 1F ).

In the epididymis, histological analysis by hematoxylin-eosin staining revealed that the sperm count was lower in Phf7 ^tKO than in the control group (Figure 1G, 1H). Sperm from Phf7 ^tKO mice showed lower motility than the control group (Figure 1I). In IVF experiments, Phf7 ^tKO sperm showed a lower fertilization rate than control sperm (Figure 1J). By subsequently observing sperm morphology, it could be seen that the majority of Phf7 ^tKO sperm had rounder and larger heads than the control group (Figure 1K). This indicates that Phf7 ^tKO sperm have less condensed chromatin in the head compared to the control group.

During the sperm maturation process, the nuclei of round spermatids undergo condensation by chromatin condensation involving histone-protamine exchange. Sperm with less condensed heads are manifested by abnormal ratios of histones and protamines in the chromatin. Therefore, we measured the amounts of histones and protamines in sperm. Higher amounts of histones were detected in Phf7 ^tKO sperm compared to the control group (Fig. 1L, 1M). Furthermore, more PRM1 and less PRM2 were observed in Phf7 ^tKO sperm (Fig. 1M). Next, we analyzed spermatids in which histone-protamine exchange occurs. To investigate whether histones remain in condensed spermatids in which histones are not normally detected, we performed co-immunostaining of histone H3 with PRM1, PRM2, and TNP2 in testis sections. In the stage VII-VIII lavage duct of control mice, H3 was not detected in condensed spermatids expressing PRM1 and PRM2, whereas in Phf7 ^tKO mice, spermatids expressing both H3 and PRM1 and PRM2 were observed (Figure 1N and Figure 8C). Condensed spermatids in the stage I lavage duct of control mice express TNP2 but not H3. However, some condensed spermatids in Phf7 ^tKO mice express both H3 and TNP2 (Figure 8D). When histone protein levels were analyzed in spermatid fractions, histone levels decreased as development progressed in spermatids of control mice, but not in Phf7 ^tKO mice (Figure 1O). Thus, histones were removed from condensed spermatids of control mice, whereas histones remained in condensed spermatids of Phf7 ^tKO mice. These data indicate that Phf7 ^tKO males have a defect in histone-protamine exchange during sperm maturation, resulting in infertility due to reduced sperm count and abnormal sperm morphology.

Example 2. Elucidation that PHF7 acts as an E3 ubiquitin ligase for lysine 14 of histone H3 In this study, we observed a decrease in the H3 band below 25 kDa in testicular cells of Phf7 ^tKO mice compared to control mice (Figure 2A). To determine whether the upwardly migrating band was ubiquitinated H3, we performed immunoprecipitation with H3 antibody in testicular cell extracts and then probed with Ub antibody. Ubiquitinated H3 of the same size was detected in control testicular cell extracts, while the amount of ubiquitinated H3 was decreased in Phf7 ^tKO testicular cells (Figure 2B).

Based on the results of H3 ubiquitination, we further analyzed the possibility that PHF7 acts as an E3 ubiquitin ligase for H3. Specifically, we overexpressed PHF7 and ubiquitin in HEK293T cells to confirm H3 ubiquitination by PHF7. Interestingly, in the group containing PHF7 and Hismax-Ub, we observed 28 kDa Hismax-Ub-H3 and 22 kDa endogenous H3ub (Figure 2C). Although it is known that PHF7 induces H2A K119ub, we observed that H2A K119ub increased in the presence of PHF7. H2B and H4 were not ubiquitinated by overexpression of PHF7 (Figure 2C). We performed an in vitro ubiquitination assay of H3 using recombinant nucleosomes. To this end, we screened for E2 ubiquitin-conjugating enzymes and found that UbcH5B and 5C are the E2 enzymes involved in PHF7-mediated ubiquitination of H3 (Fig. 2D). Under the same conditions, PHF7 ubiquitinated H2A K119, H2B, and H4, but ubiquitination of H2B K120 was not detected using the H2B K120ub antibody (Fig. 2D). Addition of PHF7, E1, UbcH5B/C, and Ub increased H3 ubiquitination in a time-dependent manner (Fig. 2E). We then identified the residues involved in PHF7-mediated H3 ubiquitination. In vitro ubiquitination experiments using deletion mutants of H3 indicate that ubiquitination occurs within the N-terminus of H3 (Fig. 2F). LC-MS/MS of H3ub obtained in an in vitro ubiquitination experiment showed that the ubiquitinated residue of H3 was K14 (Figure 9A). Furthermore, when mutations were made, only the K14R mutation of H3 was not ubiquitinated by PHF7 (Figure 2G). The K14 residue of H3 was also confirmed in an in vitro ubiquitination experiment (Figure 2H). Furthermore, LC-MS/MS analysis of ubiquitinated proteins in extracts of HEK293T cells overexpressing PHF7 confirmed that overexpression of PHF7 increased the amount of modified H3K14 peptides compared to the control group (Figure 9B). LC-MS/MS using testicular cells from the control group and Phf7 ^tKO mice showed a 30% decrease in H2A and H2B ubiquitination (Figure 9C). However, LC-MS/MS analysis failed to detect H3K14ub in testicular cells. This is presumably because H3K14ub was present in low amounts or in a small proportion of sperm cells. These data indicate that PHF7 has E3 ubiquitin ligase activity at lysine 14 of H3.

PHF7 has a PHD domain and a RING domain. Previous studies have shown that the N-terminal PHD domain of PHF7 specifically binds to H3K4me2, and the RING domain of PHF7 acts on E3 ubiquitin ligase activity. To examine whether the PHD and RING domains of PHF7 are required for H3 ubiquitination, deletion mutations of each domain were made and it was found that deletion of each domain could not induce H3 ubiquitination (Figure 3A). PHF7 mutations that destroyed conserved amino acid residues in the RING and PHD domains lost E3 ubiquitin ligase activity (Figures 3B and 3C). The RING domain mutation of PHF7 could not ubiquitinate H3 in vitro either (Figure 3D). In summary, the PHD and RING domains of PHF7 are required for H3 ubiquitination, and disruption of each domain resulted in a loss of H3 ubiquitination by PHF7.

Example 3. Generation and Analysis of C160A Knock-in Mice with No Enzymatic Activity of PHF7 To investigate whether the E3 ubiquitin ligase activity of PHF7 is important in spermatogenesis, we generated PHF7 knock-in (KI) mice (hereafter Phf7 ^CA/CA ) with a C160A mutation in the RING domain that abolished the enzymatic activity of PHF7 ( FIG. 3E ). When control (Phf7 ^+/CA ) and Phf7 ^CA/CA males were mated with wild-type females, Phf7 ^CA/CA males were infertile compared to control males ( FIG. 3F ). Control males showed fertility similar to wild-type males, indicating that the C160A mutation is recessive. Although the testes of Phf7 ^CA/CA mice did not show any obvious defects, a low number of sperm was observed in the epididymis of Phf7 ^CA/CA mice (Figure 10A and B). Sperm from Phf7 ^CA/CA mice showed abnormal morphology, low motility, and low fertilization capacity compared to controls (Figures 3G, 3H, and 10C). Similar to sperm from Phf7 ^tKO mice, sperm and sperm cells from Phf7 ^CA/CA mice showed defective histone removal during the sperm maturation process (Figures 3J-3L), indicating that Phf7 ^CA/CA mice have a similar phenotype to Phf7 ^tKO mice. Importantly, H3 ubiquitination was reduced in testicular cells from Phf7 ^CA/CA mice compared to controls (Figure 3M). Taken together, this data indicates that the E3 ubiquitin ligase activity of PHF7 is important for H3 ubiquitination during the sperm maturation process.

Example 4. E3 ubiquitin ligase activity of PHF7 is important for histone removal after H4 hyperacetylation. In Drosophila, PHF7 is known to play a role in regulating the transcription of subordinate genes in germ cells. To investigate whether PHF7 deletion induces transcript changes at the gene level during mouse spermatogenesis, transcripts were analyzed in testicular cells of control and Phf7 ^tKO mice using 10X genomics analysis. The single-cell RNA sequencing results were highly reproducible in two biological repeated experiments, and well classified into known cell populations from spermatogonia to spermatids (Figure 4A, Figure 11). The cell populations show that the composition of cells involved in spermatogenesis is similar between the control and Phf7 ^tKO mice. However, surprisingly, we could not find any significant differences in gene expression patterns within each population between the two mice (Figure 4B), indicating that PHF7 deficiency does not significantly change transcripts during mouse spermatogenesis. On this basis, we analyzed the changes in chromatin condensation during the sperm maturation process.

Therefore, we first analyzed the expression of Tnp and Prm. As a proxy for the single-cell RNA sequencing results, qRT-PCR analysis showed that the mRNA levels of these genes were not significantly different (Figure 4C). However, immunoblot analysis using sperm cell extracts showed that the protein levels of TNP1/2 and PRM2 were reduced in Phf7 ^tKO and Phf7 ^CA/CA mice compared to controls (Figures 4D, 4E). Next, we analyzed H4 hyperacetylation, an epigenetic event that occurs before histone-protamine exchange in elongated spermatids. Surprisingly, spermatids from Phf7 ^tKO and Phf7 ^CA/CA mice showed significantly higher levels of H4 acetylation than those from controls (Figures 4F, 4G). To investigate how PHF7 deficiency increased H4 acetylation in the testis, we performed immunostaining experiments on testis sections. In mice, the spermatogenesis cycle consists of 12 stages, and after stage 12, it becomes stage 1 of the next cycle (Figure 12). After meiosis, the sperm maturation process is divided into 16 steps. Spermatogonia at the base of the lavage duct migrate inward as the spermatogenesis process progresses12, and condensed spermatids exist in the lavage duct at stages 1-8 (steps 12-16), and elongated spermatids exist in the lavage duct at stages 9-11 (steps 9-11). In the control testes, H4 acetylation first appears in elongated spermatids at step 9 in the lavage duct at stage 9, and decreases in condensed spermatids at step 12 in the lavage duct at stage 12, but in the testes of Phf7 ^tKO and Phf7 ^CA/CA mice, H4 acetylation remains in condensed spermatids at steps 13-16 in the lavage duct at stages 1-8, where H4 acetylation is not normally present (Figures 12B, C). H4 hyperacetylation occurring in elongating spermatids induces histone removal, which reduces the level of H4 acetylation in condensing spermatids. For more precise verification, H4ac was co-immunostained with condensing spermatid markers TNP2 (step 11-14 spermatids, stage 11-2), PRM1 (step 11-16 spermatids, stage 11-8), and PRM2 (step 14-16 spermatids, stage 2-8). In the control group, H4ac and PRM2 on testis sections showed reciprocal expression, but in the testes of Phf7 ^tKO and Phf7 ^CA/CA mice, H4ac was detected in all lavage ducts and co-stained with PRM2 (Figure 4H). TNP2 and PRM1 are expressed from step 11 spermatids (stage 11), so in the control group, the two proteins co-stained with H4ac only in step 11-12 spermatids (stage 11-12). However, in Phf7 ^tKO and Phf7 CA ^/CA mice, H4ac is expressed in all lavage ducts, so all spermatids expressing TNP2 and PRM1 co-stained with H4ac (Figure 13A, B). We observed that histones remained in condensed spermatids in Phf7 ^tKO and Phf7 ^CA/CA mice, so H4ac remains in condensed spermatids in Phf7 ^tKO and Phf7 ^CA/CA mice.

To investigate the relationship between PHF7 and H4ac, we generated Flag-PHF7 KI mice (hereafter Phf7 ^Flag ) (Figure 14A). Expression of Flag-PHF7 protein was confirmed by immunoblotting analysis using testicular cells from Phf7 ^Flag/+ mice (Figure 14B). When tissue immunostaining was performed with Flag antibody, Flag-PHF7 was detected in the nuclei of step 11-12 spermatids from step 11 to step 2 wash ducts (Figure 14C, D). When co-immunostaining for H4ac and Flag was performed in testicular sections from ^{Phf7 Flag/+} mice, Flag-PHF7 was expressed after H4ac induction and expressed like H4ac in step 11-12 spermatids (Figure 14E). The timing of PHF7 expression indicates that PHF7 deficiency does not affect the induction of H4 hyperacetylation.

Taken together, abnormally persistent H4 acetylation in condensed spermatids from Phf7 ^tKO and Phf7 ^CA/CA mice may be due to incomplete histone removal during sperm maturation, indicating that there is a defect in histone removal following H4 hyperacetylation in Phf7 ^tKO and Phf7 ^CA/CA mice (Figure 4I).

Example 5. PHF7 deficiency induces BRDT reduction in early condensed spermatids, leading to incomplete histone removal. To investigate the molecular mechanism of why histone removal was disrupted after H4 hyperacetylation in Phf7 ^tKO and Phf7 ^CA/CA mice, we first measured the levels of BRDT, a histone removal factor that recognizes H4 acetylation. Surprisingly, there was no difference in the protein and mRNA levels of BRDT in spermatids of Phf7 ^tKO and Phf7 ^CA/CA mice, and they were lower than those of the control group (Figures 5A and 5B). To determine at which stage of sperm maturation BRDT was reduced in Phf7 ^tKO and Phf7 ^CA/CA mice, tissue immunostaining was performed for BRDT and H4ac. Although BRDT is known to be detected from spermatocytes to elongating spermatids in mice and spermatozoa in humans, we confirmed that BRDT was expressed up to step 12 spermatids in control mice and its expression was decreased in step 13 spermatids (Fig. 5C, 5D). H4ac was co-stained with BRDT in the nuclei of step 11 and step 12 spermatids, and their levels were decreased in step 13 spermatids (Fig. 5E). Surprisingly, we observed that the levels of BRDT were decreased in step 12 spermatids in Phf7 ^tKO and Phf7 ^CA/CA mice, and H4ac remained more abundant in the nuclei of step 12 and step 13 spermatids (Fig. 5C-5E). Histones are removed from late elongating spermatids to early condensing spermatids, and H4ac is not detected in step 13 spermatids. These data indicate that co-expression of H4ac and BRDT by step 12 spermatids is important for histone removal, and that the E3 ubiquitin ligase activity of PHF7 is required to maintain BRDT levels in step 12 spermatids. Therefore, we hypothesized that defects in PHF7 led to failure to maintain BRDT levels in step 12 spermatids, which resulted in defective histone removal.

Example 6. E3 ubiquitin ligase activity of PHF7 is important for BRDT stability To analyze the relationship between PHF7 and BRDT in spermatids after meiosis, we first confirmed the co-expression of PHF7 and BRDT in spermatids of Phf7 ^Flag/+ mice. Histoimmunostaining results showed that PHF7 and BRDT were co-expressed in step 11-12 spermatids (Figure 6A). The BET family, which includes BRD2, 3, and 4, has an SPOP degron between BD1 and BD2, and its stability is regulated by Cul3-SPOP E3 ubiquitin ligase. Importantly, we found that BRDT, a member of the same family, has an SPOP degron sequence (Figure 6B). Immunoblot results after overexpression of BRDT and SPOP in HEK293T cells showed that SPOP-mediated BRDT degradation was suppressed by MG132 treatment (Figure 6C). BRDT mutants lacking the SPOP degron sequence were not affected by SPOP, indicating that Cul3-SPOP is an E3 ubiquitin ligase that degrades BRDT.

To examine whether PHF7 affects BRDT stability, we overexpressed BRDT, SPOP, and PHF7 in HEK293T cells. PHF7 WT, but not PHF7 C160A, increased BRDT protein levels by attenuating SPOP-mediated degradation (Fig. 6D). BRD4 and PTEN, known substrates of SPOP, were not increased by PHF7, indicating that PHF7 did not affect SPOP activity. SPOP and Cul3 were expressed in spermatids, and BRD4 and PTEN in PHF7 mutant spermatids were not significantly different from the control group (Fig. 6E). Next, a ubiquitination assay showed that only WT, but not PHF7 C160A, attenuated ubiquitination of BRDT (Fig. 6F). Cell immunostaining analysis showed that BRDT, SPOP, and PHF7 were co-expressed in the nucleus (Figure 6G). In vitro ubiquitination assays showed that PHF7 itself did not attenuate the ubiquitination of BRDT induced by SPOP (Figure 6H). This indicates that PHF7 does not directly regulate the ubiquitination of BRDT. Taken together, this data indicates that the E3 ubiquitin ligase activity of PHF7 is important for maintaining BRDT stability by attenuating SPOP-induced degradation of BRDT.

To investigate how PHF7 affects BRDT protein stability, we first confirmed the binding of PHF7 to BRDT. However, PHF7 does not directly bind to BRDT (Fig. 7A), so we decided to examine whether PHF7-mediated histone ubiquitination is related to the regulation of BRDT. In vitro pull-down assays using GST-BRDT and histone ubiquitination or H4ac-bearing nucleosomes showed that PHF7-mediated histone ubiquitination weakly increased the binding of H4ac-bearing nucleosomes to BRDT (Fig. 7B). Nevertheless, PHF7-mediated histone ubiquitination and H4ac-bearing nucleosomes, but not single nucleosomes, suppressed SPOP-induced BRDT ubiquitination (Fig. 7C). This data indicates that PHF7-mediated histone ubiquitination interacts with BRDT in the presence of H4ac and regulates BRDT ubiquitination. It is speculated that after H4 hyperacetylation in elongating spermatids, BRDT recognizes H4ac and PHF7-mediated histone ubiquitination contributes to the stabilization of BRDT in early condensing spermatids and promotes histone removal (Figure 7D).

In Drosophila, PHF7 is known to regulate the determination of germ cells as progenitors by activating male-specific gene expression in early germ cells. In mice, previous studies have focused on PHF7-mediated H2Aub in round spermatids, and postulated that RNF8-mediated H2Aub and H2Bub occur in elongating spermatids, a process required for histone-protamine exchange.

In this study, we demonstrated that H3K14ub is another substrate of PHF7 and is important for histone-protamine exchange during sperm maturation. After H4 hyperacetylation in elongating spermatids, PHF7-mediated histone ubiquitination contributes to the stabilization of BRDT in combination with this, i.e., PHF7 plays an important role in maintaining BRDT in early condensed spermatids. Deficiency of PHF7 induces a deficiency of H3K14ub, which in turn reduces the amount of BRDT protein in early condensed spermatids, inducing the development of abnormal spermatozoa with remaining histones. The present invention proposes that histone ubiquitination promotes the removal of histones for the abundant BRDT histone-protamine exchange on chromatin.

Although the illustrative embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concepts of the present application as defined in the following claims also fall within the scope of the present application.

All technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art to which the present invention pertains, unless otherwise defined. The contents of all publications referenced herein are incorporated herein by reference.

Claims (3)

providing a eukaryotic cell expressing PHF7 (PHD finger protein 7), SPOP and BRDT (Bromdomain testis-specific) proteins;
contacting the cell with a test substance that is expected to inhibit the ubiquitination activity of PHF7 to the lysine 14 residue of histone 3 (H3);
measuring the degree of ubiquitination of the H3 protein in the cells after the contact; and, if the degree of ubiquitination of the H3 protein is reduced in the cells contacted with the test substance compared to the cells not contacted in the measuring step, selecting the test substance as a histone-protamine exchange inhibitor required for sperm chromatin condensation,
The cells are provided as a non-human animal model,
The measuring step may further include measuring ubiquitination of a BRDT protein or a concentration of the BRDT protein in sperm cells of the animal model;
A method for screening a sperm chromatin condensation inhibitor, comprising the step of selecting the test substance as a histone-chromatin exchange inhibitor required for sperm nuclear condensation if the BRDT protein ubiquitination is increased compared to cells not treated with the test substance, or if the concentration of the BRDT protein is decreased compared to cells not treated with the test substance. The method for screening a sperm chromatin condensation inhibitor according to claim 1, wherein the cells are HEK293T cells that overexpress PHF7. The method for screening a sperm chromatin condensation inhibitor according to claim 1 or 2 , wherein the sperm chromatin condensation inhibitor is used as a male contraceptive.