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• W2892893971 abstract "•Nut is a post-meiotically expressed gene that is critical for male fertility•Nut recruits p300 and/or CBP to enhance histone H4K5 and H4K8 acetylation•Nut-mediated histone hyperacetylation is required for histone-to-protamine transition Nuclear protein in testis (Nut) is a universal oncogenic driver in the highly aggressive NUT midline carcinoma, whose physiological function in male germ cells has been unclear. Here we show that expression of Nut is normally restricted to post-meiotic spermatogenic cells, where its presence triggers p300-dependent genome-wide histone H4 hyperacetylation, which is essential for the completion of histone-to-protamine exchange. Accordingly, the inactivation of Nut induces male sterility with spermatogenesis arrest at the histone-removal stage. Nut uses p300 and/or CBP to enhance acetylation of H4 at both K5 and K8, providing binding sites for the first bromodomain of Brdt, the testis-specific member of the BET family, which subsequently mediates genome-wide histone removal. Altogether, our data reveal the detailed molecular basis of the global histone hyperacetylation wave, which occurs before the final compaction of the male genome. Nuclear protein in testis (Nut) is a universal oncogenic driver in the highly aggressive NUT midline carcinoma, whose physiological function in male germ cells has been unclear. Here we show that expression of Nut is normally restricted to post-meiotic spermatogenic cells, where its presence triggers p300-dependent genome-wide histone H4 hyperacetylation, which is essential for the completion of histone-to-protamine exchange. Accordingly, the inactivation of Nut induces male sterility with spermatogenesis arrest at the histone-removal stage. Nut uses p300 and/or CBP to enhance acetylation of H4 at both K5 and K8, providing binding sites for the first bromodomain of Brdt, the testis-specific member of the BET family, which subsequently mediates genome-wide histone removal. Altogether, our data reveal the detailed molecular basis of the global histone hyperacetylation wave, which occurs before the final compaction of the male genome. In mammals, unique physiological, genome-wide histone hyperacetylation has been observed, associated with a near-total histone eviction that occurs during the late stages of spermatogenesis. In this context, an outstanding issue is the understanding of the causes and consequences of this histone hyperacetylation and, more precisely, of its role in histone eviction (Goudarzi et al., 2014Goudarzi A. Shiota H. Rousseaux S. Khochbin S. Genome-scale acetylation-dependent histone eviction during spermatogenesis.J. Mol. Biol. 2014; 426: 3342-3349Crossref PubMed Scopus (64) Google Scholar). After meiosis, haploid cells, named spermatids, engage in one of the most dramatic known chromatin remodeling and genome reorganization processes, which essentially takes place in cells known as elongating and condensing spermatids. It consists of the genome-wide removal of histones and their replacement by small basic proteins, transition proteins (TPs) and protamines (Prms) (Barral et al., 2017Barral S. Morozumi Y. Tanaka H. Montellier E. Govin J. de Dieuleveult M. Charbonnier G. Couté Y. Puthier D. Buchou T. et al.Histone variant H2A.L.2 guides transition protein-dependent protamine assembly in male germ cells.Mol. Cell. 2017; 66: 89-101Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Gaucher et al., 2010Gaucher J. Reynoird N. Montellier E. Boussouar F. Rousseaux S. Khochbin S. From meiosis to postmeiotic events: the secrets of histone disappearance.FEBS J. 2010; 277: 599-604Crossref PubMed Scopus (136) Google Scholar). Spermatids are produced following two successive meiotic divisions of spermatocytes, which are continuously generated from the progenitor cells known as spermatogonia. During the past decades, we have developed strategies to discover the molecular basis of this genome-wide histone hyperacetylation taking place in late haploid male germ cells (Goudarzi et al., 2014Goudarzi A. Shiota H. Rousseaux S. Khochbin S. Genome-scale acetylation-dependent histone eviction during spermatogenesis.J. Mol. Biol. 2014; 426: 3342-3349Crossref PubMed Scopus (64) Google Scholar). These strategies included a series of structural and functional studies of the testis-specific bromodomain-containing BET factor Brdt. These investigations showed that Brdt’s first bromodomain specifically recognizes histone H4 bearing the simultaneous acetylation of K5 and K8 (Goudarzi et al., 2016Goudarzi A. Zhang D. Huang H. Barral S. Kwon O.K. Qi S. Tang Z. Buchou T. Vitte A.-L. He T. et al.Dynamic competing histone H4 K5K8 acetylation and butyrylation are hallmarks of highly active gene promoters.Mol. Cell. 2016; 62: 169-180Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, Miller et al., 2016Miller T.C.R. Simon B. Rybin V. Grötsch H. Curtet S. Khochbin S. Carlomagno T. Müller C.W. A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT.Nat. Commun. 2016; 7: 13855Crossref PubMed Scopus (72) Google Scholar, Morinière et al., 2009Morinière J. Rousseaux S. Steuerwald U. Soler-López M. Curtet S. Vitte A.-L. Govin J. Gaucher J. Sadoul K. Hart D.J. et al.Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.Nature. 2009; 461: 664-668Crossref PubMed Scopus (340) Google Scholar, Sasaki et al., 2009Sasaki K. Ito T. Nishino N. Khochbin S. Yoshida M. Real-time imaging of histone H4 hyperacetylation in living cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 16257-16262Crossref PubMed Scopus (111) Google Scholar) and somehow mediates the eviction of histones (Gaucher et al., 2012Gaucher J. Boussouar F. Montellier E. Curtet S. Buchou T. Bertrand S. Hery P. Jounier S. Depaux A. Vitte A.-L. et al.Bromodomain-dependent stage-specific male genome programming by Brdt.EMBO J. 2012; 31: 3809-3820Crossref PubMed Scopus (184) Google Scholar). In the frame of these studies, we also became interested in a testis-specific gene of unknown function, nuclear protein in testis (NUT), which is involved in a cancer known as NUT midline carcinoma (NMC). NMC is characterized by a series of chromosomal translocations that fuse the NUT gene located on chromosome 15q14 to partner genes, most frequently one of the double bromodomain proteins of the BET family, BRD4 (67% of cases) or BRD3 (25% of cases). Therefore, in more than 90% of NMC cases, NUT is fused to a double bromodomain BET factor (French, 2012French C.A. Pathogenesis of NUT midline carcinoma.Annu. Rev. Pathol. 2012; 7: 247-265Crossref PubMed Scopus (189) Google Scholar). Because of the testis-specific nature of NUT expression and its cooperation with somatic members of BETs, BRD3 and BRD4 in NMC, we hypothesized that a possible cooperation between NUT and BRDT in the physiological setting of spermatogenesis could occur and be involved in the establishment and management of histone hyperacetylation. Molecular investigations of the oncogenic activity of the fusion protein BRD4-NUT revealed that it mediates the creation of hyperacetylated chromatin domains. In cells expressing BRD4-NUT, a feed-forward loop drives histone acetylation propagation (Reynoird et al., 2010Reynoird N. Schwartz B.E. Delvecchio M. Sadoul K. Meyers D. Mukherjee C. Caron C. Kimura H. Rousseaux S. Cole P.A. et al.Oncogenesis by sequestration of CBP/p300 in transcriptionally inactive hyperacetylated chromatin domains.EMBO J. 2010; 29: 2943-2952Crossref PubMed Scopus (138) Google Scholar). This BRD4-NUT-driven histone acetylation is constrained by cellular deacetylases (Reynoird et al., 2010Reynoird N. Schwartz B.E. Delvecchio M. Sadoul K. Meyers D. Mukherjee C. Caron C. Kimura H. Rousseaux S. Cole P.A. et al.Oncogenesis by sequestration of CBP/p300 in transcriptionally inactive hyperacetylated chromatin domains.EMBO J. 2010; 29: 2943-2952Crossref PubMed Scopus (138) Google Scholar, Schwartz et al., 2011Schwartz B.E. Hofer M.D. Lemieux M.E. Bauer D.E. Cameron M.J. West N.H. Agoston E.S. Reynoird N. Khochbin S. Ince T.A. et al.Differentiation of NUT midline carcinoma by epigenomic reprogramming.Cancer Res. 2011; 71: 2686-2696Crossref PubMed Scopus (150) Google Scholar) and remains limited to the nuclear topologically associated domains (TADs) (Alekseyenko et al., 2015Alekseyenko A.A. Walsh E.M. Wang X. Grayson A.R. Hsi P.T. Kharchenko P.V. Kuroda M.I. French C.A. The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains.Genes Dev. 2015; 29: 1507-1523Crossref PubMed Scopus (118) Google Scholar), leading to the creation of hyperacetylated chromatin domains. The discovery of the molecular basis of the activity of the fusion protein BRD4-NUT in NMC and of a role for NUT in inducing enhanced histone acetylation in NMC cancer cells prompted us to propose that Nut could also have a role in inducing the genome-wide histone hyperacetylation in post-meiotic male germ cells. To explore this hypothesis, we designed a series of experiments aiming to address the role of Nut during spermatogenesis at a molecular level. The investigations reported here reveal mechanisms underlying the obscure process of histone H4 hyperacetylation and its molecular link to the genome-wide histone eviction. These data also demonstrate that the functional cooperation of Nut, p300 and/or CBP, and a BET factor that is created following the BRD4-NUT gene fusion in NMC also exists in the natural setting of Nut’s action, the post-meiotic spermatogenic cells. The sequence of events triggered by this interaction becomes oncogenic when it operates “off context.” The fusion partner of BRD4, NUT, was identified as a gene expressed in testis. However, until now, no information was available on its tissue-specific activity or on the pattern of its expression in testis during male germ cell differentiation. Using publicly available transcriptomic data from different human and mouse tissues, we found that in both human and mouse, NUT/Nut is exclusively expressed in testis (Figure S1). We prepared RNA and protein extracts from developing mouse testes to precisely define the timing of Nut gene expression and Nut protein accumulation during spermatogenesis. Post-meiotic haploid round spermatids first appear at 20 days post-partum (dpp). Traces of Nut mRNA (Figure 1A) and protein (Figure 1B) also appear at day 20. Both Nut mRNA and protein strongly accumulate at 25 dpp, when late round and early elongating spermatids are produced. In situ co-detection of Nut, along with the acrosome on spermatogenic cell preparations, shows that Nut is not detectable in early round spermatids, as defined by the size of their acrosomes, but instead accumulates in mid-round, late round, and early elongating spermatid populations (Figure 1C, left panel). The observed timing of Nut accumulation in round and early elongating spermatids corresponds to when histone H4 hyperacetylation is known to first occur (Goudarzi et al., 2014Goudarzi A. Shiota H. Rousseaux S. Khochbin S. Genome-scale acetylation-dependent histone eviction during spermatogenesis.J. Mol. Biol. 2014; 426: 3342-3349Crossref PubMed Scopus (64) Google Scholar). This observation prompted us to co-detect Nut and H4K5ac (acetyl), a critical histone mark in post-meiotic cells (Gaucher et al., 2012Gaucher J. Boussouar F. Montellier E. Curtet S. Buchou T. Bertrand S. Hery P. Jounier S. Depaux A. Vitte A.-L. et al.Bromodomain-dependent stage-specific male genome programming by Brdt.EMBO J. 2012; 31: 3809-3820Crossref PubMed Scopus (184) Google Scholar, Goudarzi et al., 2016Goudarzi A. Zhang D. Huang H. Barral S. Kwon O.K. Qi S. Tang Z. Buchou T. Vitte A.-L. He T. et al.Dynamic competing histone H4 K5K8 acetylation and butyrylation are hallmarks of highly active gene promoters.Mol. Cell. 2016; 62: 169-180Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Figure 1C (left panels) shows that the accumulation of Nut is associated with the induction of histone H4K5 acetylation in differentiating spermatids. Additional in situ co-detection of Nut and H4K5ac confirms this strict correlation between the accumulation of Nut and the occurrence of enhanced H4K5 acetylation. Only background H4K5ac could be detected in spermatocytes that do not express Nut compared to Nut-expressing round spermatids (Figure 1C, right panel). The question therefore arose of whether Nut could be a direct player in the induced acetylation of histone H4 in early elongating spermatids. To evaluate the function of Nut in post-meiotic spermatogenic cells, we set up a strategy based on the deletion of Nut exon 2 to generate NutKO/KO mice (Nut knockout [Nut KO] mice) (Figure S2). Western blots using our antibody on total testis extracts demonstrated the absence of Nut in spermatogenic cells of Nut KO mice (Figure 2A, left panel). Nut KO mice harbored smaller testes and were sterile (Figure 2A, middle and right panels). Cauda epididymis from Nut KO mice showed the absence of spermatozoa (Figure 2B), demonstrating an arrest of spermatogenesis at earlier stages. To precisely define spermatogenesis stages affected by the absence of Nut, we prepared histological sections of paraffin-embedded testes from wild-type (WT) and Nut KO mice. In Nut KO mice testes, spermatogenesis seems to occur normally until the appearance of condensing spermatids. Although round spermatids could be easily detected in both wild-type and Nut KO testes, no condensing spermatids were found in Nut KO testes (Figure 2C, DAPI panels). Immunohistochemistry on these sections using an anti-Prm1 antibody showed that in the absence of Nut, the Prm1-expressing spermatids do not undergo nuclear elongation and mostly remain round (Figure 2C, Prm1 panels). Because Nut accumulation is associated with the induction of histone H4 acetylation (Figure 1C), which is associated with the replacement of histones by TPs and protamines (Gaucher et al., 2012Gaucher J. Boussouar F. Montellier E. Curtet S. Buchou T. Bertrand S. Hery P. Jounier S. Depaux A. Vitte A.-L. et al.Bromodomain-dependent stage-specific male genome programming by Brdt.EMBO J. 2012; 31: 3809-3820Crossref PubMed Scopus (184) Google Scholar, Goudarzi et al., 2016Goudarzi A. Zhang D. Huang H. Barral S. Kwon O.K. Qi S. Tang Z. Buchou T. Vitte A.-L. He T. et al.Dynamic competing histone H4 K5K8 acetylation and butyrylation are hallmarks of highly active gene promoters.Mol. Cell. 2016; 62: 169-180Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), we focused on the process of histone-to-protamine replacement. Accordingly, TH2B, which is the major spermatogenic cell histone H2B variant (Montellier et al., 2013Montellier E. Boussouar F. Rousseaux S. Zhang K. Buchou T. Fenaille F. Shiota H. Debernardi A. Héry P. Curtet S. et al.Chromatin-to-nucleoprotamine transition is controlled by the histone H2B variant TH2B.Genes Dev. 2013; 27: 1680-1692Crossref PubMed Scopus (154) Google Scholar), was co-detected, along with histone-replacing proteins TP1, TP2, Prm1, and Prm2. Figure 3 shows that although TH2B was never observed in wild-type cells expressing histone-replacing proteins, in Nut KO spermatids, TH2B was co-detected with TP1, TP2, Prm1, and Prm2. These observations demonstrate that even though the histone-replacing proteins are expressed in the absence of Nut, they are incapable of replacing histones. To further demonstrate the defective histone replacement in the Nut KO spermatids, we made the hypothesis that the accumulation of TPs and protamines in the absence of histone replacement should leave an imprint on nucleosomes, especially on the more accessible DNA linker regions. The interaction of these highly basic proteins with linker DNA should protect DNA linker regions against micrococcal nuclease (MNase) digestion and therefore generate DNA fragments longer than the canonical 147 base pairs (bp). To test this hypothesis, we prepared nuclei from fractionated round-elongating spermatids from wild-type and Nut KO testes and submitted them to extensive digestion with MNase to generate nucleosomal monomers. We then used paired-end sequencing to accurately determine DNA fragment lengths at the highest (base pair) resolution following two independent experiments (different mice and different fractionations). Both experiments revealed an enrichement of nucleosomal DNA fragments with lengths longer than the canonical 147 bp in Nut KO spermatids compared to wild-type spermatids (Figures 4A and 4B ). Altogether, the co-immunodetection of TH2B and histone-replacing proteins, as well as the accumulation of nucleosomes with longer linker DNA in Nut KO spermatids, strongly support an impairment of histone replacement in the absence of Nut. The defective histone replacement observed in the absence of Nut was reminiscent of the impaired TP and protamine assembly previously detected in mouse spermatogenic cells expressing a Brdt mutant lacking its first bromodomain (BD1) (Gaucher et al., 2012Gaucher J. Boussouar F. Montellier E. Curtet S. Buchou T. Bertrand S. Hery P. Jounier S. Depaux A. Vitte A.-L. et al.Bromodomain-dependent stage-specific male genome programming by Brdt.EMBO J. 2012; 31: 3809-3820Crossref PubMed Scopus (184) Google Scholar) (Figure S3), suggesting a possible functional link between Nut and Brdt’s first bromodomain in histone replacement. Because Brdt’s BD1 binds to histone H4 acetylated on K5 and K8 (Goudarzi et al., 2016Goudarzi A. Zhang D. Huang H. Barral S. Kwon O.K. Qi S. Tang Z. Buchou T. Vitte A.-L. He T. et al.Dynamic competing histone H4 K5K8 acetylation and butyrylation are hallmarks of highly active gene promoters.Mol. Cell. 2016; 62: 169-180Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, Miller et al., 2016Miller T.C.R. Simon B. Rybin V. Grötsch H. Curtet S. Khochbin S. Carlomagno T. Müller C.W. A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT.Nat. Commun. 2016; 7: 13855Crossref PubMed Scopus (72) Google Scholar, Morinière et al., 2009Morinière J. Rousseaux S. Steuerwald U. Soler-López M. Curtet S. Vitte A.-L. Govin J. Gaucher J. Sadoul K. Hart D.J. et al.Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.Nature. 2009; 461: 664-668Crossref PubMed Scopus (340) Google Scholar, Sasaki et al., 2009Sasaki K. Ito T. Nishino N. Khochbin S. Yoshida M. Real-time imaging of histone H4 hyperacetylation in living cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 16257-16262Crossref PubMed Scopus (111) Google Scholar), and Brdt’s BD1 is necessary for histone-to-protamine replacement (Gaucher et al., 2012Gaucher J. Boussouar F. Montellier E. Curtet S. Buchou T. Bertrand S. Hery P. Jounier S. Depaux A. Vitte A.-L. et al.Bromodomain-dependent stage-specific male genome programming by Brdt.EMBO J. 2012; 31: 3809-3820Crossref PubMed Scopus (184) Google Scholar), the impairment of histone removal in the absence of Nut (Figures 3 and S3) could be attributed to the absence of functional Brdt or to a defective histone acetylation specifically at H4K5 and H4K8. A western blot on extracts from fractionated post-meiotic round and early elongating spermatids showed no change in the level of Brdt expression in the absence of Nut (Figure 5A). This result, along with our observation that Nut accumulation in mid- and late round and early elongating spermatids corresponds to the induction of histone hyperacetylation (Figure 1C), prompted us to perform a comparative analysis of the level of histone acetylation on histones extracted from round and early elongating spermatids from wild-type and Nut KO testes. Consequently, we sought an unbiased approach to quantitatively monitor histone acetylation in the wild-type and Nut KO round and early elongating spermatids. In vitro isotopic labeling followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis was performed to quantify histone post-translational modifications (PTMs). Histone extracts were digested with trypsin, and the resulting peptides were propionylated using light (12C6) and heavy (13C6) propionic anhydride. The light and heavy propionylated tryptic peptides were mixed in equal amounts and analyzed by HPLC-MS/MS. The quantification of histone acetylation revealed a decrease in histone H4 and H2A acetylation, with a remarkable downregulation of acetylation at H4K5 and H4K8, as well as of H2AK5 and H2AK9 (Figure 5A, lower panel; Tables S1 and S2). The downregulation of acetylation, especially at the critical H4K5 and H4K8 sites, in Nut KO round and early elongating spermatids compared to the wild-type spermatids was confirmed by western blotting, with the corresponding antibodies on independently prepared round and early elongating spermatid-enriched fractions (Figure 5B). Because none of the H3 tail lysines analyzed here by mass spectrometry were significantly affected by the absence of Nut, H3K9 acetylation was additionally checked by immunoblotting, showing only a slight decrease in Nut KO spermatids (Figure 5B). These results show that the expression of Nut in round and early elongating spermatids (Figure 1C) is required for histone H4 hyperacetylation. In addition, the impairment of Nut-mediated acetylation of H4 demonstrates the inability of Brdt’s BD1 to bind chromatin, explaining the similarity between Nut KO and Brdtdelta BD1/delta BD1 phenotypes (Figure S3). Our previous work on NMC identified p300 and/or CBP as the major cellular histone acetyltransferases (HATs) recruited by the NUT moiety of the BRD4-NUT fusion protein in a NMC cell line or upon transfection (Reynoird et al., 2010Reynoird N. Schwartz B.E. Delvecchio M. Sadoul K. Meyers D. Mukherjee C. Caron C. Kimura H. Rousseaux S. Cole P.A. et al.Oncogenesis by sequestration of CBP/p300 in transcriptionally inactive hyperacetylated chromatin domains.EMBO J. 2010; 29: 2943-2952Crossref PubMed Scopus (138) Google Scholar). We therefore hypothesized that when expressed in its physiological setting, spermatids, Nut could interact with p300 and/or CBP and use a similar mechanism to enhance histone acetylation. To verify this hypothesis, we performed two independent sets of experiments. First, the round and early elongating spermatid fractions from wild-type and Nut KO testes were used to obtain and compare the corresponding transcriptomes and to identify genes whose expression depends on Nut. The absence of Nut was found to be associated with the downregulation of 1,184 genes and the upregulation of 499 genes (using a cutoff of 1.5 for the absolute log2 fold change value and a t test p value, after adjustment for multiple tests by Benjamini-Hochberg, of p < 0.05) (Figure 6A). To ensure the observed changes in gene expression in the absence of Nut were not due to changes in the cell-type composition of the Nut KO round and elongating cell fraction compared to the same fraction of wild-type cells, we performed an additional analysis. By comparing wild-type meiotic and post-meiotic cell populations, we identified genes that are normally activated in wild-type post-meiotic cells. The observation that only a fraction of these genes was downregulated in Nut KO post-meiotic cells was supportive of a specific action of Nut on gene expression as opposed to the loss of particular cell types. The expression of a significant number of the genes, 317, including the Prm1-encoding gene, that are normally activated in wild-type post-meiotic cells did not significantly change in the absence of Nut (Figures S4A and S4B), demonstrating that our transcriptomic data from wild-type and Nut KO spermatids could be compared, because they were obtained from two comparable cell populations. We then used a series of stage-specific transcriptomes that were produced in wild-type male germ cells from our previous studies (Boussouar et al., 2014Boussouar F. Goudarzi A. Buchou T. Shiota H. Barral S. Debernardi A. Guardiola P. Brindle P. Martinez G. Arnoult C. et al.A specific CBP/p300-dependent gene expression programme drives the metabolic remodelling in late stages of spermatogenesis.Andrology. 2014; 2: 351-359Crossref PubMed Scopus (23) Google Scholar, Montellier et al., 2013Montellier E. Boussouar F. Rousseaux S. Zhang K. Buchou T. Fenaille F. Shiota H. Debernardi A. Héry P. Curtet S. et al.Chromatin-to-nucleoprotamine transition is controlled by the histone H2B variant TH2B.Genes Dev. 2013; 27: 1680-1692Crossref PubMed Scopus (154) Google Scholar) and the present work to establish the normal pattern of these Nut-regulated genes during spermatogenesis. Figure S4C shows that the genes that require Nut to be active (downregulated genes in Nut KO) are normally activated in post-meiotic cells, with the highest activity in late round and early elongating spermatids. In a previous work, we identified genes that are specifically responsive to post-meiotic p300 and/or CBP variations in spermatids (Boussouar et al., 2014Boussouar F. Goudarzi A. Buchou T. Shiota H. Barral S. Debernardi A. Guardiola P. Brindle P. Martinez G. Arnoult C. et al.A specific CBP/p300-dependent gene expression programme drives the metabolic remodelling in late stages of spermatogenesis.Andrology. 2014; 2: 351-359Crossref PubMed Scopus (23) Google Scholar). We used these data to define whether Nut regulates gene expression through p300 and/or CBP by comparing our two Nut- and p300 and/or CBP-dependent transcriptomes, both obtained in comparable post-meiotic cell populations. We looked for a significant enrichment of Nut-regulated genes in the lists of p300 and/or CBP-regulated genes, which would suggest the involvement of p300 and/or CBP in gene regulation by Nut. Gene set enrichment analysis (GSEA) was used to look for the expression in Nut KO spermatids of genes whose expression in spermatids had previously been found to be affected by a targeted post-meiotic deletion of p300 and CBP (Boussouar et al., 2014Boussouar F. Goudarzi A. Buchou T. Shiota H. Barral S. Debernardi A. Guardiola P. Brindle P. Martinez G. Arnoult C. et al.A specific CBP/p300-dependent gene expression programme drives the metabolic remodelling in late stages of spermatogenesis.Andrology. 2014; 2: 351-359Crossref PubMed Scopus (23) Google Scholar). Although following this latter approach, the inheritance of both p300 and CBP from preceding stages led to only a small decrease in the enzyme levels in post-meiotic cells, it was enough to observe a change in the expression of a specific set of genes whose expression is highly dependent on, and sensitive to, p300 and CBP dosage (Boussouar et al., 2014Boussouar F. Goudarzi A. Buchou T. Shiota H. Barral S. Debernardi A. Guardiola P. Brindle P. Martinez G. Arnoult C. et al.A specific CBP/p300-dependent gene expression programme drives the metabolic remodelling in late stages of spermatogenesis.Andrology. 2014; 2: 351-359Crossref PubMed Scopus (23) Google Scholar). Figure 6B shows a clear enrichment of the p300 and/or CBP-regulated genes among the genes up- and downregulated in Nut KO mice, supporting the idea that Nut-p300 and/or CBP, while directing the global histone acetylation, also affects the expression of late active genes before the general shutdown of transcription in elongating spermatids. The question arises of whether Nut uses CBP and p300 in spermatids as it does in NMC cells or if other HATs are also involved. To answer this question, in an additional set of experiments, we prepared extracts from fractionated wild-type and Nut KO round and early elongating spermatids, which were submitted to anti-Nut immunoprecipitation and a proteomic analysis in two independent assays. The aim of this experiment was two-fold: first, to confirm the presence of p300 and CBP in association with Nut, and second, to see whether additional cellular acetyltransferases interact with Nut in post-meiotic cells undergoing histone hyperacetylation. To maximize the chance of detecting any HAT associated with Nut, we used non-stringent immunoprecipitation conditions and performed the experiment twice on independent mice and fractionated cells. As expected from non-stringent immunoprecipitation conditions, many proteins were identified as specifically enriched in Nut wild-type cells and were observed in both experiments. However, in an effort to focus on proteins relevant to the present study, especially HATs, we chose to highlight the transcription and/or chromatin and/or RNA and/or metabolism-related proteins. Table S3 lists the proteins that were only found in the wild-type cell extracts or enriched at least 5 times in these extracts compared to Nut KO cell extracts. Despite the non-stringent immunoprecipitation conditions, p300 and CBP were the only known HATs found to be exclusively or significantly enriched in the Nut immunoprecipitates in wild-type extracts compared to Nut KO extracts (Figure 6C). In the Nut-dependent proteome, we also found TH2B-H2A.L.2 and transition protein 2 (TP2), shown to allow protamines to efficiently invade nucleosomes at the time of histone-to-protamine replacement (Barral et al., 2017Barral S. Morozumi Y. Tanaka H. Montellier E. Govin J. de Dieuleveult M. Charbonnier G. Couté Y. Puthier D. Buchou T. et al.Histone variant H2A.L.2 guides transition protein-dependent protamine assembly in male germ cells.Mol. Cell. 2017; 66: 89-101Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) (Figure 6C). Because the generation of these nucleosomal transitional states also involves H4 acetylation and Brdt's BD1, these findings reinforce the hypothesis that Nut could also be a player in the process of acetylation-dependent histone replacement. A proteomic analysis of BRD4-NUT fusion partners from a human cell line generated similar data, with p300 and CBP as the only BR" @default.
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• W2892893971 title "Nut Directs p300-Dependent, Genome-Wide H4 Hyperacetylation in Male Germ Cells" @default.
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• W2892893971 doi "https://doi.org/10.1016/j.celrep.2018.08.069" @default.
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