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BRD4 dynamics reveal novel acrosome-dependent chromatin reorganization during post-meiotic mammalian sperm development

ABSTRACT: During the post-meiotic phase of spermatogenesis, transcription is progressively repressed as the nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyper-acetylation and replacement of most histones with sperm-specific protamines. Although BRDT has been shown to function in transcription as well as histone removal in post-meiotic spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of mouse testes revealed BRD4 in a complete ring around the nuclei of spermatids containing hyper-acetylated histones. The BRD4 ring lies directly adjacent to the acroplaxome, or the cytoskeletal base of the acrosome, and does not form in acrosomal mutant mice. ChIP sequencing in round spermatids revealed enrichment of BRD4 and acetylated histone H3 and H4 at the promoters of active genes. GO Term analysis showed that BRD4 and BRDT bind to distinct subsets of spermatogenesis-specific genes. Association of BRD4 with Cyclin T1 decreases as spermatogenesis progresses despite a persistence of association with acetylated H4. Moreover, acetylated histones are removed from the condensing spermatid nucleus in a wave following the progressing acrosome. These data provide evidence for an interesting mechanism in which BRD4 and perhaps acetylated histones are removed from the spermatid genome via the progressing acrosome as transcription is repressed. Single replicates each of Brd4, H3K9me3, H3K9ac, H4K5ac, H4K8ac, H4K12ac, H4K16ac, H4Kac (pan-acetyl antibody), and input in mouse round spermatid cells; input is used to control for local sonication efficiency bias.

ORGANISM(S): Mus musculus

SUBMITTER: Gregory Donahue

PROVIDER: E-GEOD-56526 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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BRD4 dynamics reveal novel acrosome-dependent chromatin reorganization during post-meiotic mammalian sperm development

Project description:During the post-meiotic phase of spermatogenesis, transcription is progressively repressed as the nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyper-acetylation and replacement of most histones with sperm-specific protamines. Although BRDT has been shown to function in transcription as well as histone removal in post-meiotic spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of mouse testes revealed BRD4 in a complete ring around the nuclei of spermatids containing hyper-acetylated histones. The BRD4 ring lies directly adjacent to the acroplaxome, or the cytoskeletal base of the acrosome, and does not form in acrosomal mutant mice. ChIP sequencing in round spermatids revealed enrichment of BRD4 and acetylated histone H3 and H4 at the promoters of active genes. GO Term analysis showed that BRD4 and BRDT bind to distinct subsets of spermatogenesis-specific genes. Association of BRD4 with Cyclin T1 decreases as spermatogenesis progresses despite a persistence of association with acetylated H4. Moreover, acetylated histones are removed from the condensing spermatid nucleus in a wave following the progressing acrosome. These data provide evidence for an interesting mechanism in which BRD4 and perhaps acetylated histones are removed from the spermatid genome via the progressing acrosome as transcription is repressed.

2015-03-06 | GSE56526 | GEO

Genome-wide analysis of the distribution of the testis-specific double bromodomain protein BRDT reveals distinct roles in pachytene spermatocyte and round spermatids

Project description:BRDT, a member of the BET family of double bromodomain-containing proteins, is expressed uniquely in the testis from pachytene spermatocytes through round spermatids, and is essential for spermatogenesis in the mouse. Although BRDT is known to bind to acetylated lysines in chromatin, it is not known where in the genome BRDT binds or whether the sites vary during the complex stages of differentiation in which it is expressed. Herein, we use ChIP-Seq to identify genome-wide BRDT binding sites in chromatin from mouse male germ cells at two distinct stages of differentiation. We identified 5452 BRDT-bound genomic loci in pachytene spermatocytes and 5618 BRDT-bound genomic loci in round spermatids. Roughly two thirds of BRDT binding sites were located in genes and BRDT binding correlated with highly transcribed genes. BRDT binding sites also overlapped with several histone modifications or variants that are associated with active transcription. Motif analysis revealed that BRDT-bound regions are enriched for consensus sequences for the MYB, RFX, ETS and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE and RFX in round spermatids. Taken together, our data suggest that BRDT regulates distinct downstream target genes and may have unique functions in the meiotic and spermiogenic transcription programs.

2021-01-26 | GSE98489 | GEO

PHF7 Modulates BRDT Stability and Histone-to-Protamine Exchange during Spermiogenesis

Project description:Spermatogenesis is a complex process of sperm generation, including mitosis, meiosis, and spermiogenesis. During spermiogenesis, histones in post-meiotic spermatids are removed from chromatin and replaced by protamines. Although histone-to-protamine exchange is important for sperm nuclear condensation, the underlying regulatory mechanism is still poorly understood. Here, we identify PHD finger protein 7 (PHF7) as an E3 ubiquitin ligase for histone H3K14 in post-meiotic spermatids. Generation of Phf7-deficient mice and Phf7 C160A knockin mice with impaired E3 ubiquitin ligase activity reveals defects in histone-to-protamine exchange caused by dysregulation of histone removal factor Bromodomain, testis-specific (BRDT) in early condensing spermatids. Surprisingly, E3 ubiquitin ligase activity of PHF7 on histone ubiquitination leads to stabilization of BRDT by attenuating ubiquitination of BRDT. Collectively, our findings identify PHF7 as a critical factor for sperm chromatin condensation and contribute to mechanistic understanding of fundamental phenomenon of histone-to-protamine exchange and potential for drug development for the male reproduction system.

2020-07-28 | GSE136752 | GEO

BRD4 assists elongation of both coding and enhancer RNAs guided by histone acetylation

Project description:In serum-starved and re-fed mouse fibroblast, nascent RNA-seq analysis showed that the BET inhibitor JQ1 antagonized a process regulating PIC formation and a downstream process involved in progressive elongation. To specifically address the role of BRD4 and its interactions with acetylated histones and P-TEFb, YFP-tagged BRD4 proteins (wild type and mutant BRD4) were stably expressed in cells, endogenous BRD4 of which was knocked down by shRNA (shBRD4). Nascent RNA-seq analysis showed that BRD4 facilitated transcript elongation in a manner dependent on acetylated histone interaction but not P-TEFb. Furthermore, the role of BRD4 in enhancer activity was evaluated by mapping the intergenic distributions of Pol II, CDK9, H3K27 acetylation (Ac) and H4 Ac as well as BRD4 by ChIP-seq analysis. The results suggest that the role of intergenic BRD4 on nearby gene expression is mediated through enhanced synthesis of eRNA.

2014-11-10 | GSE58731 | GEO

Bromodomain-dependent stage-specific male genome programming by Brdt [ChIP-Seq]

Project description:Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential meiotic genes and repressing a M-bM-^@M-^\progenitor cellsM-bM-^@M-^] gene expression program, while M-bM-^@M-^\primingM-bM-^@M-^] a post-meiotic gene group for further activation. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for BrdtM-bM-^@M-^Ys first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome. Examination of Brdt binding on chromatin in meiotic (spermatocytes) and post-meiotic (round spermatids) male germ cells from adult wild type mice

2012-08-30 | E-GEOD-39908 | biostudies-arrayexpress

Tsga8 is required for spermatid morphogenesis and male fertility in mice

Project description:During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility.

2021-04-16 | GSE147805 | GEO

Expression data from purified control and BrdtΔBD1 round spermatids.

Project description:Brdt is a testis specific member of a family of chromatin interacting proteins. All of the family members have been shown to regulate transcription. Brdt is highly expressed in round spermatids, and may play a role in transcriptional regulation in these cells. We investigated transcriptional changes in mutant round spermatids that were homozygous for a mutation in which the first bromodomain of Brdt was removed. Round spermatids were purified from seven adult animals of each genotype for each Affymetrix microarray. Purity of round spermatids was assessed by propidium iodide staining.

2011-10-20 | E-GEOD-33132 | biostudies-arrayexpress

Nut interactome in round/elongating spermatids

Project description:NUT, nuclear protein in testis is the universal fusion partner of BRD4 in the highly aggressive NUT Midline Carcinoma (NMC), but its physiological function was unknown. Here we show that Nut is exclusively expressed in post-meiotic spermatogenic cells, at the time of genome-wide histone hyperacetylation. Inactivation of Nut induces a spermatogenesis arrest at the histone-to-protamine replacement stage, leading to male infertility. Subsequent molecular investigations show that Nut sustains global histone H4 hyperacetylation in post-meiotic cells. Additionally, Nut mediates a p300/CBP-dependent gene expression program and, by enhancing acetylation of H4 at both K5 and K8 sites, provides binding sites for the first bromodomain of Brdt, which drives histone removal. Nut’s major function is therefore to use the ubiquitous HATs p300/CBP to direct a cell-type specific histone hyperacetylation. Its ectopic activity in NMC recreates a forced p300-induced histone hyperacetylation / bromodomain binding loop that normally operates in post-meiotic spermatogenic cells.

2018-10-02 | PXD008727 | Pride

Bromodomain-dependent stage-specific male genome programming by Brdt [Illumina BeadArray]

Project description:Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post-meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis-specific gene expression program. In meiotic cells, Brdt initiates a genuine histone acetylation-guided programming of the genome by activating essential meiotic genes and repressing a “progenitor cells” gene expression program, while “priming” a post-meiotic gene group for further activation. At post-meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt’s first bromodomain to direct the genome-wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome. Total RNA obtained from testes from prepuberal mice at 17dpp and at 20dpp were compared between Brdt-/- and Brdt+/- (17dpp), between Brdt-/- and Brdt +/+ (20dpp) and between BrdtBD1del and Brdt wt mice (20dpp). In each experiments, 6 replicates of each genotype and condition were used.

2012-08-30 | E-GEOD-39909 | biostudies-arrayexpress

Characterization of TP2 interactome

Project description:Spermiogenesis is marked by an almost genome-wide histone-to-protamine transition. Protamines are sperm-specific, small and highly positively charged proteins mainly responsible for genome compaction at this step. According to previous investigations from our lab and other groups, it is known that just before their removal, histones become hyperacetylated, which creates binding sites for the first bromodomain of a testis-specific member of the BET double bromodomain protein family, Brdt. Brdt’s first bromodomain is necessary for transition proteins (TPs) and PRMs incorporation and subsequent histone removal. Histone hyperacetylation, specifically on H4 lysine residues 5 and 8 which are recognized by Brdt, is controlled by a post-meiotic enhancer of CBP/p300 acetyltransferase activity, known as Nut. The process of TP assembly itself is dependent on a prior incorporation of a histone variant known as H2A.L.2, expressed in the same cells and at the same time as TPs. To characterize the proteins associating with TP2, we performed a mass spectrometry-based proteomics analysis of TP2 co-immunoprecipitations elutes from condensing spermatids extract. The immunoprecipitation was performed using extract from stages 12-16 spermatids from wild-type and H2A.L.2-KO mice. In parallel, immunopurifications were performed using a non-specific antibody (pre-immune goat serum) to allow the identification of proteins specifically purified with TP2 as opposed to proteins non-specifically precipitated.

2022-02-14 | PXD031665 | Pride

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