DOT1L regulates chromatin reorganization and gene expression during sperm differentiation.

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.

Battle of the Sex Chromosomes: Competition between X and Y Chromosome-Encoded Proteins for Partner Interaction and Chromatin Occupancy Drives Multicopy Gene Expression and Evolution in Muroid Rodents.

Transmission distorters (TDs) are genetic elements that favor their own transmission to the detriments of others. Slx/Slxl1 (Sycp3-like-X-linked and Slx-like1) and Sly (Sycp3-like-Y-linked) are TDs, which have been coamplified on the X and Y chromosomes of Mus species. They are involved in an intragenomic conflict in which each favors its own transmission, resulting in sex ratio distortion of the progeny when Slx/Slxl1 versus Sly copy number is unbalanced. They are specifically expressed in male postmeiotic gametes (spermatids) and have opposite effects on gene expression: Sly knockdown leads to the upregulation of hundreds of spermatid-expressed genes, whereas Slx/Slxl1-deficiency downregulates them. When both Slx/Slxl1 and Sly are knocked down, sex ratio distortion and gene deregulation are corrected. Slx/Slxl1 and Sly are, therefore, in competition but the molecular mechanism remains unknown. By comparing their chromatin-binding profiles and protein partners, we show that SLX/SLXL1 and SLY proteins compete for interaction with H3K4me3-reader SSTY1 (Spermiogenesis-specific-transcript-on-the-Y1) at the promoter of thousands of genes to drive their expression, and that the opposite effect they have on gene expression is mediated by different abilities to recruit SMRT/N-Cor transcriptional complex. Their target genes are predominantly spermatid-specific multicopy genes encoded by the sex chromosomes and the autosomal Speer/Takusan. Many of them have coamplified with not only Slx/Slxl1/Sly but also Ssty during muroid rodent evolution. Overall, we identify Ssty as a key element of the X versus Y intragenomic conflict, which may have influenced gene content and hybrid sterility beyond Mus lineage since Ssty amplification on the Y predated that of Slx/Slxl1/Sly.

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes.

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

Genetic Factors Affecting Sperm Chromatin Structure.

Spermatozoa genome has unique features that make it a fascinating field of investigation: first, because, with oocyte genome, it can be transmitted generation after generation; second, because of genetic shuffling during meiosis, each spermatozoon is virtually unique in terms of genetic content, with consequences for species evolution; and finally, because its chromatin organization is very different from that of somatic cells or oocytes, as it is not based on nucleosomes but on nucleoprotamines which confer a higher order of packaging. Histone-to-protamine transition involves many actors, such as regulators of spermatid gene expression, components of the nuclear envelop, histone-modifying enzymes and readers, chaperones, histone variants, transition proteins, protamines, and certainly many more to be discovered.In this book chapter, we will present what is currently known about sperm chromatin structure and how it is established during spermiogenesis, with the aim to list the genetic factors that regulate its organization.

Rescue of Sly Expression Is Not Sufficient to Rescue Spermiogenic Phenotype of Mice with Deletions of Y Chromosome Long Arm.

Mice with deletions of the Y-specific (non-PAR) region of the mouse Y chromosome long arm (NPYq) have sperm defects and fertility problems that increase proportionally to deletion size. Mice with abrogated function of NPYq-encoded gene Sly (sh367 Sly-KD) display a phenotype similar to that of NPYq deletion mutants but less severe. The milder phenotype can be due to insufficient Sly knockdown, involvement of another NPYq gene, or both. To address this question and to further elucidate the role of Sly in the infertile phenotype of mice with NPYq deletions, we developed an anti-SLY antibody specifically recognizing SLY1 and SLY2 protein isoforms and used it to characterize SLY expression in NPYq- and Sly-deficient mice. We also carried out transgene rescue by adding Sly1/2 transgenes to mice with NPYq deletions. We demonstrated that SLY1/2 expression in mutant mice decreased proportionally to deletion size, with ~12% of SLY1/2 retained in shSLY sh367 testes. The addition of Sly1/2 transgenes to mice with NPYq deletions rescued SLY1/2 expression but did not ameliorate fertility and testicular/spermiogenic defects. Together, the data suggest that Sly deficiency is not the sole underlying cause of the infertile phenotype of mice with NPYq deletions and imply the involvement of another NPYq gene.