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.

Return of the forgotten hero: the role of Y chromosome-encoded Zfy in male reproduction.

The Y-linked zinc finger gene ZFY is conserved across eutherians and is known to be a critical fertility factor in some species. The initial studies of the mouse homologues, Zfy1 and Zfy2, were performed using mice with spontaneous Y chromosome mutations and Zfy transgenes. These studies revealed that Zfy is involved in multiple processes during spermatogenesis, including removal of germ cells with unpaired chromosomes and control of meiotic sex chromosome inactivation during meiosis I, facilitating the progress of meiosis II, promoting spermiogenesis, and improving assisted reproduction outcomes. Zfy was also identified as a key gene in Y chromosome evolution, protecting this chromosome from extinction by serving as the executioner responsible for meiosis surveillance. Studies with targeted Zfy knock-outs revealed that mice lacking both homologues have severe spermatogenic defects and are infertile. Based on protein structure and in vitro assays, Zfy is expected to drive spermatogenesis as a transcriptional regulator. The combined evidence documents that the presence of at least one Zfy homologue is required for male fertility and that Zfy2 plays a more prominent role. This knowledge reinforces the importance of these factors for mouse spermatogenesis and informs our understanding of the human ZFY variants, which are homologous to the mouse Zfy1 and Zfy2.

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.

Correction: A Genetic Basis for a Postmeiotic X Versus Y Chromosome Intragenomic Conflict in the Mouse.

[This corrects the article DOI: 10.1371/journal.pgen.1002900.].

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.

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.

Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging.

In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with extensive deletions of non-pairing Y chromosome long arm (NPYq) are infertile and produce sperm with grossly misshapen heads, abnormal chromatin packaging and DNA damage. The NPYq-encoded multi-copy gene Sly controls the expression of sex chromosome genes after meiosis and Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has been shown to be the underlying cause of the sperm head anomalies and infertility associated with NPYq gene loss, but it was not known whether it recapitulates sperm DNA damage phenotype. We produced and examined mice with transgenically (RNAi) silenced Sly and demonstrated that these mice have increased incidence of sperm with DNA damage and poorly condensed and insufficiently protaminated chromatin. We also investigated the contribution of each of the two Sly-encoded transcript variants and noted that the phenotype was only observed when both variants were knocked down, and that the phenotype was intermediate in severity compared with mice with severe NPYq deficiency. Our data demonstrate that Sly deficiency is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions and point to SLY proteins involvement in chromatin reprogramming during spermiogenesis, probably through their effect on the post-meiotic expression of spermiogenic genes. Considering the importance of the sperm epigenome for embryonic and fetal development and the possibility of its inter-generational transmission, our results are important for future investigations of the molecular mechanisms of this biologically and clinically important process.

A genetic basis for a postmeiotic X versus Y chromosome intragenomic conflict in the mouse.

Intragenomic conflicts arise when a genetic element favours its own transmission to the detriment of others. Conflicts over sex chromosome transmission are expected to have influenced genome structure, gene regulation, and speciation. In the mouse, the existence of an intragenomic conflict between X- and Y-linked multicopy genes has long been suggested but never demonstrated. The Y-encoded multicopy gene Sly has been shown to have a predominant role in the epigenetic repression of post meiotic sex chromatin (PMSC) and, as such, represses X and Y genes, among which are its X-linked homologs Slx and Slxl1. Here, we produced mice that are deficient for both Sly and Slx/Slxl1 and observed that Slx/Slxl1 has an opposite role to that of Sly, in that it stimulates XY gene expression in spermatids. Slx/Slxl1 deficiency rescues the sperm differentiation defects and near sterility caused by Sly deficiency and vice versa. Slx/Slxl1 deficiency also causes a sex ratio distortion towards the production of male offspring that is corrected by Sly deficiency. All in all, our data show that Slx/Slxl1 and Sly have antagonistic effects during sperm differentiation and are involved in a postmeiotic intragenomic conflict that causes segregation distortion and male sterility. This is undoubtedly what drove the massive gene amplification on the mouse X and Y chromosomes. It may also be at the basis of cases of F1 male hybrid sterility where the balance between Slx/Slxl1 and Sly copy number, and therefore expression, is disrupted. To the best of our knowledge, our work is the first demonstration of a competition occurring between X and Y related genes in mammals. It also provides a biological basis for the concept that intragenomic conflict is an important evolutionary force which impacts on gene expression, genome structure, and speciation.

Generation and Characterization of a Transgenic Mouse That Specifically Expresses the Cre Recombinase in Spermatids.

Spermiogenesis is the step during which post-meiotic cells, called spermatids, undergo numerous morphological changes and differentiate into spermatozoa. Thousands of genes have been described to be expressed at this stage and could contribute to spermatid differentiation. Genetically-engineered mouse models using Cre/LoxP or CrispR/Cas9 are the favored approaches to characterize gene function and better understand the genetic basis of male infertility. In the present study, we produced a new spermatid-specific Cre transgenic mouse line, in which the improved iCre recombinase is expressed under the control of the acrosomal vesicle protein 1 gene promoter (Acrv1-iCre). We show that Cre protein expression is restricted to the testis and only detected in round spermatids of stage V to VIII seminiferous tubules. The Acrv1-iCre line can conditionally knockout a gene during spermiogenesis with a > 95% efficiency. Therefore, it could be useful to unravel the function of genes during the late stage of spermatogenesis, but it can also be used to produce an embryo with a paternally deleted allele without causing early spermatogenesis defects.

The multicopy gene Sly represses the sex chromosomes in the male mouse germline after meiosis.

Studies of mice with Y chromosome long arm deficiencies suggest that the male-specific region (MSYq) encodes information required for sperm differentiation and postmeiotic sex chromatin repression (PSCR). Several genes have been identified on MSYq, but because they are present in more than 40 copies each, their functions cannot be investigated using traditional gene targeting. Here, we generate transgenic mice producing small interfering RNAs that specifically target the transcripts of the MSYq-encoded multicopy gene Sly (Sycp3-like Y-linked). Microarray analyses performed on these Sly-deficient males and on MSYq-deficient males show a remarkable up-regulation of sex chromosome genes in spermatids. SLY protein colocalizes with the X and Y chromatin in spermatids of normal males, and Sly deficiency leads to defective repressive marks on the sex chromatin, such as reduced levels of the heterochromatin protein CBX1 and of histone H3 methylated at lysine 9. Sly-deficient mice, just like MSYq-deficient mice, have severe impairment of sperm differentiation and are near sterile. We propose that their spermiogenesis phenotype is a consequence of the change in spermatid gene expression following Sly deficiency. To our knowledge, this is the first successful targeted disruption of the function of a multicopy gene (or of any Y gene). It shows that SLY has a predominant role in PSCR, either via direct interaction with the spermatid sex chromatin or via interaction with sex chromatin protein partners. Sly deficiency is the major underlying cause of the spectrum of anomalies identified 17 y ago in MSYq-deficient males. Our results also suggest that the expansion of sex-linked spermatid-expressed genes in mouse is a consequence of the enhancement of PSCR that accompanies Sly amplification.

Testicular germ cell-specific lncRNA, Teshl, is required for complete expression of Y chromosome genes and a normal offspring sex ratio.

Heat shock factor 2 (HSF2) regulates the transcription of the male-specific region of the mouse Y chromosome long arm (MSYq) multicopy genes only in testes, but the molecular mechanism underlying this tissue specificity remains largely unknown. Here, we report that the testicular germ cell-specific long noncoding RNA (lncRNA), NR_038002, displays a characteristic spatiotemporal expression pattern in the nuclei of round and elongating spermatids. NR_038002-knockout male mice produced sperm with abnormal head morphology and exhibited reduced fertility accompanied by a female-biased sex ratio in offspring. Molecular analyses revealed that NR_038002 interacts with HSF2 and thereby activates expression of the MSYq genes. We designate NR_038002 as testicular germ cell-specific HSF2-interacting lncRNA (Teshl). Together, our study is the first to demonstrate that the testis specificity of HSF2 activity is regulated by the lncRNA Teshl and establishes a Teshl-HSF2-MSYq molecular axis for normal Y-bearing sperm qualities and consequent balanced offspring sex ratio.

LINE-1 transcription in round spermatids is associated with accretion of 5-carboxylcytosine in their open reading frames.

Chromatin of male and female gametes undergoes a number of reprogramming events during the transition from germ cell to embryonic developmental programs. Although the rearrangement of DNA methylation patterns occurring in the zygote has been extensively characterized, little is known about the dynamics of DNA modifications during spermatid maturation. Here, we demonstrate that the dynamics of 5-carboxylcytosine (5caC) correlate with active transcription of LINE-1 retroelements during murine spermiogenesis. We show that the open reading frames of active and evolutionary young LINE-1s are 5caC-enriched in round spermatids and 5caC is eliminated from LINE-1s and spermiogenesis-specific genes during spermatid maturation, being simultaneously retained at promoters and introns of developmental genes. Our results reveal an association of 5caC with activity of LINE-1 retrotransposons suggesting a potential direct role for this DNA modification in fine regulation of their transcription.

Molecular Mechanisms of Trophoblast Dysfunction Mediated by Imbalance between STOX1 Isoforms.

STOX1 is a transcription factor involved in preeclampsia and Alzheimer disease. We show that the knock-down of the gene induces rather mild effect on gene expression in trophoblast cell lines (BeWo). We identified binding sites of STOX1 shared by the two major isoforms, STOX1A and STOX1B. Profiling gene expression of cells overexpressing either STOX1A or STOX1B, we identified genes downregulated by both isoforms, with a STOX1 binding site in their promoters. Among those, STOX1-induced Annexin A1 downregulation led to abolished membrane repair in BeWo cells. By contrast, overexpression of STOX1A or B has opposite effects on trophoblast fusion (acceleration and inhibition, respectively) accompanied by syncytin genes deregulation. Also, STOX1A overexpression led to abnormal regulation of oxidative and nitrosative stress. In sum, our work shows that STOX1 isoform imbalance is a cause of gene expression deregulation in the trophoblast, possibly leading to placental dysfunction and preeclampsia.

The multi-copy mouse gene Sycp3-like Y-linked (Sly) encodes an abundant spermatid protein that interacts with a histone acetyltransferase and an acrosomal protein.

Deletion analysis has established that genes on the Y chromosome are essential for normal sperm production in humans, mice, and Drosophila. In mice, long-arm deletions have an impact on spermiogenesis, with the most extensive deletions resulting in severe sperm head malformations and infertility. Intriguingly, smaller deletions are compatible with fertility but result in a distorted sex ratio in favor of females, and recently it was found that Y long-arm deletions are also associated with a marked upregulation of several X-encoded and Y-encoded spermatid-expressed genes. The mouse Y long arm encodes a number of distinct transcripts, each of which derives from multiple gene copies. Of these multicopy genes, the recently described Sly has been favored as the gene underlying the spermiogenic defects associated with Y long-arm deletions. To assess the candidacy of Sly, the expression of this gene was examined in the testis at the transcript and protein levels. Sly is transcribed after the first meiotic division in secondary spermatocytes and round spermatids and encodes two transcript variants, Sly_v1 and Sly_v2 (proteins referred to as SLY1 and SLY2). We raised an antibody against SLY1 which detected the protein in round and early elongating spermatids, where it is predominantly cytoplasmic. Yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLY1 interacts with the acrosomal protein DKKL1, the histone acetyltransferase KAT5 (also known as TIP60), and the microtubule-associated protein APPBP2. Together, these data suggest SLY1 may be involved in multiple processes during spermiogenesis, including the control of gene expression and the development or function of the acrosome.

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.

Differential Sperm Motility Mediates the Sex Ratio Drive Shaping Mouse Sex Chromosome Evolution.

The mouse sex chromosomes exhibit an extraordinary level of copy number amplification of postmeiotically expressed genes [1, 2], driven by an "arms race" (genomic conflict) between the X and Y chromosomes over the control of offspring sex ratio. The sex-linked ampliconic transcriptional regulators Slx and Sly [3-7] have opposing effects on global transcription levels of the sex chromosomes in haploid spermatids via regulation of postmeiotic sex chromatin (PMSC) [8-11] and opposing effects on offspring sex ratio. Partial deletions of the Y chromosome (Yq) that reduce Sly copy number lead to global overexpression of sex-linked genes in spermatids and either a distorted sex ratio in favor of females (smaller deletions) or sterility (larger deletions) [12-16]. Despite a large body of work studying the role of the sex chromosomes in regulating spermatogenesis (recent reviews [17-20]), most studies do not address differential fertility effects on X- and Y-bearing cells. Hence, in this study, we concentrate on identifying physiological differences between X- and Y-bearing sperm from Yq-deleted males that affect their relative fertilizing ability and consequently lead to sex ratio skewing. We show that X- and Y-bearing sperm in these males have differential motility and morphology but are equally able to penetrate the cumulus and fertilize the egg once at the site of fertilization. The altered motility is thus deduced to be the proximate cause of the skew. This represents the first demonstration of a specific difference in sperm function associated with sex ratio skewing.

A Decade of Exploring the Mammalian Sperm Epigenome: Paternal Epigenetic and Transgenerational Inheritance.

The past decade has seen a tremendous increase in interest and progress in the field of sperm epigenetics. Studies have shown that chromatin regulation during male germline development is multiple and complex, and that the spermatozoon possesses a unique epigenome. Its DNA methylation profile, DNA-associated proteins, nucleo-protamine distribution pattern and non-coding RNA set up a unique epigenetic landscape which is delivered, along with its haploid genome, to the oocyte upon fertilization, and therefore can contribute to embryogenesis and to the offspring health. An emerging body of compelling data demonstrates that environmental exposures and paternal lifestyle can change the sperm epigenome and, consequently, may affect both the embryonic developmental program and the health of future generations. This short review will attempt to provide an overview of what is currently known about sperm epigenome and the existence of transgenerational epigenetic inheritance of paternally acquired traits that may contribute to the offspring phenotype.

Systematic quantitative analysis of H2A and H2B variants by targeted proteomics.

BACKGROUND: Histones organize DNA into chromatin through a variety of processes. Among them, a vast diversity of histone variants can be incorporated into chromatin and finely modulate its organization and functionality. Classically, the study of histone variants has largely relied on antibody-based assays. However, antibodies have a limited efficiency to discriminate between highly similar histone variants. RESULTS: In this study, we established a mass spectrometry-based analysis to address this challenge. We developed a targeted proteomics method, using selected reaction monitoring or parallel reaction monitoring, to quantify a maximum number of histone variants in a single multiplexed assay, even when histones are present in a crude extract. This strategy was developed on H2A and H2B variants, using 55 peptides corresponding to 25 different histone sequences, among which a few differ by a single amino acid. The methodology was then applied to mouse testis extracts in which almost all histone variants are expressed. It confirmed the abundance profiles of several testis-specific histones during successive stages of spermatogenesis and the existence of predicted H2A.L.1 isoforms. This methodology was also used to explore the over-expression pattern of H2A.L.1 isoforms in a mouse model of male infertility. CONCLUSIONS: Our results demonstrate that targeted proteomics is a powerful method to quantify highly similar histone variants and isoforms. The developed method can be easily transposed to the study of human histone variants, whose abundance can be deregulated in various diseases.

FOXL2 in the pituitary: molecular, genetic, and developmental analysis.

FOXL2 is a forkhead transcription factor expressed in the eye, ovary, and pituitary gland. Loss of function mutations in humans and mice confirm a functional role for FOXL2 in the eye and ovary, but its role in the pituitary is not yet defined. We report that FOXL2 colocalizes with the glycoprotein hormone alpha-subunit (alphaGSU) in quiescent cells of the mouse pituitary from embryonic d 11.5 through adulthood. FOXL2 is expressed in essentially all gonadotropes and thyrotropes and a small fraction of prolactin-containing cells during pregnancy, but not somatotropes or corticotropes. The coincident expression patterns of FOXL2 and alphaGSU suggested that the alphaGSU gene (Cga) is a downstream target of FOXL2. We demonstrate that FOXL2 regulates mouse Cga transcription in gonadotrope-derived (alphaT3-1, LbetaT2), thyrotrope-derived (alphaTSH) and heterologous (CV-1) cells in a context-dependent manner. In addition, a FOXL2-VP16 fusion protein is sufficient to stimulate ectopic Cga expression in transgenic animals. Normal FOXL2 expression requires the transcription factors Lhx3 and Lhx4 but not of Prop1. Thus, FOXL2 expression is affected by mutations in early pituitary developmental regulatory genes, and its expression precedes that of genes necessary for gonadotrope-specific development such as Egr1 and Sf1 (Nr5a1). These data place FOXL2 in the hierarchy of pituitary developmental control and suggest a role in regulation of Cga gene expression.

SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation.

Sperm differentiation requires unique transcriptional regulation and chromatin remodeling after meiosis to ensure proper compaction and protection of the paternal genome. Abnormal sperm chromatin remodeling can induce sperm DNA damage, embryo lethality and male infertility, yet, little is known about the factors which regulate this process. Deficiency in Sly, a mouse Y chromosome-encoded gene expressed only in postmeiotic male germ cells, has been shown to result in the deregulation of hundreds of sex chromosome-encoded genes associated with multiple sperm differentiation defects and subsequent male infertility. The underlying mechanism remained, to date, unknown. Here, we show that SLY binds to the promoter of sex chromosome-encoded and autosomal genes highly expressed postmeiotically and involved in chromatin regulation. Specifically, we demonstrate that Sly knockdown directly induces the deregulation of sex chromosome-encoded H2A variants and of the H3K79 methyltransferase DOT1L. The modifications prompted by loss of Sly alter the postmeiotic chromatin structure and ultimately result in abnormal sperm chromatin remodeling with negative consequences on the sperm genome integrity. Altogether our results show that SLY is a regulator of sperm chromatin remodeling. Finally we identified that SMRT/N-CoR repressor complex is involved in gene regulation during sperm differentiation since members of this complex, in particular TBL1XR1, interact with SLY in postmeiotic male germ cells.