Debra J. Wolgemuth

The double bromodomain and extra-terminal domain (BET) proteins are critical epigenetic readers that bind to acetylated histones in chromatin and regulate transcriptional activity and modulate changes in chromatin structure and organization. The testis-specific BET member, BRDT, is essential for the normal progression of spermatogenesis as mutations in the Brdt gene result in complete male sterility. Although BRDT is expressed in both spermatocytes and spermatids, loss of the first bromodomain of BRDT leads to severe defects in spermiogenesis without overtly compromising meiosis. In contrast, complete loss of BRDT blocks the progression of spermatocytes into the first meiotic division, resulting in a complete absence of post-meiotic cells. Although BRDT has been implicated in chromatin remodeling and mRNA processing during spermiogenesis, little is known about its role in meiotic processes. Here we report that BRDT is an essential regulator of chromatin organization and reprograming during prophase I of meiosis. Loss of BRDT function disrupts the epigenetic state of the meiotic sex chromosome inactivation in spermatocytes, affecting the synapsis and silencing of the X and Y chromosomes. We also found that BRDT controls the global chromatin organization and histone modifications of the chromatin attached to the synaptonemal complex. Furthermore, the homeostasis of crossover formation and localization during pachynema was altered, underlining a possible epigenetic mechanism by which crossovers are regulated and differentially established in mammalian male genomes. Our observations reveal novel findings about the function of BRDT in meiosis and provide insight into how epigenetic regulators modulate the progression of male mammalian meiosis and the formation of haploid gametes. BRDT, a testis-specific member of the bromodomain and extra-terminal (BET) subfamily of epigenetic reader proteins, is essential for the generation of male gametes. In post-meiotic cells, BRDT is involved in chromatin organization and transcriptional regulation through its first bromodomain motif, as loss of the BD1 results in a truncated BRDT protein that fully interrupts the differentiation of the germ cells during the process of spermiogenesis. Complete loss of BRDT function results in an arrest during meiotic prophase with no cells progressing into post-meiotic stages. However, neither the specific role of BRDT in meiosis nor the pathways affected by its depletion are known. We investigated how BRDT controls meiosis by examining its subcellular localization during prophase I as well as the meiotic consequences observed with the loss of BRDT function. BRDT localizes throughout the chromatin of autosomes and sex chromosomes in a dynamic pattern during pachynema and diplonema. Loss of BRDT severely disrupts the epigenetic reprograming and silencing of transcription of the sex chromosomes, the global and regional chromatin configuration, and the formation and localization of crossovers in spermatocytes. Thus, BRDT regulates key meiotic processes that determine the genetic and epigenetic homeostasis of the male gamete.

We have shown that E-type cyclins are key regulators of mammalian male meiosis. Depletion of cyclin E2 reduced fertility in male mice due to meiotic defects, involving abnormal pairing and synapsis, unrepaired DNA, and loss of telomere structure. These defects were exacerbated by additional loss of cyclin E1, and complete absence of both E-type cyclins produces a meiotic catastrophe. Here, we investigated the involvement of E-type cyclins in maintaining telomere integrity in male meiosis. Spermatocytes lacking cyclin E2 and one E1 allele (E1+/-E2-/-) displayed a high rate of telomere abnormalities but can progress to pachytene and diplotene stages. We show that their telomeres exhibited an aberrant DNA damage repair response during pachynema and that the shelterin complex proteins TRF2 and RAP2 were significantly decreased in the proximal telomeres. Moreover, the insufficient level of these proteins correlated with an increase of γ-H2AX foci in the affected telomeres and resulted in telomere associations involving TRF1 and telomere detachment in later prophase-I stages. These results suggest that E-type cyclins are key modulators of telomere integrity during meiosis by, at least in part, maintaining the balance of shelterin complex proteins, and uncover a novel role of E-type cyclins in regulating chromosome structure during male meiosis.

Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis. Understanding the control of meiosis is fundamental to deciphering the origin of male infertility. Although the mechanisms controlling meiosis are poorly understood, key regulators of mitosis, such as cyclins, appear to be critical. In this regard, male mice deficient for cyclin E2 exhibit subfertility and defects in spermatogenesis; however, neither the stages of germ cell differentiation affected nor the responsible mechanisms are known. We investigated how E-type cyclins control male meiosis by examining their expression in spermatogenesis and the consequences that multiple deletions of Ccne1 and Ccne2 alleles produce. Loss of Ccne2 expression increases cyclin E1 levels as a compensatory effect, but there are still meiotic defects and subfertility. Further, loss of one Ccne1 allele in the absence of cyclin E2 results in infertility as does loss of the remaining Ccne1 allele, but with even more severe meiotic abnormalities. We further found that cyclin E1 is involved in sex chromosome synapsis while E2 is involved with homologous pairing and chromosome and telomere integrity. These processes and structures were severely disrupted in absence of both cyclin E1 and E2, uncovering new roles for the E-type cyclins in regulating male meiosis.

The RAD9A-RAD1-HUS1 (9-1-1) complex is a PCNA-like heterotrimeric clamp that binds damaged DNA to promote cell cycle checkpoint signaling and DNA repair. While various 9-1-1 functions in mammalian somatic cells have been established, mounting evidence from lower eukaryotes predicts critical roles in meiotic germ cells as well. This was investigated in 2 recent studies in which the 9-1-1 complex was disrupted specifically in the mouse male germline through conditional deletion of Rad9a or Hus1. Loss of these clamp subunits led to severely impaired fertility and meiotic defects, including faulty DNA double-strand break repair. While 9-1-1 is critical for ATR kinase activation in somatic cells, these studies did not reveal major defects in ATR checkpoint pathway signaling in meiotic cells. Intriguingly, this new work identified separable roles for 9-1-1 subunits, namely RAD9A- and HUS1-independent roles for RAD1. Based on these studies and the high-level expression of the paralogous proteins RAD9B and HUS1B in testis, we propose a model in which multiple alternative 9-1-1 clamps function during mammalian meiosis to ensure genome maintenance in the germline.

Summary In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIβ-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.

Cyclins are key regulators of the mammalian cell cycle, functioning primarily in concert with their catalytic partners, the cyclin-dependent kinases (Cdks). While their function during mitosis in somatic cells has been extensively documented, their function during both mitosis and meiosis in the germ line is poorly understood. From the perspective of cell cycle regulation there are several aspects of mammalian spermatogenesis that suggest unique modes of regulation and hence, possible unique functions for the cyclins. This review will summarize our current understanding of cyclin expression and function in the male germ line, with particular focus on the A and E type cyclins in the mouse model. While the focus is on mammalian spermatogenesis, we note contrasts with similar functions in the female germ line when relevant and also draw upon observations in other model systems to provide further insight.

Cyclin A1 is a male germ cell-specific cell cycle regulator that is essential for spermatogenesis. It is unique among the cyclins by virtue of its highly restricted expression in vivo, being present in pachytene and diplotene spermatocytes and not in earlier or later stages of spermatogenesis. To begin to understand the molecular mechanisms responsible for this narrow window of expression of the mouse cyclin A1 (Ccna1) gene, we carried out a detailed analysis of its promoter. We defined a 170-bp region within the promoter and showed that it is involved in repression of Ccna1 in cultured cells. Within this region we identified known cis-acting transcription factor binding sequences, including an Sp1-binding site and two GATA1-binding sites. Neither Sp1 nor GATA1 is expressed in pachytene spermatocytes and later stages of germ cell differentiation. Sp1 is readily detected at earlier stages of spermatogenesis. Site-directed mutagenesis demonstrated that neither factor alone was sufficient to significantly repress expression driven by the Ccna1 promoter, while concurrent binding of Sp1, and most likely GATA1 and possibly additional factors was inhibitory. Occupancy of Sp1 on the Ccna1 promoter and influence of GATA1-dependent cis-acting elements was confirmed by ChIP analysis in cell lines and most importantly, in spermatogonia. In contrast with many other testis-specific genes, the CpG island methylation status of the Ccna1 promoter was similar among various tissues examined, irrespective of whether Ccna1 was transcriptionally active, suggesting that this regulatory mechanism is not involved in the restricted expression of Ccna1.

Members of the BET (bromodomain and extra terminal motif) family of proteins have been shown to be chromatin-interacting regulators of transcription. We previously generated a mutation in the testis-specific mammalian BET gene Brdt (bromodomain, testis-specific) that yields protein lacking the first bromodomain (BRDT^ΔBD1) and observed disrupted spermiogenesis and male sterility. To determine whether BRDT^ΔBD1 protein results in altered transcription, we analyzed the transcriptomes of control versus Brdt^ΔBD1/ΔBD1 round spermatids. Over 400 genes showed statistically significant differential expression, and among the up-regulated genes, there was an enrichment of RNA splicing genes. Over 60% of these splicing genes had transcripts that lacked truncation of their 3′-untranslated region (UTR) typical of round spermatids. We selected four of these genes to characterize: Srsf2, Ddx5, Hnrnpk and Tardbp. The 3′-UTRs of Srsf2, Ddx5 and Hnrnpk mRNAs were longer in mutant round spermatids and resulted in reduced protein levels. Tardbp was transcriptionally up-regulated and a splicing shift toward the longer variant was observed. All four splicing proteins were found to complex with BRDT in control and mutant testes. We thus suggest that, along with modulating transcription, BRDT modulates gene expression as part of the splicing machinery. These modulations alter 3′-UTR processing in round spermatids; importantly, the BD1 is essential for these functions.