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Title: MOF influences meiotic expansion of H2AX phosphorylation and spermatogenesis in mice}
Article:
Abstract:
Three waves of H2AX phosphorylation( γH2AX) have been observed in male meiotic prophase I: the first is ATM-dependent and occurs at leptonema, while the second and third are ATR-dependent, occuring at zygonema and pachynema, respectively. The third wave of H2AX phosphorylation marks and silences unsynapsed chromosomes. Little is known about H2AX phosphorylation expands to chromatin-wide regions in spermatocytes. Here, we report that histone acetyltransferase( HAT) MOF is involved in all three waves of H2AX phosphorylation expansion. Germ cell-specific deletion of Mof in spermatocytes by Stra8-Cre( Mof cKO) caused global loss of H4K16ac. In leptotene and zygotene spermatocytes of cKO mice, the γH2AX signals were observed only along the chromosomal axes, and chromatin-wide H2AX phosphorylation was lost. In almost 40% of early-mid pachytene spermatocytes from Mof cKO mice, γH2AX and MDC1 were detected along the unsynapsed axes of the sex chromosomes, but failed to expand, which consequently caused meiotic sex chromosome inactivation( MSCI) failure. Furthermore, though RAD51 was proficiently recruited to double-strand break( DSB) sites, defects in DSB repair and crossover formation were observed in Mof cKO spermatocytes, indicating that MOF facilitates meiotic DSB repair after RAD51 recruitment. We propose that MOF regulates male meiosis and is involved in the expansion of all three waves of H2AX phosphorylation from the leptotene to pachytene stages, initiated by ATM and ATR, respectively.
Introduction:
Meiosis, the process that generates haploid daughter cells, is critical for sexual reproduction in eukaryotes. The key to normal meiotic progression is the fidelity of chromosome interactions, during which homologous chromosomes undergo pairing, synapsis, recombination and proper segregation. The formation and repair of programmed DNA double strand breaks( DSBs) are essential for chromosome dynamics in meiotic prophase I. These DSBs are repaired through two pathways, a synthesis-dependent strand annealing( SDSA) pathway to form non-crossover products and the classical pathway through double Holiday Junction( dHJ) formation to create both crossover and non-crossover products[1, 2]. The DSBs that persist until pachynema are more likely to be repaired through the classical pathway to generate crossovers[3]. The mismatch repair complex MLH1-MLH3 is required for the resolution of dHJs to form the class I crossovers that account for approximately 90% of crossovers in mice[4]. The dynamics of γH2AX distribution is one of the major characteristics of meiosis and is commonly used as a marker for meiotic progression. In somatic cells, the phosphorylation of H2AX after DSB formation is first initiated by ATM and/or DNA-PK[5], then expanded by the recruitment of MDC1, which is facilitated by MOF mediated H4K16ac[6–8]. Despite its well-known role in the DNA damage response( DDR), many reports highlight that γH2AX should not be considered simply as a DSB marker[9, 10]. In mammalian meiotic prophase I, three waves of H2AX phosphorylation can be observed, all of which are initiated and expanded through pathways that are different from those in somatic cells. During leptonema, γH2AX signals are initiated solely by ATM in a DSB-dependent manner and are distributed throughout the nucleus. This wave of γH2AX signals can be found in wild type( WT) and Atr knockout mice[11], but not in mice deficient for Atm or Spo11[12]. The second wave is initiated by ATR at DSBs. This wave of H2AX phosphorylation can be observed in Atm-deficient spermatocytes at early-mid zygonema and is lost when Spo11 or Atr is deleted[11–13]. The third wave is also mediated by ATR and marks chromatin associated with unsynapsed axes at the zygotene/pachytene transition[14, 15]. Unlike the first two waves of H2AX phosphorylation expansion at leptonema and zygonema, the third wave can be observed in Spo11-deficient spermatocytes[12]. This wave is responsible for meiotic silencing of unsynapsed chromosomes( MSUC), and occurs on the unpaired regions of the sex chromosomes, resulting in meiotic sex chromosome inactivation( MSCI) and formation of the sex body[16]. Mutations causing defective expansion of H2AX phosphorylation in pachytene spermatocytes lead to MSCI failure. This triggers the MSCI checkpoint before late pachynema, which subsequently leads to apoptosis of pachytene spermatocytes[16]. This later wave of H2AX phosphorylation is reported to be initiated by the localization of BRCA1 to the unsynapsed axes[17], which recruits ATR for H2AX phosphorylation and MDC1 for γH2AX signal expansion[11, 18]. MDC1 also contributes to the DDR in somatic cells[7, 19]. However, spreading of H2AX phosphorylation in leptotene and zygotene spermatocytes is unaltered in Mdc1-/- spermatocytes[18], and is therefore regulated by unknown mechanisms. MOF is a major HAT for histone H4 lysine 16( H4K16) in somatic cells, and belongs to the MYST family of acetyltransferases[20]. Unlike most histone modifications, acetylation of H4K16 is unique in regulating higher order chromatin structures beyond the level of nucleosomes, and removes the natural barrier to damaged DNA sites to facilitate repair protein access to DNA breaks[21, 22]. Consistent with this, MOF is essential for the recruitment of MDC1[23], a key step in the expansion of H2AX phosphorylation after DSB formation in somatic cells. It has also been reported that MOF is indispensable for the recruitment of BRCA1 and 53BP1 to damage sites after DSB formation[23]. In addition to its known function in recruitment of DDR factors, MOF is essential during DSB repair. In somatic cells, Mof knockout results in persistent DSBs and reduces homologous recombination( HR) efficiency, which can be rescued by HDAC inhibition[23, 24], demonstrating a crucial role for H4K16ac in HR. H4K16ac is also detected in meiotic cells, and deacetylation of H4K16ac during oocyte maturation is critical for proper chromosome segregation[25]. However, the meiotic function of MOF and H4K16ac in male germ cells has not been reported. In this study, we generated male mice with germ cell-specific Mof deletion mediated by Stra8-Cre( Mof cKO mice). We show that MOF is involved in the expansion of chromatin-wide H2AX phosphorylation in leptonema and zygonema. MDC1 recruitment and MSCI are also disturbed in Mof deleted pachytene spermatocytes, with absence of XY-H2AX phosphorylation expansion, which may partly contribute to the germ cell loss observed in Mof cKO mice. RAD51 foci persist in Mof cKO spermatocytes until diplonema and are associated with a significant reduction in crossover markers and chiasma formation. Together, these data indicate that the expansion of different waves of H2AX phosphorylation, efficient DSB repair, and the formation of crossovers during male meiosis are affected by MOF, thus highlighting novel roles for this protein.
Discussion:
As a major HAT for H4K16, MOF is well known for its functional role in chromatin remodelling, the DDR and DSB repair. Previous work on HDACs found that H4K16ac deacetylation is critical for proper chromosome segregation and kinetochore function[25]. Mof has been found to be indispensable for embryonic stem cell development and its deficiency results in mouse embryonic lethality[35, 36]. Our recent study also revealed a role for MOF and H4K16ac in oocyte development[37]. However, Mof function in male meiotic cells, especially in the three waves of meiosis-specific H2AX phosphorylation expansion and programmed DSB repair, has not been studied. In this study, we report that Mof deletion in spermatocytes using Stra8-Cre causes global loss of H4K16ac as well as defective spermatogenesis. In Mof cKO mice, the γH2AX signal failed to expand chromatin-wide in leptotene and zygotene spermatocytes, and to the sex body region in about 40% of early-mid pachytene spermatocytes. We also observed persistent RAD51 foci, as well as a significant reduction in crossover number, in Mof cKO spermatocytes, consistent with a defect in chiasma formation in Mof cKO mice. To be noted, we also observed some tubules with no spermatocytes in cKO testes, which may due to the loss of Mof in some spermatogonia, and consequently cause germ cell loss before the initiation of meiosis. Indeed, we did find that Mof specific deletion in spermatogonia leads to completely loss of differentiated spermatogonia( S11 Fig). Our study demonstrates for the first time that one major function of Mof in meiotic prophase I is to facilitate the three waves of H2AX phosphorylation expansion. Mof deletion blocked the chromatin-wide expansion of H2AX phosphorylation in most leptotene spermatocytes. However, γH2AX foci could still be observed, indicating that Mof is not required for the initiation of H2AX phosphorylation at this stage( Fig 4). A similar pattern of γH2AX staining was observed in Mof cKO zygotene spermatocytes( Fig 4). We suggest that MOF is involved in the expansion, but not initiation, of the first two waves of H2AX phosphorylation. These two waves of chromatin-wide H2AX phosphorylation expansion are preserved in Mdc1-deficient mice[18] and thus MDC1-independent. The third wave of H2AX phosphorylation expansion is well known for its specific localization and essential role in pachytene spermatocytes. This wave is responsible for MSCI[13]. During the zygotene/pachytene transition, MSCI is initiated by the recruitment of BRCA1 to the axes of Synaptonemal Complexes( SCs)[18, 38, 39], which consequently recruits ATR and MDC1, leading to phosphorylation and expansion of H2AX[18]. We found that MOF influences the expansion of H2AX phosphorylation during pachynema by recruiting MDC1 to the sex chromosomes. Notably, some Mof cKO pachytene spermatocytes exhibit normal γH2AX / MDC1 recruitment, suggesting that Mof deletion does not occur in all germ cells. We suspect that some cells that complete pachynema are those with residual MOF. This possibility is supported by the finding that a comparably higher rate of positive H4K16ac staining in surviving Mof cKO diplotene cells compared with pachytene cells( S2C and S2D Fig). The rest of the cells that complete pachynema may come from those pachytene cells with MDC1 recruitment and the expansion of H2AX phosphorylation despite the loss of MOF. This possibility is supported by the fact that H3K4me3, RNA Pol II( S12 Fig) and MDC1( S13 Fig) staining are similar to controls in the surviving Mof cKO diplotene cells, most of which are H4K16ac negative( S2D Fig). Though H2AX phosphorylation has been reported to facilitate early DSB repair, especially the recruitment of RAD51, we found no defects in these processes in Mof cKO spermatocytes( S7 Fig). This result might be explained by the observation that formation of γH2AX foci remains undisturbed in leptotene and zygotene spermatocytes after Mof deletion. Notably, at pachynema more RAD51 and DMC1 foci were observed in Mof cKO spermatocytes than in controls, suggesting a defect in HR after strand invasion. This is consistent with the decreased number of crossovers in Mof cKO spermatocytes, indicating that MOF may participate in processing HR intermediates. One possible mechanism could be via migration of dHJs. It has been reported that H3K4me3 mediated by PRDM9 may facilitate the dHJ migration by creating an extended nucleosome-depleted region( NDR), which restricts the localization of crossovers[40, 41]. MOF can recognize H3K4me3 by its chromodomain[42, 43], raising the possibility that MOF may recognize the H3K4me3 sites modified by PRDM9[41], and initiate H4K16ac around that region to stabilize the NDR. Despite their importance in expansion of H2AX phosphorylation, Mof and H4K16ac are present at comparably low levels at leptonema and zygonema. We suggest that low levels of Mof and H4K16ac during these stages is sufficient to ensure accessibility of DDR factors, as observed in somatic cells[23, 42, 44], and that higher levels are required at pachynema for DSB repair after stand invasion.
Phosphorylation of H2AX is a well-established step in the DNA damage response of somatic cells , and also occurs in germ cells during meiosis . During spermatogenesis , H2AX phosphorylation regulates the formation and repair of programmed DSBs and meiotic silencing of unpaired chromosomes . A critical step in these processes is spreading of H2AX phosphorylation along the chromosomes beyond the initiation site , which occurs by a poorly characterized mechanism . Here , we report that MOF is involved in the proper expansion of H2AX phosphorylation during meiosis . MOF also influences meiotic sex chromosome inactivation by facilitating early pachytene recruitment of MDC1 , but is dispensable for BRCA1 or ATR recruitment to axial elements . Furthermore , MOF is involved in proper meiotic recombination and male germ cell development . As well as identifying a new MOF-dependent step for regulating expansion of H2AX phosphorylation , these findings have broad implications for the role of epigenetic modifications in meiosis and human reproductive health .