ARID1A governs silencing of sex-linked transcription during male meiosis in the mouse [RNA-seq]

We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. The germ cell-specific depletion of ARID1A resulted in a pachynema arrest and failure to repress sex-linked genes indicating a defective MSCI. Consistent with this defect, mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. By investigating potential mechanisms underlying these anomalies, we identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed dramatic shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that does not co-localize with DMC1 (DNA Meiotic Recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences sex chromosome gene regulation and DNA repair during meiosis I. RNA-seq was performed to determine the influence of ARID1A on meiotic transcription during pachynema.RNA-seq was performed in quadruplicate on RNA obtained from frozen pellets of Arid1aWT and Arid1acKO pachytene spermatocytes purified by Sta-Put gravity sedimentation.本研究提供证据表明，BAF（BRG1/BRM相关因子，BRG1/BRM Associated Factor）染色质重塑复合物参与减数分裂性染色体失活（meiotic sex chromosome inactivation，MSCI）。通过免疫荧光（immunofluorescence，IF）实验，我们观察到推定的BAF DNA结合亚基ARID1A（AT富集相互作用结构域1a，AT-rich Interaction Domain 1a）在减数第一次分裂双线期的雄性性染色体上呈现富集现象。生殖细胞特异性敲除ARID1A会导致粗线期阻滞，并无法抑制性连锁基因的表达，提示MSCI存在缺陷。与该缺陷相符，突变型性染色体上出现了异常的延伸型RNA聚合酶II（RNA polymerase II）富集，同时通过ATAC-seq检测到染色质整体可及性升高。为探究上述异常现象的潜在分子机制，我们发现ARID1A可促进组蛋白变体H3.3在性染色体上的优先富集——这是MSCI的已知标志性特征。缺失ARID1A后，性染色体上的H3.3富集水平显著降低，与常染色体相近。通过CUT&RUN技术进行的高分辨率分析显示，在ARID1A缺失后，性染色体上H3.3的结合位点发生了显著变化：从离散的基因间区域和宽泛的基因体结构域转向启动子区域。多个性连锁位点出现了异位的H3.3结合，且这些位点并不与DMC1（DNA减数分裂重组酶1，DNA Meiotic Recombinase 1）共定位。该结果提示，ARID1A对于DMC1定位到未联会的性染色单体是必需的。综上，ARID1A介导的H3.3定位调控可影响减数第一次分裂过程中性染色体的基因表达调控与DNA修复。本研究通过RNA测序（RNA-seq）探究ARID1A对粗线期减数分裂转录的影响。实验采用四次生物学重复，以通过Sta-Put重力沉降法纯化得到的Arid1a野生型（Arid1aWT）与Arid1a条件性敲除（Arid1acKO）粗线期精母细胞的冻存沉淀提取RNA进行测序。