Gcn5-mediated histone acetylation governs nucleosome dynamics in spermiogenesis [RNA-Seq]

During mammalian spermatogenesis, male germ cells undergo dramatic reorganization of chromatin, whereby 90-99% of histones are evicted and replaced by protamines. This reorganization prominently features histone acetylation to loosen chromatin structure. Given the potential role of retained histones in fertility and early embryonic development, the genomic location of retained nucleosomes is of great interest. However, the ultimate position and mechanisms underlying nucleosome eviction/retention are poorly understood, including several studies utilizing MNase-seq methodologies, but reporting remarkably dissimilar locations. Here, we utilized ATAC-seq to determine the location of retained nucleosomes in mouse sperm and found enrichment at promoters, but also retention at inter- and intragenic regions, and repetitive elements. We further investigated nucleosome eviction/retention by generating pre-meiotic, germ cell specific, conditional knockout mice for the histone acetyltransferase Gcn5, a key histone acetyltransferase. Gcn5cKO germ cells exhibited abnormal chromatin dynamics during spermiogenesis, including diminished nucleosome eviction leading to increased histone retention in sperm. These mice exhibited severe reproductive phenotypes: abnormal sperm production, sperm morphology, and ultimately, male infertility. Our findings demonstrate Gcn5 mediated histone acetylation promotes chromatin accessibility and nucleosome eviction in spermiogenesis, and that loss of histone acetylation leads to defects that disrupt male fertility and potentially, early embryogenesis. Includes RNA-seq from four stages of spermatogenesis (meiotic spermatid, round spermatid, elongating spermatid, mature sperm) in three separate genetic backgrounds: SV129E mice (1 replicate each), C57BL6 GCN5+/+ mice (2 replicates each) and C57BL6 GCN5-/- mice (Stra8-CRE homozygous flox'd alleles) (2 replicates each)在哺乳动物精子发生过程中，雄性生殖细胞的染色质（chromatin）会发生剧烈重塑：90%~99%的组蛋白（histone）被移除并替换为鱼精蛋白（protamine）。该重塑过程的显著特征为组蛋白乙酰化（histone acetylation），以松解染色质结构。鉴于滞留组蛋白在生育能力与早期胚胎发育中潜在的关键作用，滞留核小体（retained nucleosome）的基因组定位备受关注。然而，核小体移除与滞留的最终位点及其背后的调控机制仍不甚明确：尽管已有多项研究采用微球菌核酸酶测序（MNase-seq）技术开展相关分析，但得到的定位结果差异显著。本研究通过转座酶可及性测序（ATAC-seq）解析了小鼠精子中滞留核小体的定位特征，发现其不仅富集于启动子（promoter）区域，同时也滞留于基因间区（intergenic region）、基因内区（intragenic region）以及重复元件（repetitive element）区域。为进一步探究核小体的移除与滞留机制，我们构建了减数分裂前生殖细胞特异性条件性敲除小鼠，靶向敲除关键组蛋白乙酰转移酶（histone acetyltransferase）Gcn5。Gcn5条件性敲除（Gcn5cKO）的生殖细胞在精子形成（spermiogenesis）过程中出现染色质动态异常，具体表现为核小体移除能力减弱，进而导致精子中组蛋白滞留量升高。此类敲除小鼠表现出严重的生殖表型：精子生成异常、精子形态异常，最终引发雄性不育。本研究结果证实，Gcn5介导的组蛋白乙酰化可在精子形成过程中促进染色质可及性与核小体移除；而组蛋白乙酰化的缺失会引发相关缺陷，进而破坏雄性生育能力并可能影响早期胚胎发育。本数据集包含三种遗传背景下的RNA测序（RNA-seq）数据，涵盖精子发生的四个阶段：减数分裂精细胞（meiotic spermatid）、圆形精细胞（round spermatid）、伸长型精细胞（elongating spermatid）与成熟精子（mature sperm）；三种遗传背景分别为SV129E小鼠（各1个重复样本）、C57BL6 GCN5+/+小鼠（各2个重复样本）以及C57BL6 GCN5-/-小鼠（即携带Stra8-CRE的纯合loxP侧翼等位基因小鼠，各2个重复样本）