Paternal histone H2A variant incorporation and function before and after fertilization in C. elegans [RNA-seq]

During spermatogenesis, chromatin repackaging transforms germ cells into sperm and transmits paternal epigenetic information to embryos. We have found distinct functions in these processes for two C. elegans H2A variants, HTAS-1, a sperm-specific paralog, and HTZ-1, a conserved H2A.Z homolog. HTAS-1 activates and represses gene expression less than two-fold, indicating HTAS-1 does not globally silence genes. In contrast, during spermatogenesis HTZ-1 regulates different germline genes compared to HTAS-1. During oogenesis, HTZ-1 strongly represses sperm gene expression over six-fold and activates oocyte and sex-independent genes, revealing tissue-specific modes of HTZ-1 regulation. ChIP-seq analysis of sperm chromatin shows HTZ-1 is present in 75% of HTAS-1 targets. HTZ-1 is incorporated at promoters of targets not occupied by HTAS-1 but is depleted in promoters of genes also occupied by HTAS-1. Instead HTAS-1 and HTZ-1 incorporate within gene bodies of shared targets. Post- fertilization, paternally-contributed HTAS-1 regulates the expression of a large number of genes in mixed-staged embryos. Correspondingly, loss of HTAS-1 causes decreased fertility and delayed development. Our studies indicate H2A variants have distinct incorporation mechanisms during sperm formation and tissue-specific functions in gene expression and fertility. We performed short-read RNA-sequencing on HTZ-1 and HTAS-1 knockouts and wildtype controls. For HTAS-1, we examined knockouts vs wildtype expression in male gonads and mixed embryos. For HTZ-1, we compared expression between wildtypes and knockouts in male and hermaphrodite germlines.

在精子发生过程中，染色质重包装会将生殖细胞转化为精子，并将父本表观遗传信息传递至胚胎。我们发现秀丽隐杆线虫（C. elegans）的两种组蛋白H2A变体在该过程中具备独特功能：其一为精子特异性旁系同源蛋白HTAS-1，其二为保守的H2A.Z同源蛋白HTZ-1。HTAS-1对基因表达的激活与抑制幅度均不足两倍，这表明HTAS-1并不会全局性沉默基因。与之相反，在精子发生过程中，HTZ-1所调控的生殖系基因与HTAS-1存在显著差异。在卵子发生过程中，HTZ-1可将精子相关基因的表达抑制六倍以上，同时激活卵母细胞相关基因与性别非依赖型基因，这揭示了HTZ-1调控模式的组织特异性。对精子染色质开展的染色质免疫共沉淀测序（ChIP-seq）分析显示，HTZ-1结合于75%的HTAS-1靶基因位点。HTZ-1会被整合至未被HTAS-1结合的靶基因启动子区域，但在同时被HTAS-1结合的基因启动子区域中则呈现耗竭状态。与之相反，HTAS-1与HTZ-1均会整合至共有靶基因的基因本体区域内。受精后，父本来源的HTAS-1可调控多阶段混合胚胎中大量基因的表达。相应地，HTAS-1功能缺失会导致生育能力下降以及发育延迟。本研究表明，组蛋白H2A变体在精子形成过程中具备独特的整合机制，并在基因表达调控与生育能力方面发挥组织特异性功能。我们对HTZ-1与HTAS-1敲除样本及野生型对照样本开展了短读长RNA测序（short-read RNA-sequencing）。针对HTAS-1，我们检测了雄性性腺与混合胚胎中敲除样本与野生型样本的基因表达差异。针对HTZ-1，我们比较了雄性与雌雄同体生殖系中野生型样本与敲除样本的基因表达水平。