KDM6A/UTX promotes spermatogenic gene expression across generations but is dispensable for male fertility

Paternal chromatin undergoes extensive structural and epigenetic changes during mammalian spermatogenesis, producing sperm with an epigenome optimized for the transition to embryogenesis. Lysine demethylase 6a (KDM6A, also called UTX) promotes gene activation in part via demethylation of H3K27me3, a developmentally important repressive modification abundant throughout the epigenome of spermatogenic cells and sperm. We previously demonstrated increased cancer risk in genetically wild type mice derived from a paternal germ line lacking Kdm6a (Kdm6a cKO), indicating a role for KDM6A in regulating heritable epigenetic states. However, the regulatory function of KDM6A during spermatogenesis is not known. Here, we show that Kdm6a is transiently expressed in spermatogenesis, with RNA and protein expression largely limited to late spermatogonia and early meiotic prophase. Kdm6a cKO males do not have defects in fertility or the overall progression of spermatogenesis. However, hundreds of genes are deregulated upon loss of Kdm6a in spermatogenic cells, with a strong bias towards downregulation coinciding with the time when Kdm6a is expressed. Misregulated genes encode factors involved in chromatin organization and regulation of repetitive elements, and a subset of these genes was persistently deregulated in the male germ line across two generations of offspring of Kdm6a cKO males. Genome-wide epigenetic profiling revealed broadening of H3K27me3 peaks in differentiating spermatogonia of Kdm6a cKO mice, suggesting that KDM6A demarcates H3K27me3 domains in the male germ line. Our findings highlight KDM6A as a transcriptional activator in the mammalian male germ line that is dispensable for spermatogenesis but important for safeguarding gene regulatory state intergenerationally. bulk RNA-seq, single cell RNA-seq, ATAC-seq, and CUT&Tag for H3K27me3 in control and Kdm6a knockout spermatogenic cells and testis哺乳动物精子发生过程中，父本染色质会经历广泛的结构与表观遗传重塑，最终产生表观基因组适配胚胎发生过渡的成熟精子。赖氨酸去甲基化酶6a（Lysine demethylase 6a, KDM6A，又称UTX）可通过对组蛋白H3第27位赖氨酸三甲基化（H3K27me3）的去甲基化作用部分促进基因激活；H3K27me3是一种发育过程中至关重要的抑制性修饰，在生精细胞与精子的表观基因组中广泛富集。我们此前的研究证实，源自缺失Kdm6a的父本生殖细胞系（Kdm6a条件性敲除，Kdm6a cKO）的遗传野生型小鼠癌症风险升高，表明KDM6A在调控可遗传表观遗传状态中发挥作用。然而，目前尚不清楚KDM6A在精子发生过程中的调控功能。本研究发现，Kdm6a在精子发生中呈瞬时表达特征，其RNA与蛋白质表达主要局限于晚期精原细胞与减数分裂早前期。Kdm6a cKO雄性小鼠的生育能力与精子发生整体进程均未出现异常。然而，生精细胞中Kdm6a缺失会导致数百个基因表达失调，且表达失调以基因下调为主，该趋势恰好与Kdm6a的表达时段相吻合。失调基因所编码的蛋白因子参与染色质组织与重复序列调控，其中部分基因在Kdm6a cKO雄性小鼠的两代子代的雄性生殖系中持续存在表达失调。全基因组表观遗传谱分析显示，Kdm6a cKO小鼠的分化型精原细胞中H3K27me3峰域出现扩展，提示KDM6A可在雄性生殖系中界定H3K27me3结构域。本研究结果表明，KDM6A是哺乳动物雄性生殖系中的转录激活因子：其对精子发生并非必需，但可保障代际间基因调控状态的稳定。本研究采用的实验技术包括：针对对照与Kdm6a敲除生精细胞、睾丸样本的批量RNA测序（bulk RNA-seq）、单细胞RNA测序（single cell RNA-seq）、转座酶可及性测序（ATAC-seq），以及针对H3K27me3的切割与标签测序（CUT&Tag）