运用ATAC-seq技术分析染色质可接近性对犏牛初级精母细胞基因表达的调控作用

To elucidate the molecular mechanism of male sterility in cattle-yak is the key task to solve the problem of yak crossbreeding and to resolve the mechanism of reproductive isolation between species. In our testis transcriptome analysis, it was found that gene expression was disordered during the leptotene-zygotene spermatocyte (LS-ZS) to pachytene spermatocyte (PS) transition in cattle-yak. Using ATAC-seq approach reveals that dynamic reorganization of chromatin accessibility underlies diverse transcriptomes during spermatogenesis. The aim of this project is to investigate the regulatory mechanism of chromatin accessibility on gene expression during chromosome interactions, synapsis, and DNA recombination in primary spermatocytes of cattle-yak. The LS-ZS and PS in adult testis will be purified by fluorescence-activated cell sorting (FACS) and then for ATAC-seq and RNA-seq sequencing. The different of chromatin accessibility and transcriptomes in primary spermatocytes between cattle-yak and yak will be analyzed. The effect of DNA methylation levels and transcription factor binding levels in the accessible chromatin regions on gene expression will be investigated. The results will provide basic data and new ideas for exploring the molecular mechanism of male sterility in cattle-yak from the perspective of epigenetics, and have important theoretical significance and potential application value for revealing the mechanism of reproductive isolation between species and promoting the crossbreeding in yaks.

阐明普通牛与牦牛杂交后代犏牛雄性不育的分子机理，是解决牦牛杂交育种难题和解析种间生殖隔离机制的关键。课题组先前的转录组分析发现，犏牛睾丸中初级精母细胞的细线期-偶线期（LS-ZS）向粗线期（PS）转变阶段基因表达发生紊乱。研究表明，染色质可接近性动态重组是精子发生过程基因转录调控的基础。本项目利用荧光激活细胞分选技术（FACS）分选出成年期睾丸的LS-ZS和PS进行ATAC-seq和RNA-seq测序，分析犏牛和牦牛间初级精母细胞的染色质可接近性差异与转录组差异，并研究关键染色质可接近区域的DNA甲基化水平和转录因子结合水平对基因表达的影响，从染色质可接近性角度探讨犏牛初级精母细胞染色体配对、联会和DNA重组过程基因表达的调控机制。结果将为从表观遗传角度探索犏牛雄性不育的分子机制提供基础数据和新思路，对揭示种间生殖隔离机制和推动牦牛杂交育种具有重要的理论意义和潜在的应用价值。

普通牛与牦牛种间杂交后代犏牛在产肉和产奶性能上具有明显杂种优势，但雄性不育使得杂种优势无法通过横交固定传承，阻碍了牦牛种间杂交育种。阐明普通牛与牦牛杂交后代犏牛雄性不育的分子机理，是解决牦牛杂交育种难题和解析种间生殖隔离机制的关键。本项目分析比较了犏牛和普通牛、牦牛间睾丸组织的细胞类型、基因表达、染色质可接近性、DNA甲基化与piRNA生成的差异。组织学分析表明，犏牛雄性不育主要表现为粗线期精母细胞生成受阻。RNA-seq分析显示犏牛睾丸中精子发生、Wnt信号、细胞凋亡等信号通路发生大量基因表达紊乱；犏牛中精原细胞标记基因表达升高，减数分裂细线期/偶线期初级精母细胞特异表达的联会相关基因显著下降，而粗线期初级精母细胞极显著下降，圆形精子细胞、长形精子细胞特异表达的基因几乎不表达，结合单细胞转录组测序（scRNA-seq）数据提示犏牛生精受阻过程可能发生在细线期/偶线期向粗线期精母细胞转变阶段；基于差异表达基因的转录因子富集分析和ATAC-seq技术，揭示AMYB可能是参与粗线期精母细胞基因表达阻滞的关键转录因子；利用WGBS结合sRNA-seq发现，犏牛睾丸组织中PIWIL1、TDRD1、DDX4、PLD6、MAEL等piRNA生成基因启动子发生DNA超甲基化，进而抑制piRNA生成基因表达和粗线期piRNA的生成，提示DNA超甲基化抑制piRNA生成也是参与粗线期精母细胞阻滞的关键因素。本研究明确了犏牛生精受阻的关键阶段，挖掘出参与粗线期精母细胞基因表达阻滞的关键转录因子，为理解犏牛雄性不育的分子机理提供了新见解，但对驱动犏牛初级精母细胞减数分裂失败的关键因素有待后续进一步探索。