Transcriptome analysis of early and late pachytene spermatocytes in WT and Zfp541-KO mice (Smart-Seq2)

The DSB-machinery, which induces the programmed DNA double-strand breaks (DSBs) in leptotene and zygotene stages during meiosis, needs to be kept in silence after the initiation of pachytene stage to prevent the activation of DSB checkpoint that may lead to meiotic arrest or apoptosis of germ cells. However, the mechanisms underlying this repression remain largely unknown. Here, we report that ZFP541, a germ cell-specific zinc finger protein, is responsible for the suppression of DSBs formation at late pachytene. Lack of Zfp541 in mice leads to generation of DSBs in late pachytene spermatocytes by DSB formation related-proteins and causes male infertility due to meiotic failure. Plated-based scRNA-seq of Zfp541-/- spermatocytes revealed that ZFP541 negatively regulates many meiotic prophase genes, including genes for DSB formation and their upstream transcriptional regulators, in late pachytene spermatocytes. These results were confirmed by 10x single-cell RNA-seq data on spermatogenesis of Zfp541-/- testes, which suggested that Zfp541 is required for repressing the activation of pre-pachytene gene expression programs from early to late pachytene. ZFP541 ChIP-seq on pachytene and diplotene spermatocytes demonstrated that ZFP541 occupies the promoters of meiosis initiators (e.g., Meiosin and Rxra) and a subset of their downstream genes to repress their transcription, and thus prevent the reactivation of pre-pachytene gene expression programs in pachytene spermatocytes. Thus, our results not only revealed the role of ZFP541 in maintaining the repression of pre-pachytene transcriptional programs in pachytene spermatocytes but also provide new insight into the regulation of meiotic progression by timely turning off pre-pachytene genes. Overall design: To identify differentially expressed genes (DEGs) in Zfp541-KO spematocytes, single-cell RNA-seq was performed on synchronized spermatocytes using a modified Smart-Seq2 method (Chen et al., 2018). Briefly, 2-dpp mice were pipette fed WIN 18,446 (MP) for 7 consecutive days. At Day 8 of WIN 18,446 treatments, these animals were received an injection of RA (Sigma), and then left to recover. We collected testes enriched for early (260 hours after retinoic acid treatment) and late pachytene (360 hours after retinoic acid treatment) cells from WT and Zfp541-KO mice.

在减数分裂的细线期与偶线期阶段，负责诱导程序性DNA双链断裂（DNA double-strand breaks，DSBs）的DSB形成复合体，需在粗线期启动后保持沉默，以避免激活DSB检查点，进而引发生殖细胞的减数分裂阻滞或细胞凋亡。
然而，该沉默过程的潜在分子机制目前仍鲜为人知。
本研究发现，一种生殖细胞特异性锌指蛋白ZFP541，可在粗线期晚期抑制DSB的形成。
小鼠体内Zfp541基因缺失后，粗线期晚期精母细胞会被DSB形成相关蛋白重新诱导产生DSB，并因减数分裂失败引发雄性不育。
对Zfp541基因敲除（Zfp541-/-）精母细胞的基于培养板的单细胞RNA测序（single-cell RNA-seq，scRNA-seq）分析显示，ZFP541可在粗线期晚期精母细胞中负调控大量减数分裂前期基因，其中包括DSB形成相关基因及其上游转录调控因子。
针对Zfp541基因敲除小鼠睾丸生精过程的10x单细胞RNA测序数据验证了上述结果，该数据表明，Zfp541是维持从粗线期早期到晚期抑制前粗线期基因表达程序激活所必需的因子。
对粗线期与双线期精母细胞开展的ZFP541染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing，ChIP-seq）分析显示，ZFP541可结合减数分裂起始因子（如Meiosin与Rxra）及其部分下游基因的启动子区域以抑制其转录，从而阻止粗线期精母细胞中前粗线期基因表达程序的重新激活。
综上，本研究不仅揭示了ZFP541在维持粗线期精母细胞中前粗线期转录程序沉默的作用，还为通过适时关闭前粗线期基因以调控减数分裂进程提供了新的研究视角。
整体实验设计：
为鉴定Zfp541基因敲除（Zfp541-KO）精母细胞中的差异表达基因（differentially expressed genes，DEGs），本研究采用改良的Smart-Seq2方法对同步化精母细胞开展单细胞RNA测序（Chen等，2018）。
具体而言，将出生后2日龄（2-day post-partum, 2-dpp）的小鼠连续7天通过移液器饲喂WIN 18,446（MP）。在WIN 18,446处理的第8天，给这些小鼠注射视黄酸（RA，Sigma公司产品），随后使其恢复。
我们分别在视黄酸处理后260小时（富集粗线期早期细胞）与360小时（富集粗线期晚期细胞）时，从野生型（WT）与Zfp541基因敲除小鼠中收集睾丸组织。