Identifying the Molecular Function of the Y-linked Mouse Zinc Finger Proteins ZFY1 and ZFY2

项目摘要 小鼠锌指蛋白ZFY 1和ZFY 2是雄性生育所必需的。尽管人类ZFY的生殖作用 尚未确定，Y-衍生的Zfy转基因在添加到具有以下特征的小鼠模型中时改善精子发生： Y染色体贡献有限。具体来说，Zfy（1）恢复了粗线期的质量控制检查点 （2）促进第二次减数分裂和产生单倍体圆形精子细胞; 改善精子生成和辅助生殖结果。Nakasuji等人和沃德实验室最近生产了Zfy 1/2 双基因敲除（DKO）小鼠，两组均观察到完全丧失生育能力和严重缺陷， 精子发生同源物促进男性生育能力的机制仍然未知，但基于 其预测的蛋白质序列和在人类培养细胞中的体外测定ZFY被广泛认为是一种转录本， 因子Zfy 1/2 DKO小鼠生殖细胞的转录组和蛋白质组分析将有助于确定哪些基因是 由ZFY 1和ZFY 2调节，并且两种蛋白质的生化功能可以通过基于抗体的测定来确定 例如免疫沉淀（IP）和染色质免疫沉淀（ChIP）。拟议项目将调查 ZFY的分子功能，假设ZFY 1和ZFY 2有助于精子发生和男性生育力， 调节一系列生殖相关基因的表达。这一假设将在两个具体目标中得到检验。 在具体目标1中，我们将对初级精母细胞（ps）、次级精母细胞（ps）和次级精母细胞（ps）进行转录组和蛋白质组分析。 Zfy 1/2 DKO雄性的精母细胞（ss）和圆形精子细胞（rs），以确定哪些基因在 没有ZFY。这将涉及通过荧光激活细胞分选（FACS）分离Zfy 1/2 DKO生殖细胞，提取 从每种细胞类型中提取RNA和蛋白质，然后进行RNA-seq和质谱（MS）。潜在下游 然后从DKO小鼠中失调的基因中鉴定ZFY 1和ZFY 2的候选物。在具体目标2中，抗体- 将进行基于的测定以发现ZFY 1和ZFY 2在小鼠睾丸中的生化功能。这将需要 首先建立检测ZFY 1和ZFY 2蛋白的方法。因此，在目标2.1中，受精卵将被CRISPR/Cas9靶向 技术来创建敲入（KI）小鼠模型，独立于每种同源物，其中靶向ZFY将具有C- 末端HA标签（XYZfy 1-HA和XYZfy 2-HA）。在目标2.2中，我们将确认我们可以特异性识别ZFY 1和ZFY 2 蛋白质，以及任何潜在的结合伙伴，免疫沉淀和液相色谱法，然后 串联质谱法（IP/LC/MS）。最后，在目标2.3中，来自KI小鼠的纯化生精细胞将用于 ChIP-PCR，以确定ZFY 1和ZFY 2是否调节中鉴定的选定下游候选物的表达。 具体目标1。此外，将进行ChIP-seq以在全基因组范围内鉴定ZFY 1和ZFY 2靶标。拟建项目 将促进对两种已知生育因子ZFY 1和ZFY 2在小鼠中的功能的理解。可能导致 在ZFY同源物的靶点中鉴定新的育性基因，这反过来可以为我们了解 同源人ZFY同种型。

Project Summary The mouse zinc finger proteins ZFY1 and ZFY2 are essential for male fertility. Although human ZFY’s reproductive role has not yet been determined, the Y-derived Zfy transgenes improve spermatogenesis when added to mouse models with limited Y chromosome contribution. Specifically, Zfy (1) reinstates quality control checkpoints during the pachytene stage and metaphase I of meiosis, (2) promotes the second meiotic division and production of haploid round spermatid, and (3) improves spermiogenesis and assisted reproduction outcome. Nakasuji et al. and the Ward Lab recently produced Zfy1/2 double knock-out (DKO) mice, with both groups observing a complete loss of fertility and severe defects in spermatogenesis. The mechanism by which the homologues contribute to male fertility remains unknown but based on its predicted protein sequence and in vitro assays in human cultured cells ZFY is widely believed to be a transcription factor. Transcriptome and proteome analyses of germ cells from Zfy1/2 DKO mice will help identify which genes are regulated by ZFY1 and ZFY2, and the biochemical function of both proteins can be determined by antibody-based assays such as immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP). The proposed project will investigate the molecular function of ZFY, with the hypothesis that ZFY1 and ZFY2 contribute to spermatogenesis and male fertility by regulating the expression of a cascade of reproduction-related genes. This hypothesis will be tested in two specific aims. In Specific Aim 1, we will perform transcriptome and proteome analyses of primary spermatocytes (ps), secondary spermatocytes (ss), and round spermatid (rs) from Zfy1/2 DKO males to determine which genes are dysregulated in the absence of ZFY. This will involve isolating Zfy1/2 DKO germ cells via fluorescence-activated cell sorting (FACS), extracting RNA and protein from each cell type, and then performing RNA-seq and mass spectrometry (MS). Potential downstream candidates of ZFY1 and ZFY2 will then be identified from the genes dysregulated in DKO mice. In Specific Aim 2, antibody- based assays will be performed to discover the biochemical function of ZFY1 and ZFY2 in the mouse testis. This will require first generating a method to detect ZFY1 and ZFY2 proteins. Thus, in Aim 2.1, zygotes will be targeted with CRISPR/Cas9 technology to create knock-in (KI) mouse models, independently for each homologue, in which targeted ZFY will have a C- terminal HA tag (XYZfy1-HA and XYZfy2-HA). In Aim 2.2, we will confirm that we can specifically recognize ZFY1 and ZFY2 proteins, as well as any potential binding partners, with immunoprecipitation and liquid chromatography followed by tandem mass spectrometry (IP/LC/MS). Finally, in Aim 2.3, purified spermatogenic cells from the KI mice will be used for ChIP-PCR to determine whether ZFY1 and ZFY2 regulate expression of selected downstream candidates identified in Specific Aim 1. Additionally, ChIP-seq will be done to identify ZFY1 and ZFY2 targets genome-wide. The proposed project will advance understanding of the function of two known fertility factors, ZFY1 and ZFY2, in mice. It may lead to identification of new fertility genes among targets of ZFY homologues, which could in turn inform our knowledge on the homologous human ZFY isoform.