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

鉴定 Y 连锁小鼠锌指蛋白 ZFY1 和 ZFY2 的分子功能

基本信息

批准号：
10749409
负责人：
Hayden Robert Holmlund
金额：
$ 3.37万
依托单位：
UNIVERSITY OF HAWAII AT MANOA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-04 至 2026-08-03
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10749409
关键词：
Address Amino Acid Sequence Antibodies Award Binding Biochemical Biological Assay C-terminal CRISPR/Cas technology Cell Separation Clinical Collaborations Cultured Cells Defect Detection Development F Factor Fellowship Fertility Fertilization in Vitro Fluorescence-Activated Cell Sorting Genes Genetic Germ Cells Haploidy Homologous Gene Human Immunoprecipitation Infertility Investigation Japan Knock-in Mouse Knock-out Knockout Mice Knowledge Link Liquid Chromatography Male Infertility Mass Spectrum Analysis Meiosis Metaphase Methods Molecular Morphogenesis Mus Nucleic Acid Regulatory Sequences Outcome Pachytene Stage Paper Pathway interactions Play Production Protein Isoforms Proteins Proteome Publishing Quality Control RNA Regulation Reporter Reproduction Role Spermatids Spermatocytes Spermatogenesis Spermatogenic Cell Spermiogenesis Tail Testing Testis Time Training Transcript Transgenes Transgenic Organisms Variant Y Chromosome ZFY protein Zinc Fingers assisted reproduction candidate identification cell type chromatin immunoprecipitation experience experimental study gene function genome editing genome-wide improved in vitro Assay insight male male fertility mouse model protein aminoacid sequence reproductive sperm cell sperm function tandem mass spectrometry transcription factor transcriptome transcriptome sequencing visiting scholar ward zygote

项目摘要

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.

项目摘要小鼠锌指蛋白ZFY1和ZFY2对于雄性生育至关重要。尽管人类ZFY的复制角色具有尚未确定，Y衍生的ZFY转基因在添加到具有有限的Y染色体贡献。具体而言，ZFY（1）在Pachytene阶段恢复了质量控制检查点（2）促进了第二个减数分裂分裂和单倍体圆形精子的产生，以及（3）改善精子生成并辅助繁殖结果。 Nakasuji等。病房实验室最近生产了ZFY1/2 双重敲除（DKO）小鼠，两组都观察精子发生。同源物促进男性生育能力的机制仍然未知，但基于它预测的蛋白质序列和在人培养细胞中的体外测定ZFY被普遍认为是转录因素。来自ZFY1/2 DKO小鼠的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ZFY1和ZFY2调节，两种蛋白质的生化功能可以通过基于抗体的测定确定例如免疫沉淀（IP）和染色质免疫沉淀（CHIP）。拟议的项目将调查 ZFY的分子功能，假设ZFY1和ZFY2有助于通过调节级联相关基因级联的表达。该假设将以两个具体的目的进行检验。在特定目标1中，我们将对原代精子细胞（PS）进行转录组和蛋白质组分析 ZFY1/2 DKO雄性的精子细胞（SS）和圆形精子（RS），以确定哪些基因在该基因中失调缺乏ZFY。这将涉及通过荧光激活的细胞分选（FACS）分离ZFY1/2 DKO生殖细胞，从而提取每种细胞类型的RNA和蛋白质，然后进行RNA-Seq和质谱法（MS）。潜在的下游然后，将从DKO小鼠失调的基因中鉴定ZFY1和ZFY2的候选物。在特定的目标2中，抗体 - 将进行基于基于的测定，以发现小鼠睾丸中ZFY1和ZFY2的生化功能。这将需要首先生成一种检测ZFY1和ZFY2蛋白的方法。在AIM 2.1中，Zygotes将以CRISPR/CAS9为目标为每个同源物独立于目标ZFY具有C-的技术而独立于创建敲入（KI）的型鼠标模型的技术端子HA标签（XyzFy1-Ha和XyzFy2-Ha）。在AIM 2.2中，我们将确认我们可以明确识别ZFY1和ZFY2 蛋白质以及任何潜在的结合伙伴，具有免疫沉淀和液相色谱法，然后是串联质谱法（IP/LC/MS）。最后，在AIM 2.3中，Ki小鼠的纯化精子细胞将用于 CHIP-PCR确定ZFY1和ZFY2是否调节选定的下游候选者的表达特定的目标1。此外，将进行芯片序列以识别全基因组的ZFY1和ZFY2靶标。拟议的项目将进一步了解小鼠中两个已知的生育因子ZFY1和ZFY2的功能。它可能导致识别ZFY同源物靶标之间的新生育基因，这反过来又可能为我们的了解同源的人ZFY同工型。