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.

项目概要小鼠锌指蛋白 ZFY1 和 ZFY2 对于男性生育能力至关重要，尽管人类 ZFY 的生殖作用对男性生育能力至关重要。尚未确定，Y 衍生的 Zfy 转基因在添加到小鼠模型中时可改善精子发生具体来说，Zfy (1) 在粗线期恢复质量控制检查点。和减数分裂中期 I，(2) 促进第二次减数分裂和单倍体圆形精子细胞的产生，以及 (3) Nakasuji 等人和 Ward 实验室最近生产了 Zfy1/2。双敲除（DKO）小鼠，两组均观察到完全丧失生育能力和严重的缺陷同源物促进男性生育力的机制仍然未知，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对 Zfy1/2 DKO 小鼠生殖细胞的转录组和蛋白质组分析将有助于识别哪些基因。受 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来确定例如免疫沉淀（IP）和染色质免疫沉淀（ChIP）拟议的项目将研究 ZFY 的分子功能，假设 ZFY1 和 ZFY2 通过以下方式促进精子发生和男性生育能力该假设将在两个特定目标中得到检验。在具体目标 1 中，我们将对初级精母细胞 (ps)、次级精母细胞进行转录组和蛋白质组分析。来自 Zfy1/2 DKO 雄性的精母细胞 (ss) 和圆形精子细胞 (rs)，以确定哪些基因在这将涉及通过荧光激活细胞分选 (FACS) 分离 Zfy1/2 DKO 生殖细胞，提取来自每种细胞类型的 RNA 和蛋白质，然后进行 RNA 测序和潜在下游质谱 (MS)。然后将从 DKO 小鼠中失调的基因中鉴定 ZFY1 和 ZFY2 的候选者，在特定目标 2 中，抗体-。将进行基于检测的实验来发现 ZFY1 和 ZFY2 在小鼠睾丸中的生化功能。首先生成一种检测 ZFY1 和 ZFY2 蛋白的方法，因此，在 Aim 2.1 中，受精卵将被 CRISPR/Cas9 靶向。技术来创建敲入 (KI) 小鼠模型，每个同源物独立，其中目标 ZFY 将具有 C- 终端HA标签（XYZfy1-HA和XYZfy2-HA）在目标2.2中，我们将确认我们可以特异性识别ZFY1和ZFY2。蛋白质以及任何潜在的结合伴侣，通过免疫沉淀和液相色谱法，然后最后，在目标 2.3 中，来自 KI 小鼠的纯化生精细胞将用于分析。 ChIP-PCR 确定 ZFY1 和 ZFY2 是否调节所选下游候选物的表达具体目标 1. 此外，将进行 ChIP-seq 来识别全基因组范围内的 ZFY1 和 ZFY2 靶标。将促进对两种已知生育因子 ZFY1 和 ZFY2 在小鼠中的功能的了解。在 ZFY 同源物的靶标中鉴定出新的育性基因，这反过来又可以告诉我们关于同源人类 ZFY 亚型。