Investigating the role of bromodomain-containing proteins in the production of viable spermatozoa and male fertility

研究含溴结构域蛋白在活精子产生和男性生育能力中的作用

• 批准号: 10615696
• 负责人: Paula Elaine Cohen
• 金额: $ 38.68万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615696
• 关键词: Alleles; Assisted Reproductive Technology; Binding; Binding Sites; Bromodomain; Chromatin; Chromatin Structure; Chromosome Pairing; Complex; DNA Packaging; DNA Polymerase II; Data; Defect; Development; Differentiated Gene; Ensure; Environment; Equus caballus; Event; Failure; Fertility; Gene Expression Profile; Gene Expression Regulation; Gene Silencing; Genes; Genetic Recombination; Genetic Transcription; Genome; Genome Mappings; Histone Acetylation; Histones; Human; Immunofluorescence Immunologic; Infertility; Knockout Mice; Machine Learning; Male Infertility; Mediating; Meiosis; Meiotic Prophase I; Modeling; Molecular; Morphogenesis; Morphology; Mus; Mutant Strains Mice; Mutation; Nature; Nuclear; Pathway interactions; Pattern; Play; Process; Production; Protamines; Proteins; Repression; Role; SPO11 gene; Sampling; Seminal fluid; Sperm Head; Spermatids; Spermatocytes; Spermatogenesis; Spermiogenesis; Stratification; Testing; Testis; Time; Transcriptional Regulation; gene repression; genetic corepressor; genome-wide; insight; male; male fertility; men; molecular subtypes; mutant; promoter; protein function; recruit; sperm analysis; sperm cell; sperm morphology; sperm viability; tool

Spermatogenesis is a highly unique differentiation process that involves complex mechanisms of gene regulation, particularly at the level of the transcriptional machinery. The complexity of this process is underscored by the fact that 30% of male infertility is attributable to sperm morphology defects and/or poor semen quality. Bromodomain-containing (BD) proteins are critical regulators of transcription, which act by binding acetylated histone residues at their target loci and recruiting the appropriate transcriptional regulators. Mutations in the genes encoding three BD proteins (BRDT, BRD4 and BRWD1), or that of their interacting proteins, result in morphologically abnormal sperm in mice, and have been associated with poor semen quality and infertility in men. We hypothesize that BRDT, BRD4, and BRWD1 play interconnected roles during meiotic prophase I and spermiogenesis, two key stages of spermatogenesis during which stringent transcriptional regulation is exerted, to ensure appropriate transcriptional control and chromatin compaction leading to the production of morphologically normal sperm. We propose the following model: (1) during meiotic prophase I, BRDT ensures appropriate temporo-spatial control of transcriptional repression to allow for the events of recombination and synapsis; (2) then, upon entry into spermiogenesis, BRDT aids in chromatin compaction during histone-to-protamine exchange, by shutting down transcription across the genome; (3) at the same time, BRD4 and BRWD1 are required to overcome the transcriptional silencing imposed by BRDT specifically at genes essential for spermatid development. Studies herein will test this integrated model of BD action in mice and men. In Aim 1, we will elucidate the role of BRDT in mediating progressive transcriptional shut down during meiotic prophase I using a mouse mutant lacking Brdt. We will examine meiotic progression in wildtype and mutant spermatocytes, and will define the genome-wide distribution of BD proteins and components of the transcriptional machinery. We will ask whether BRDT function is dependent on synapsis or recombination. In Aim 2, we will explore the role of BRWD1 and BRD4 in overcoming BRDT-mediated repression and ensuring expression of critical spermatid differentiation genes. We will investigate how BD proteins co-operate to ensure the correct chromatin environment is in place to allow for the progressive nuclear compaction that arises due to the histone-to-protamine exchange. In Aim 3, we will map the genome-wide distribution of BRDT and BRWD1, histone acetylation, and transcription in human testis, with matched analysis of sperm morphology. By combining these data with analysis of sperm chromatin compaction in wildtype and BD knockout mice, we will develop machine learning tools that permit stratification of sperm from infertile men based on BD protein function and chromatin compaction. These studies are the first to elucidate the coordinated roles of BD proteins in ensuring normal sperm morphology and we will apply our understanding of this transcriptional regulation to defining the causes for sperm defects in infertile men.

精子发生是一个非常独特的分化过程，涉及复杂的基因机制 调节，特别是在转录机制水平上。这个过程的复杂度是 30% 的男性不育症可归因于精子形态缺陷和/或精子质量差，这一事实强调了这一点 精液质量。含溴结构域 (BD) 蛋白是转录的关键调节因子，其作用是 在其目标位点结合乙酰化组蛋白残基并招募适当的转录调节因子。 编码三种 BD 蛋白（BRDT、BRD4 和 BRWD1）的基因突变，或其相互作用的基因突变 蛋白质，导致小鼠精子形态异常，并与精液质量差有关 以及男性不育症。我们假设 BRDT、BRD4 和 BRWD1 在 减数分裂前期 I 和精子发生，精子发生的两个关键阶段，在此期间严格 发挥转录调控作用，以确保适当的转录控制和染色质 压缩导致产生形态正常的精子。我们提出以下模型： (1) 在减数分裂前期 I，BRDT 确保转录抑制的适当时空控制 允许重组和突触事件发生； (2) 然后，在进入精子发生时，BRDT 有助于 通过关闭组蛋白与鱼精蛋白交换期间的染色质压缩 基因组； (3)同时需要BRD4和BRWD1来克服转录沉默 BRDT 专门针对精子细胞发育必需的基因施加。本文的研究将对此进行测试 小鼠和男性 BD 作用的综合模型。在目标 1 中，我们将阐明 BRDT 在调解中的作用 使用缺乏 Brdt 的小鼠突变体在减数分裂前期 I 进行性转录关闭。我们将 检查野生型和突变型精母细胞的减数分裂进程，并将定义全基因组 BD 蛋白和转录机制组件的分布。我们会询问 BRDT 是否 功能依赖于突触或重组。在目标 2 中，我们将探讨 BRWD1 和 BRD4 在 克服 BRDT 介导的抑制并确保关键精子细胞分化基因的表达。我们 将研究 BD 蛋白如何合作以确保正确的染色质环境到位，以允许 由于组蛋白与鱼精蛋白的交换而产生的逐渐核压缩。在目标 3 中，我们将 绘制人类睾丸中 BRDT 和 BRWD1、组蛋白乙酰化和转录的全基因组分布图， 与精子形态的匹配分析。通过将这些数据与精子染色质分析相结合 在野生型和 BD 敲除小鼠中进行压实，我们将开发允许分层的机器学习工具 基于 BD 蛋白功能和染色质压缩的不育男性精子。这些研究是 首先阐明 BD 蛋白在确保正常精子形态方面的协调作用，我们将应用 我们对这种转录调控的理解可以确定不育男性精子缺陷的原因。