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

精子发生是一个非常独特的分化过程，涉及复杂的基因调控机制， 调控，特别是在转录机制的水平。这个过程的复杂性在于 30%的男性不育症可归因于精子形态缺陷和/或精子发育不良， 精液质量含溴结构域（BD）蛋白是转录的关键调节因子，其通过以下方式起作用： 结合乙酰化组蛋白残基在其靶基因座并募集适当的转录调节因子。 编码三种BD蛋白（BRDT、BRD 4和BRWD 1）的基因突变，或它们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 和男性不育。我们假设BRDT、BRD 4和BRWD 1在这一过程中发挥着相互关联的作用。 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 转录调控，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 允许重组和突触的事件;（2）然后，进入精子发生，BRDT帮助 在组蛋白-鱼精蛋白交换过程中，通过关闭跨膜的转录， （3）BRD 4和BRWD 1是克服转录沉默的关键基因 由BRDT特异性地施加在精子细胞发育所必需的基因上。本研究将对此进行测试 在小鼠和人中BD作用的综合模型。在目标1中，我们将阐明BRDT在介导 使用缺乏Brdt的小鼠突变体在减数分裂前期I期间进行性转录关闭。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蛋白的分布和转录机制的组成部分。我们会问BRDT是否 功能依赖于突触或重组。在目标2中，我们将探讨BRWD 1和BRD 4在以下方面的作用： 克服BRDT介导的阻遏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， 由于组蛋白-鱼精蛋白交换而引起的进行性核致密化。在目标3中，我们 绘制人类睾丸中BRDT和BRWD 1、组蛋白乙酰化和转录的全基因组分布图， 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基因敲除小鼠中，我们将开发允许分层的机器学习工具， 基于BD蛋白功能和染色质致密化的不育男性精子。这些研究是 首先阐明BD蛋白在确保正常精子形态中的协调作用，我们将应用 我们对这种转录调控的理解有助于确定不育男性精子缺陷的原因。

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.