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

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.