Higher order chromatin organization during spermatogenesis

Spermatogenesis is a complex process in which diploid male germ cell progenitors undergo meiosis and then differentiate further to form mature haploid sperm. The zinc finger protein CTCF, in conjunction with cohesin, is essential for DNA looping in cultured cells. We have shown that CTCF, and its testis-specific paralogue CTCFL, bind similar consensus sequences but are functionally different proteins with distinct genome-wide binding patterns. We recently developed the necessary unique tools to examine the roles of CTCF and CTCFL in testis, allowing us to investigate, for the first time, fundamental mechanisms underlying higher order chromatin organization during male gametogenesis. We will employ genome-wide approaches on highly purified male germ cell populations, representing subsequent stages of spermatogenesis. Cells will be isolated by sedimentation/centrifugation-based approaches, in combination with flow cytometry with a novel marker. We will examine transcription regulation (RNA-Seq) and DNA binding (ChIP-Seq) by CTCF(L), as well as three dimensional DNA looping in loci important for spermatogenesis (3C-Seq) in the presence/absence of CTCF(L). Genome-wide experiments will be complemented by other studies, e.g. immunofluorescent analyses of testis sections. In cultured cells, the binding of, and competition between, CTCF and CTCFL is dependent on nucleosome composition and histone modifications. Thus, in vivo experiments will focus on the genome-wide binding of CTCF(L), in relation to histone (variants), cohesin, and RNA polymerase II. Combined, our studies will yield novel insight into the roles of CTCF and CTCFL during spermatogenesis, and provide information on the relationship between chromatin organization, testis-specific gene expression, imprinting and meiotic recombination.