THE APPLICATION OF HISTONE ANALYSIS FOR IDENTIFYING DISTAL REGULATORY ELEMENTS - REGULATION OF FSHR

Abstract

Mechanisms that regulate gene expression are fundamental to many complex biological processes and disease states. Genome-wide approaches that combine chromatin-immunoprecipitation (ChIP) and next-generation sequencing have greatly advanced our understanding of chromatin structure and the role that histone modification plays in transcriptional regulation. In particular, these advances revealed important associations between functional, non-coding DNAs and specific histone modifications, which have been used technically to identify numerous distal regulatory elements and furthered our knowledge of transcriptional regulation and cell-specific gene regulation. Fshr is a gene expressed only in testicular Sertoli cells and ovarian granulosa cells and its expression is critical for proper gonad function and fertility. Importantly, underlying its exquisite cell-specificity is a transcriptional mechanism limited to only two cell types, which previous studies revealed was dependent on distal regulatory elements. To help identify these elements, we used ChIP, combined with next-generation sequencing, to globally map Histone 3 Lysine 4 tri-methylation (H3K4me3) in Sertoli and myoid cells. H3K4me3 is a post-translational histone modification known to associate with distal regulatory elements and promoter regions of actively transcribed genes. Analysis of H3K4me3 enrichment profiles identified a distal site 3' to Fshr that was specific to Sertoli cells. Transient transfection analysis indicated the region represses Fshr promoter activity and in vitro binding analysis revealed that GATA-4 and an unknown protein bound to the region, implicating them in cell-specific regulation of Fshr. These studies provide evidence that Fshr is regulated by a distal regulatory element and have provided insight into the nature of these regulatory proteins. This work provides a database mapping H3K4me3 enrichment within Sertoli and peritubular myoid cells that can be used to identify new regulatory regions. Overall, these studies have furthered our knowledge of cell-specific gene regulation in Sertoli cells and provided new data that will lead to a better understanding of transcriptional regulation in Sertoli cells.