GENOME-WIDE MAPPING OF FUNCTIONAL ELEMENTS IN GERM CELLS IN VIVO

Abstract

The developmental progression of primordial germ cells (PGCs) to fully mature gametes involves extensive epigenetic changes which could be important for the gain of totipotency upon fertilization. Germ cells are also unique in that they can be reprogrammed to pluripotent embryonic germ cells (EGC) without the need for ectopic overexpression of any transcription factor if appropriate culture conditions are provided. Such plasticity to regain pluripotency or acquire totipotency could potentially be attributed to both transcriptional and epigenetic properties of germ cells, which are difficult to study on a genome-wide scale due to limited numbers of germ cells per embryo. In particular, it has been technically challenging to map histone modifications in germ cells as chromatin immunoprecipitation (ChIP) typically requires substantially high amounts of starting cell material. Using modified and optimized procedures, I purified germ cells from E13.5 male embryos, and performed chromatin immunoprecipitation (ChIP) coupled with a high-throughput sequencing technology. Genome-wide maps of H3K4me3, H3K27me3, H3K27ac and H2BK20ac have been generated for E13.5 male germ cells to enable the identification of active and poised regulatory elements, such as promoters and enhancers. In addition, expression profiling was performed on E11.5, E13.5 and E15.5 germ cells to study the transcriptional status/ changes of genes located near these regulatory elements, providing insight into the biological transitions that occur during this developmental period. Lastly, combining these datasets with motif enrichment analyses, potential germ cell regulators were identified and functionally validated in vitro.