The goal of this project is to define the genetic and epigenetic signature that distinguishes human primordial germ cells (PGCs) from human embryonic stem cells (hESCs) with a specific focus on trimethylation (me3) of histone H3 lysine (K) 27, and function of the Polycomb Group (PcG) proteins on PGC formation. Human ESCs are the only genetically malleable human cell-based model for examining human germ cell formation. Germ cell derivation from the epiblast requires a delicate balance involving expression of pluripotent genes (such as NANOG and OCT4), suppression of somatic genes (such as HOXB1 and BRACHYURY) and functional unipotency to exclusively form gametes. Loss of germ line unipotency results in germ cell tumorigenesis. Therefore, disruption of the balance of factors that regulate germ cell derivation results in adverse clinical outcomes. In previous work Dr. Clark has shown that derivation of human germ cells from hESCs varies between independently derived lines. Her preliminary data now reveals that this can be correlated with differences in transcription of germ cell specific genes in undifferentiated ESCs as well as differential methylation of H3K27me3 at promoters of germ cell-expressed loci. In this Project, Dr Clark will continue her research on the role of the PcG proteins and H3K27me3 in modulating PGC formation in the following specific Aims: 1) Evaluating H3K27me3 and X reactivation during PGC development in two lines of female hESCs; 2) Deciphering the genome wide localization of H3K27me3 in four lines of hESCs, and PGCs derived from hESCs, and 3) Determining the function of the PcG repressive complex (PCR2) in PGC formation in four independently derived lines of hESCs. Sharing results on germ cell formation from hESCs with Projects 1 and 3 are essential to understanding hESC potential because the ability to form germ line is simultaneously reversed to form neural and hematopoietic lineages (loss of pluripotency and activation of somatic cell transcriptional programs). Thus identification of hESC lines that are capable of both faithful germ cell formation and robust somatic cell differentiation would constitute the best developmental models and therapeutic tools for future research.