The pluripotency of the embryonic stem (ES) cells, i.e. the properties to proliferate indefinitely in culture and differentiate into virtually every other cell type in the body, is controlled by a handful of transcription factors, which are proteins that bind to DNA and selectively activate expression of specific genes. The main objective of this project is to understand how transcription factors mediate the selective activation of genes involved in pluripotency of the ES cells. In the previous funding periods, we demonstrated that ES cell differentiation is accompanied by dynamic modifications of the histone proteins at genomic regions where the key stem cell regulatory factors bind. In the current funding cycle, we identified a protein involved in regulating the chromatin modification state in the ES cell genome, and showed that loss of this protein results in up-regulation of more than a thousand genes in the ES cell. We also identified a protein that recognizes the chromatin modification mark at transcription factor binding sites, and showed that its genomic distribution is consistent with the role in mediating transcriptional activation in the ES cells. Finally, in order to better understand the mechanisms of transcriptional regulation, we determined the 3-dimensional genome organization in the human and mouse embryonic stem cells. We found that the chromosomes in these cells are partitioned into mega-base sized genomic domains that are largely stable and invariant through cell differentiation, and highly conserved during evolution. Results from the present research have shed new lights into the mechanisms of gene activation in ES cells, and will help us devise better strategies to manipulate the embryonic stem cells for cell-based therapeutics.