The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells. promotes H3K18 and H4 acetylation and activation or increased firing efficiency at origins.12 In humans, H4K20me2/3 recruit ORC to chromatin through direct binding with ORC1 and ORCA.13,14 Certain histone modifications also correlate with replication timing and ORC binding in P P P P P P value < 1e-300, Fig.?4E). Similarly, we did not detect enrichment of H4K20me1 levels in HeLa S3 and K562 leukemia cells at the genomic regions that in H1 hESCs the BrdU peaks overlapped with H4K20me1 (Fig. S4B). Taken together, our analysis shows NVP-ADW742 that H3K18ac, H3K56ac, and H4K20me1 are enriched at the regions of active DNA replication mainly in hESCs and correlate positively with the intensity of BrdU peaks. The H1-IMR90-Saos-2 shared peaks of BrdU are associated with high level of histone marks in hESCs and conserved DNA sequences across vertebrates To rule out that the association of histone modifications with DNA replication occur at H1-specific BrdU peaks, we divided the BrdU peaks into those that are shared between H1, IMR90, and Saos-2 cells and those that are unique to H1 (1167 vs 2813 BrdU peaks, respectively). The average levels of BrdU Gpc4 intensity was much higher in shared regions than in H1 specific regions. The shared regions also had higher levels of H3K18ac, H3K56ac, and H4K20me1 while NVP-ADW742 H1 specific regions showed low levels of histone modifications (Fig.?5A). Gene ontology analysis indicated that the shared peaks were associated with differentiation genes while H1 unique peaks were linked to embryonic development genes (Fig.?5B). Furthermore, the shared BrdU regions had higher degree of DNA sequence conservation among vertebrates compared with H1-specific BrdU peaks (Fig.?5C). Thus the shared BrdU regions account for most of NVP-ADW742 the enrichment observed for the associated histone modifications, indicating that patterns of histone modifications are indeed different at the same replicating regions between hESCs and differentiated cells. Figure?5. The BrdU peaks shared by the three cell types are preferentially associated with histone marks in hESCs and conserved DNA sequences. (A) Average levels of BrdU incorporation and histone modifications in hESCs at the center of BrdU peaks … EP300/CREBBP depletion decreases global BrdU incorporation in hESCs EP300 and CREBBP are the main HATs for H3K18 and H3K56.11,30 We therefore tested whether EP300 and CREBBP were required for DNA replication in hESCs. Since EP300 and CREBBP are largely redundant in acetylation of H3K18,30 we co-transfected siRNAs against both genes into hESCs and performed BrdU-seq (Table S1). As shown in Figure?6A, EP300 and CREBBP protein levels decreased ~85C90% upon knockdown. Levels of H3K18ac and H3K56ac also decreased dramatically after knockdown, indicating that EP300/CREBBP were responsible for the bulk of H3K18ac and H3K56ac in hESCs. Figure?6. EP300 and CREBBP HATs are required for normal pattern of BrdU incorporation in hESCs. (A) Western blots and signal quantifications of the indicated factors are NVP-ADW742 shown for control (ctrl) and KD of EP300/CREBBP in H1 hESCs. (B) Distribution … The number of BrdU peaks decreased from 5086 in control siRNA to 1755 in EP300/CREBBP KD hESCs. Depletion of EP300/CREBBP also significantly decreased BrdU blocks from 296 to 33. The remaining BrdU peaks after KD were similarly distributed across the genome with 42.2% and 49.5% of peaks in introns and distal intergenic regions, respectively (Fig.?6B). The majority (64%) of the BrdU peaks in EP300/CREBBP KD cells still occurred within DNA repeats (Table S3) and were associated with negative regulators of cell proliferation such as cyclin-dependent kinase inhibitors, genes involved in cell morphogenesis and cell fate commitment (Fig.?6C). Among the 1755 BrdU peaks in KD cells,.