Problem: Many active genes in embryonic stem cells escape detection on expression microarrays due to hybridization kinetics. Thus many active genes in ESCs await discovery. Background: Many active genes eluding detection with expression microarrays can be detected by mapping RNA-Polymerase-II (Pol-II) around gene transcription start sites. Using this method, we recently uncovered 800 novel gene activation events during photoreceptor maturation, which were previously undetected by expression microarray studies (Tummala et al, 2010). Hypothesis: A substantial number of active genes in mESCs are not active in maturing neural tissues, and this set of genes are particularly enriched for functions regulating gene expression. Research: Pluripotent mouse ESCs (feeder-free) were subjected to Pol-II ChIP-on-Chip analysis using the Affymetrix Mouse Promoter tiling array. This platform was chosen for its very high resolution (35 bp) and substantial coverage around transcription start sites. Tracks of RNA-Polymerase-II binding were mapped to the genome with Tiling Analysis Software, and associated to specific gene domains using Genpathway’s Transpath. Active genes in mESCs were compared to our database of active genes generated from immature (P2) and mature (P25) mouse neural retina (http://www.molvis.org/molvis/v16/a32/). Functional clustering of the genes was accomplished using the DAVID ontology tools @ http://david.abcc.ncifcrf.gov/ . Observations: 3029 promoter-associated RNA-Polymerase-II tracks were detected by Pol-II ChIP-on-Chip in pure mESCs, and mapped to upstream and in-gene regions of 2335 genes. Stemness genes Nanog, Pou5f1 (Oct4), Foxd3, and Sox2 were captured, validating the results. Functional analysis of mESC active genes revealed enrichment for chromatin regulatory proteins expected to be present in pluripotent dividing stem cells. The data set was enriched for genes with regulatory functions. 837 genes were unique to mESCs when compared to our active gene list derived from Pol-II ChIP-on-Chip analysis of differentiating neural retina. 158 (20.6 %) of the genes have gene regulatory roles and 8 (1%) have roles in neural plate formation and cerebellum morphogenesis. 35 of the genes are homeobox transcription factors. 132 of our mESC unique genes were not previously reported from a similar Pol-II ChIP-on-Chip analysis using a lower resolution platform. 50% of these genes have transcription factor domains, including 26 with homeobox domains. 5% of the genes have chromatin regulatory function. Conclusion: High-resolution Pol-II mapping revealed a substantial number of novel gene activation events in mESCs. Homeobox genes formed the largest single group of these genes, and many others are also transcription factors, supporting our hypothesis regarding the nature of these genes. Our results now provide a novel collection of genes for investigations of ESC maintenance and differentiation. NIH EY14626 (KPM) NIH EY14803 (O.U.) OU-Beaumont Award