PROJECT SUMMARY The overall goal of this proposal is to reveal the molecular mechanisms by which the reprogramming factors Oct4 (O), Sox2 (S), Klf4 (K), and cMyc (M) revert somatic cells to iPSCs. Conversion of somatic cells to pluripotency is the most robust example of cellular reprogramming. In many other cases of transcription factor (TF)-induced cell fate conversion, for example the direct conversion of fibroblasts to neurons, cells fail to completely silence the starting cell transcriptome. Thus, uncovering mechanisms by which OSKM inactivate the somatic program and activate the pluripotency program during reprogramming to iPSCs will reveal important principles of how cell identity is maintained and effectively converted, and enable novel approaches to make cellular reprogramming, including lineage conversions, more efficient and faithful. In preliminary studies, we determined the genomic locations of OSKM early in reprogramming of mouse fibroblasts and in the pluripotent state, which revealed a dramatic change of OSKM targets during reprogramming and engendered new hypotheses about the action of OSKM. Early in reprogramming, the essential reprogramming factors OSK only engage a small set of pluripotency enhancers and, unexpectedly, bind extensively to somatically active enhancers. At this early time point, when very few transcriptional changes occur, we observed that almost all somatic enhancers lose active chromatin marks. Explaining how somatic enhancer decommissioning may occur, we found that somatic TFs are redistributed away from somatic enhancers early in reprogramming, to other genomic sites bound by OSK. Our findings lead to the unique hypothesis that OSK orchestrate reprogramming by mediating the activation of pluripotency enhancers as well as the silencing of somatic enhancers, and we speculate that the effect of OSK on somatic TFs is similarly important for reprogramming as step-wise pluripotency enhancer activation. Another crucial step that we defined in the reprogramming process is the reactivation of the inactive X chromosome (Xi). We discovered that Xi-reactivation is associated with DNA demethylation and is one of the final steps of reprogramming. How the disassembly of this heterochromatic structure is achieved is unknown. Based on our studies and collaborations with the Zaret lab on silencing via heterochromatin and the Hochedlinger lab on post-transcriptional control, we are well positioned to unveil the mechanisms underlying OSK's step-wise engagement of pluripotency enhancers, somatic enhancer decommissioning by OSK, and Xi-reactivation, with these Aims: 1) To uncover the mechanisms by which OSK target and modulate somatic and pluripotency enhancers early in reprogramming. 2) To define the co-factors required for the step-wise selection and activation of pluripotency enhancers by OSK. 3) To delineate mechanisms of Xi-reactivation and heterochromatin disassembly. While we will perform most experiments on fibroblast repr...