PROJECT SUMMARY/ABSTRACT Tissue manufacturing for skin replacement therapy is extremely inefficient, in part due to an incomplete understanding of gene expression mechanisms regulating stem cell commitment. One such poorly understood mechanism is chromatin looping, which has been thought to serve as a template for gene expression changes during cell state transitions. This knowledge gap is a major roadblock in producing sufficient graftable keratinocytes from genetically corrected patient stem cells for treatment of debilitating skin diseases. The long- term goal of this proposal is to understand the molecular mechanisms underlying stem cell differentiation to improve skin replacement therapy. Preliminary data from the applicant’s group suggest that retinoic acid (RA) and bone morphogenic protein (BMP4) morphogens can induce stem cell-derived graftable keratinocytes through induction of the master epithelial regulator and transcription factor p63. Interestingly, p63 cannot activate downstream gene expression programs in the absence of these morphogens. Further, high dimensional chromatin analyses suggest that morphogens cause major changes in chromatin looping. Therefore, the central hypothesis is that RA and BMP4 stimulate looping between p63 binding sites and distal loci, as well as alter the p63 interactome, to facilitate p63-dependent gene expression. This proposal will test the hypothesis by pursuing two specific aims. Aim 1 will determine which regulatory regions within chromatin loops are required for p63-dependent gene expression. CRISPR/Cas9 tools, which have been well established in the applicant’s laboratory, will be used to sequentially block p63 binding sites and the loci to which they are looped. The effects of each deletion will be measured by downstream gene expression, loop formation, and cellular differentiation markers. The proposed experiments will focus on loops at TFAP2C and HES1 loci, which are known to be crucial during stem cell commitment. Aim 2 will elucidate p63 interacting proteins that are required for downstream gene expression. Candidate members of the p63 interactome will be identified using a novel proximity labeling system known as BASU. This system has been validated in the applicant’s laboratory using embryonic stem cells. The importance of each p63 interacting protein will then be evaluated using a CRISPR/Cas9 loss of function screen followed by measurements of p63-dependent gene expression and cellular differentiation. The rationale for the proposed research is that it will provide useful chromatin dynamic information which can be used to improve the current stem cell differentiation protocol for tissue replacement therapy. This proposal is innovative, because it uses novel genetic and proteomics techniques to elucidate mechanisms of chromatin loops that were previously unrecognized. The findings will be significant, because they will broaden the understanding of p63 during de...