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Date Submitted

Spring 4-29-2014

Research Mentor and Department

Dr. Cristina de Guzman Strong




The molecular mechanisms that govern enhancer-mediated chromatin remodeling for gene activation and transcription are poorly understood. Our lab has recently identified an evolutionarily conserved non-coding element named 923 as a regulatory enhancer within the Epidermal Differentiation Complex (EDC) locus. The EDC, located on human chromosome 1q21, encodes genes required for skin barrier function. Although our studies identify a mechanistic role for AP-1/c-Jun transcription factor binding to 923 in mediating EDC chromatin remodeling and relevant gene activation, residual 923 enhancer activity and chromatin looping continues to persist upon AP-1/c-Jun inhibition. Thus, the precise mechanism that links promoters in the EDC to an enhancer is currently unknown. I hypothesize a chromatin remodeling complex comprised of distinct transcription factors, histone modifications associated with active and open chromatin, and connective proteins, such as cohesin, that coalesce at 923 to coordinate enhancer activity with EDC chromatin topology and expression. To address this hypothesis, I will employ an ab initio biochemical/proteomics approach using DNA pull-down assays to identify proteins that bind to 923. Specifically, a biotinylated 923 DNA probe will be incubated with a nuclear extract from either proliferating or differentiated human keratinocytes. The DNA-protein complex will be incubated with Streptavidin Dynabeads to isolate and elute the bound proteins and will be subsequently identified by liquid chromatography-mass spectrometry (LC-MS), which employs ionization and fragmentation of proteins to yield identification based on mass to charge ratios. Preliminary results with cell fractionation methods demonstrate feasibility as distinct nuclear and cytosolic protein fractions were obtained. In addition, pilot experiments to assess efficient biotinylation of the probe identified successful labeling based on DNA enrichment after Streptavidin beads incubation, and differential EDC gene expression was identified in harvested keratinocytes. Elucidation of the molecular players in the proposed chromatin remodeling complex will further our understanding of the mechanisms that coordinate enhancer-mediated chromatin remodeling in mammalian tissue.

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