TRANSCRIPTIONAL REGULATORY AND CELL DIFFERENTIATION INFLUENCES OF AN ENDOCRINE DISRUPTING CHEMICAL

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PROJECT SUMMARY One major group of environmental toxicant that affect humans negatively are endocrine disrupting chemicals (EDCs). These chemicals interfere with the body’s natural hormone regulation leading to a range of human diseases. Our research focuses on the EDC tributyltin (TBT), a chemical frequently used as a pesticide and plastic stabilizer. TBT has major adipogenic effects when exposed in utero or in adult multipotent stem cells. Previously published data have demonstrated that TBT exposure promotes differentiation of mesenchymal stem cells (MSCs) into adipogenesis, and also increases their lipid content, representing both numerical and qualitative effects on adipocytes. Mechanistically, TBT has been found to bind to the ligand-binding domain of the peroxisome proliferator-activated receptor gamma (PPARg) transcription factor (TF), which is known to form a heterodimer with the RXR TF when activated, promoting a transcriptional reprogramming of MSCs to commit them to adipogenesis. MSCs can differentiate into a number of lineages, including muscle, bone, cartilage and fibrocystic cells. When a cell undergoes transcriptional reprogramming, the sites at which the TFs bind change, reflected by alterations of the distributions of loci of open chromatin. In this project, we propose to differentiate MSCs to both adipocytes and myocytes, initially using the cell culture conditions known to promote specific differentiation of MSCs. We will map the loci of open chromatin and test gene expression in these samples, allowing us to identify TFs mediating these differentiation pathways by searching for motif enrichment corresponding to known TF binding sites. With this information available, we can then use the same approaches to test how TBT causes transcriptional reprogramming, which should reveal whether the process is identical or involves a different set of TFs. Finally, we will apply the new CellTagging approach to test cells at multiple stages of differentiation to myocytes to test whether TBT exposure affects only undifferentiated MSCs, or can also cause transdifferentiation of cells already developing in the myogenic lineage. These new insights into the mechanism of action of TBT will be valuable in understanding how EDCs have their disease- causing effects. We will also get insights from TBT into how we a small molecule can mediate ‘epigenetic therapy’, influencing transcriptional reprogramming but in a way that is targeted to specific genomic locations. Under the mentorship of Drs. John Greally and Paul Frenette, I will accomplish these goals while developing new skills in developmental biology and genetics. Additionally I will gain valuable experiences in presenting, networking, and manuscript writing, all of which are essential as I train to become and independent investigator and physician-scientist.