TRANSCRIPTIONAL REGULATORY AND CELL DIFFERENTIATION INFLUENCES OF AN ENDOCRINE DISRUPTING CHEMICAL

项目摘要 对人类产生负面影响的一组主要环境毒物是内分泌干扰物 化学物质（EDCs）。这些化学物质会干扰人体的天然激素调节， 一系列的人类疾病。我们的研究重点是EDC三丁基锡（TBT），一种化学品 常用作杀虫剂和塑料稳定剂。TBT具有主要的脂肪形成作用， 在子宫内或在成体多能干细胞中暴露。此前公布的数据显示， 三丁基锡化合物暴露促进间充质干细胞（MSC）分化成脂肪， 也增加了他们的脂质含量，代表了数量和质量上的影响， 脂肪细胞从机制上讲，已经发现TBT与蛋白质的配体结合结构域结合。 过氧化物酶体增殖物激活受体γ（PPARg）转录因子（TF）， 已知当被激活时与RXR TF形成异二聚体，促进转录调节。 重编程MSC以使其致力于脂肪生成。MSC可以分化成许多 包括肌肉、骨骼、软骨和纤维囊性细胞。当细胞经历 转录重编程，转录因子结合的位点发生变化，反映在 开放染色质位点的分布。在这个项目中，我们建议将MSC分化为 脂肪细胞和肌细胞，最初使用已知的细胞培养条件，以促进特异性 MSC的分化。我们将绘制开放染色质的基因座，并测试这些细胞中的基因表达。 样本，使我们能够通过搜索这些分化途径来识别介导这些分化途径的TF。 对应于已知TF结合位点的基序富集。有了这些信息，我们 然后可以使用相同的方法来测试TBT如何引起转录重编程， 应该揭示该过程是相同的还是涉及不同的TF集。最后我们将 将新的CellTagging方法应用于测试分化为肌细胞的多个阶段的细胞 为了测试TBT暴露是否只影响未分化的MSC，或者也可以引起 已经在肌源性谱系中发育的细胞的转分化。这些新的见解， 三丁基锡化合物的作用机制对于了解内分泌干扰物如何致病具有重要价值- 造成影响。我们还将从TBT中了解小分子如何介导 “表观遗传疗法”，影响转录重编程，但以一种针对 特定的基因组位置在John Greally和Paul Frenette博士的指导下，我将 完成这些目标，同时发展发育生物学和遗传学的新技能。 此外，我将获得宝贵的经验，介绍，网络，和手稿写作，所有 这些都是必不可少的，因为我训练成为独立的调查员和医生科学家。

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.