TRANSCRIPTIONAL REGULATORY AND CELL DIFFERENTIATION INFLUENCES OF AN ENDOCRINE DISRUPTING CHEMICAL

项目总结 对人类有负面影响的一大类环境毒物是内分泌干扰物 化学品(EDCs)。这些化学物质会干扰人体的自然荷尔蒙调节 一系列人类疾病。我们的研究重点是EDC三丁基锡(TBT)，一种化学物质 经常用作杀虫剂和塑料稳定剂。TBT在以下情况下具有主要的成脂作用 暴露在子宫或成人多能干细胞中。之前公布的数据表明 TBT暴露促进间充质干细胞(MSCs)分化为成脂细胞， 也增加了它们的脂类含量，表现出对 脂肪细胞。从机制上讲，TBT被发现与TBT的配体结合域结合 过氧化物酶体增殖物激活受体γ(PPARg)转录因子(Tf)，是 已知当被激活时与RXR TF形成异二聚体，促进转录 对骨髓间充质干细胞进行重新编程，使其向成脂方向发展。MSCs可以分化为多个 血统，包括肌肉、骨、软骨和纤维囊性细胞。当一个细胞经历 转录重编程，转录因子结合改变的位点，反映在 开放染色质的基因座分布。在这个项目中，我们建议将MSCs区分为 脂肪细胞和肌细胞，最初使用已知的细胞培养条件促进特异性 间充质干细胞的分化。我们将绘制开放染色质的基因图谱，并测试这些基因的表达 样本，使我们能够通过搜索来识别介导这些分化途径的因子 与已知的转铁蛋白结合位点相对应的基序丰富。有了这些信息，我们 然后可以使用相同的方法来测试TBT如何导致转录重新编程，这 应该揭示这一过程是相同的，还是涉及一组不同的TF。最后，我们会 应用新的细胞标记方法测试分化为心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MSCs，或者也可能导致 已经在肌源性谱系中发育的细胞的转分化。这些新的洞察 TBT的作用机制对于了解内皮样细胞是如何发病的具有重要意义。 造成影响。我们还将从TBT中获得关于我们一个小分子如何调解的见解 ‘表观遗传疗法’，影响转录重编程，但以一种针对性的方式 特定的基因组位置。在约翰·格雷博士和保罗·弗雷内特博士的指导下，我将 在开发发育生物学和遗传学方面的新技能的同时，实现这些目标。 此外，我还将在演讲、网络和手稿写作等方面获得宝贵的经验 在我被训练成为独立的调查员和内科科学家的过程中，其中一些是必不可少的。

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.