Silencer Control of T cell Homeostasis

项目摘要/摘要 这位候选人目前是辛辛那提大学(CCHMC)博士实验室的医学博士研究生。 阿特姆·巴斯基。该提案描述了一项导致独立护理人员的研究和培训相结合的计划 在生物医学研究方面。该项目的研究目标是确定人类T细胞中的功能性消音器和 以了解它们的作用机制。超过98%的人类基因组是非编码的。据信，许多人 非编码序列是受调节的，并以细胞类型特定的方式动态利用来指示 基因表达程序。虽然增强剂在健康和包括癌症在内的疾病中得到了广泛的研究 像T-ALL一样，消音器的位置和生物功能在很大程度上是未知的。和增强剂一样，消音器也是 被认为有助于细胞类型特定的基因表达模式，因此对 在不同的细胞类型中的调节和功能。特别是，消音器对转录组的贡献 在人类T细胞中的维持，而规范抑制物(PRC2)激活消音器的机制是 未知。目前迫切需要直接研究CD4+T细胞中的功能性沉默分子。我已经建造了 一种消音器屏幕，使用否定选择方法来识别来自 T细胞中开放染色质的全基因组文库。我在Jurkat细胞的屏幕上的初步结果表明 与最近发表的消音器测试相比，功能性消声器在很大程度上处于独特的位置。 Jurkat细胞中的功能性沉默分子也富含T细胞特有的转录因子基序，并且存在于 参与T细胞稳态、激活和Th分化的基因。我假设T细胞特异性 通过主动抑制异常来维持静息T细胞的动态平衡 基因表达。在目标I中，我试图使用这一新的方法来鉴定人类CD4+T细胞中的功能沉默元件 我开发的化验方法。这项工作将贡献一份功能性消声器元件的图集，包括 富含这些元件的组蛋白标记和抑制转录因子的模式，并阐明沉默因子 受控的监管网络，以刺激假说的产生和他人的研究。在AIM II中，我会 研究典型的抑制子PRC2激活消音器的机制，并确定一些功能 抑制子序列还决定了H3K27me3介导的基因抑制。总体而言，这项工作有望 通过沉默元件调节基因的新功能数据为T细胞生物学提供新的见解。我 预计这项工作将产生一份与CD4+T细胞中沉默成分高度相关的图谱，实验验证 调节这些元件的转录因子，并支持PrC2协调的假设 沉默因子在转录抑制和H3K27me3调控转录中的作用 监管。在我的支持和经验丰富的指导团队的指导下，我希望继续开发 在我的整个职业生涯中，为患者的预后做出有意义的贡献所需的科学和临床技能 独立研究生涯。

Project Summary/Abstract The candidate is currently an MD-PhD student at the CCHMC (University of Cincinnati) in the laboratory of Dr. Artem Barski. The proposal describes a combined research and training program leading to independent carreer in biomedical research. The research goal of this project is to identify functional silencers in human T cells and to understand their mechanism of action. Over 98% of the human genome is non-coding. It is believed that many non-coding sequences are regulatory, and are dynamically utilized in a cell type specific manner to dictate programs of gene expression. While enhancers have widely been studied in health and disease including cancers like T-ALL, the location and biological function of silencers is largely unknown. Like enhancers, silencers are believed contribute to cell type specific patterns of gene expression, and thus hold unique requirements for regulation and function in different cell types. Particularly, the contribution of silencers to transcriptome maintenance in human T cells, and the mechanism for silencer activation by canonical repressors (PRC2) is unknown. There is a critical need to directly interrogate functional silencers in CD4+ T cells. I have constructed a silencer screen using a negative selection method to identify the sequences of functional (active) silencers from a genome-wide library of open chromatin in T cells. Preliminary results from my screen in Jurkat cells suggests that functional silencers are largely in unique locations compared to recently published silencer assays. Functional silencers in Jurkat cells also enrich for T cell specific transcription factor motifs, and exist nearest to genes involved in T cell homeostasis, activation, and Th differentiation. I hypothesize that a T cell specific repertoire of silencers actively maintain homeostasis of resting T cells cell by active suppression of aberrant gene expression. In Aim I, I seek to identify functional silencer elements in human CD4+ T cells using the novel assay which I have developed. This work will contribute an atlas of functional silencer elements, including patterns of histone marks and repressor transcription factors enriched at these elements, and elucidate a silencer controlled regulatory network to stimulate hypothesis generation and the research of others. In Aim II, I will investigate a mechanism of silencer activation by canonical repressor PRC2, and determine if some functional silencer sequences also dictate H3K27me3 mediated gene repression. Collectively this work is expected to provide new insights into T cell biology with novel functional data of gene regulation by silencer elements. I expect this work to generate a highly relevant atlas of silencer elements in CD4+ T cells, experimental validation of transcription factors which regulate these elements, and support for a hypothesis that PRC2 coordinates silencer function by mediating both transcriptional repression and H3K27me3 directed transcriptional regulation. Guided by my supportive and experienced mentorship team, I hope to continue developing the scientific and clinical skills necessary to make meaningful contributions to patient outcomes throughout my independent research career.