Silencer Control of T cell Homeostasis

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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.