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Mechanistic Insights Into Gene Regulation By Transcriptional Enhancers using CRISPR Genome Engineering


Transcriptional regulation is the key mechanism responsible for differential gene expression in the 300+ human cell types during both development and disease. Here I use both genomic and epigenomic tools to characterize the role of cis-regulatory elements in transcriptional regulation. In Chapter 1, I discuss the current epigenome mapping toolkit and utilities of epigenome maps. I focus particularly on mapping of DNA methylation, chromatin modification state and chromatin structures, and emphasize the use of epigenome maps to delineate human gene regulatory sequences and developmental programs. I also provide a perspective on the progress of the epigenomics field and challenges ahead. In Chapter 2, I explore the role of a large cell-type restricted regulatory element, called a super enhancer (SE), in controlling the expression of a master regulatory transcription factor of mouse embryonic stem cells. This SE is occupied by Oct4, Sox2, Nanog, and the mediator complex, and physically interacts with the Sox2 locus via DNA looping. Using a simple and highly efficient double-CRISPR genome editing strategy the entire 13-kb SE was deleted and the transcriptional defects in the resulting monoallelic and biallelic deletion clones were characterized with RNA-seq. These results showed that the SE is responsible for over 90% of Sox2 expression, and Sox2 is the only target gene along the chromosome. These results support the functional significance of a SE in maintaining the pluripotency transcription program in mouse ESCs. In Chapter 3, my work continues to dissect the impact of SE deletion on transcription from the Sox2 locus. ChIP-seq and allelic 4C -seq assays are used to understand the impact on RNA polymerase II recruitment, open chromatin, and local interactions at the Sox2 promoter. Second, the functional study of enhancers is expanded to a selection of 100 enhancers and SEs in mouse embryonic stem cells again using a CRISPR deletion strategy. The purpose of this study is to determine which enhancers predicted by chromatin signatures are functional, to identify their target gene(s), and to determine the extent to which each enhancer contributes to the expression of their target gene(s)

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