Manipulation of three-dimensional chromatin structure to regulate oncogenes in breast and ovarian cancer.

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ABSTRACT It has become increasingly apparent in recent years that the chromosomal contacts created by the three- dimensional (3D) organization of the genome are critical in the regulation of gene expression. Disruption of this structure has been strongly implicated in the vast alterations in gene expression that are observed in many cancers, and there is increasing evidence that alterations in chromatin structure surrounding key oncogenes, such as myc, may be of significant importance in both driving oncogenesis and in sustaining disease. Despite this knowledge, it remains unclear whether alterations in chromatin structure are a cause or a consequence of alterations in transcriptional activity. This is in large part due to a limited availability of tools to study single chromatin contacts. Reported methods for forcibly looping distant genomic regions have frequently involved either gross alterations of the linear DNA sequence or are technically challenging and demand significant prior knowledge of loop-mediating factors. To address these issues, I have developed a novel technology for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9) that allows for a versatile, targeted, and reversible manipulation of chromatin contacts. I have demonstrated the utility of CLOuD9 to modulate gene expression in human cells, and plan to further utilize it to investigate how differential enhancer contacts can regulate expression of myc in breast and ovarian cancer (BOC). I hypothesize that alteration of the chromatin contacts surrounding the myc locus is sufficient to alter myc expression, and thereby impact the development and progression of these cancers. In the future, I plan to investigate the mechanisms by which dysregulation of chromatin structure contributes to acquired chemoresistance on a genome-wide scale. Collectively, findings from this body of work will be critical to obtaining a more comprehensive understanding of how chromatin dynamics specifically impact transcription apparatus to facilitate oncogenesis, and may further shed light on useful ways to design novel therapies for more targeted cancer treatments.