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

摘要 近年来，越来越明显的是，由三个基因创造的染色体联系-- 基因组的三维结构在基因表达调控中起着至关重要的作用。扰乱这一点 结构与基因表达的巨大变化密切相关，在许多 癌症，越来越多的证据表明，关键癌基因周围染色质结构的变化， 例如myc，可能在推动肿瘤发生和维持疾病方面都具有重要意义。尽管 目前尚不清楚染色质结构的改变是导致还是导致 转录活性的改变。这在很大程度上是由于研究单项研究的工具有限 染色质接触。已报道的强制循环远程基因组区域的方法经常涉及 无论是对线性DNA序列的粗略更改，还是在技术上具有挑战性并需要显著的事前 环路中介因素的知识。为了解决这些问题，我开发了一种新技术 使用CRISPR-dCas9(Cloud9)重新组织染色质环路，从而实现多功能、有针对性和 染色质接触的可逆操作。我已经证明了Cloud9调节基因的效用 在人类细胞中表达，并计划进一步利用它来研究差异增强子接触如何 调节myc在乳腺癌和卵巢癌中的表达。我推测染色质的变化 Myc基因座周围的接触足以改变myc的表达，从而影响发育 以及这些癌症的进展。在未来，我计划研究调控失调的机制 染色质结构在全基因组范围内对获得性化疗耐药有贡献。总的来说，调查结果 对于更全面地了解染色质是如何 动力学特别影响转录装置以促进肿瘤的发生，并可能进一步阐明 为更有针对性的癌症治疗设计新疗法的有用方法。

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.