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

近年来，越来越明显的是，由三维结构产生的染色体接触， (3D)基因组的组织在基因表达的调节中至关重要。破坏这种 结构与基因表达的巨大变化密切相关，这些变化在许多人中观察到， 癌症，并且越来越多的证据表明，围绕关键癌基因的染色质结构的改变， 例如myc，在驱动肿瘤发生和维持疾病中可能是非常重要的。尽管 尽管如此，目前仍不清楚染色质结构的改变是导致 转录活性的改变。这在很大程度上是由于有限的工具，研究单一的 染色质接触。已报道的用于使远距离基因组区域强制成环的方法经常涉及 线性DNA序列的总体改变或者是技术上具有挑战性的，并且需要显著的先验知识。 循环介导因素的知识。为了解决这些问题，我开发了一种新技术， 使用CRISPR-dCas 9（CLOuD9）的染色质环重组，其允许多功能的、靶向的和 染色质接触的可逆操作。我已经证明了CLOuD9调节基因表达的效用。 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 调节乳腺癌和卵巢癌（BOC）中myc的表达。我假设染色质的改变 myc基因座周围的接触足以改变myc的表达，从而影响发育。 和这些癌症的进展。在未来，我计划调查的机制，失调的 染色质结构有助于在全基因组范围内获得化学抗性。总体而言， 从这一机构的工作将是至关重要的，以获得更全面的了解染色质如何 动力学特别影响转录装置，以促进肿瘤发生，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。

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.