HIJACKING OF SUPER-ENHANCERS FOR CANCER-SPECIFIC THERAPEUTICS

 描述(由申请人提供)：细胞命运由潜在的遗传密码、DNA甲基化、组蛋白修饰和高阶染色质结构分级控制，当这些结构被破坏时，可能会导致癌症。DNA编码的增强子与基因驱动启动子在染色体内形成环，通过控制转录机制的定位来调节组织特异性基因的表达。增强子是包含转录功能结合位点的不同基因组区域，可以形成跨越数十到数百个千碱基的长范围染色质环。虽然增强子和典型的位点控制区是在30多年前发现的，并通过拓扑方法在全球范围内进行了表征，但对于转录领域的专家来说，实时增强子环的形成机制仍然是一个谜。在全球范围内，增强剂通过与组织特异性的相互作用来控制细胞的命运和疾病 启动子或关键肿瘤致病基因。因此，在分子水平上更详细地了解增强剂是如何形成和调节基因表达的，可能会为通过组织特异性疗法抗击疾病提供新的途径。这项建议旨在开发一种系统，以快速诱导远程染色体内环，以审问和劫持增强子环机制。通过将CRISPR/dCas9基因组靶向技术与小分子诱导邻近相结合，我将能够控制染色体环事件，从而以癌症特异性的方式进行基因表达。这个系统将允许我们劫持活性增强子的功能复合体，并将它们物理地拴在一个抑制的或低表达的启动子上，以控制基因的表达。由于化学诱导系统允许精确的时间控制二聚化，而核酸酶死亡(DCas9)系统允许协调蛋白质靶向遗传区域，我们将能够物理地连接在细胞整个生命周期中相互作用的任何两个基因组区域。我们试图在癌症特有的环境中控制细胞抑制基因的表达。此外，我们将定义增强子形成和崩溃过程中发生的事件的生化序列，并在核组织的背景下建立染色体环事件的生物物理模型。在这项工作的高潮，我们将能够验证高阶染色质结构的争议机制，解释为什么人类基因组的某些区域非常容易改变和易位，并验证增强子景观的重新用途用于癌症特异性治疗。

 DESCRIPTION (provided by applicant): Cellular fate is hierarchically controlled by the underlying genetic code, DNA methylation, histone modifications, and higher order chromatin structure, which when disrupted can lead to cancer. Intra- chromosomal loop-formation of DNA-encoded enhancers with gene-driving promoters regulates tissue- specific gene expression by controlling the localization of transcriptional machinery. Enhancers are distinct genomic regions containing transcription faction binding sites and can form long-range chromatin loops that span tens to hundreds of kilobases. Though enhancers and canonical locus control regions were discovered over 30 years ago and are globally characterized by topological methods, the mechanisms of enhancer-loop formation in real-time remains a mystery to experts in the field of transcription. Globally, enhancers govern cell fate and disease by interacting with tissue-specific promoters or key tumor pathogenesis genes. Thus, a more detailed understanding at the molecular level of how enhancers form and mediate gene expression could provide novel inroads into combatting diseases with tissue-specific therapies. This proposal seeks to develop a system to rapidly induce long-range intra-chromosomal loops for the purpose of interrogating and hijacking enhancer looping mechanisms. By combining the CRISPR/dCas9 genome targeting technology with small-molecule induced proximity, I will be able to control chromosomal looping events, and hence gene expression in a cancer-specific manner. This system will allow us to hijack the functional complexes of an active enhancer, and physically tether them to a repressed, or low-expressing promoter to control gene expression. Because the chemical-induced system allows precise temporal control of dimerization, and the nuclease dead (dCas9) system allows for coordinated protein targeting to genetic regions, we will be able to physically tether any two genomic regions that interact throughout the life of a cell. We seek to control the expression of cell-arresting genes in a cancer-specific environment. Further, we will both define the biochemical sequence of events that occur during enhancer formation and collapse, and develop of biophysical model of chromosomal looping events in the context of nuclear organization. At the culminating of this work, we will be able to validate the controversia mechanics of higher-order chromatin structure, explain why certain regions of the human genome are highly susceptible to alteration and translocation, and validate the repurposing of enhancer landscapes for cancer-specific therapies.