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.
Enhancers regulate gene expression in a cell-type specific manner by forming long-range chromosomal loops with promoters of key identity-defining genes have been shown to play a major role in cancer. Within the context of enhancer function, one of the most important contributors to gene regulation is a DNA looping event, which recruits transcriptional co-activators to the promoter of a key gene. The goal of this project is to provide a complete mechanistic characterization of enhancer looping kinetics in real-time, yield insights into enhancer-dependent genome organization, and validate cancer- specific hijacking of enhancers for therapeutic purposes using novel chemical tools, recently developed.
| Kadoch, Cigall; Williams, Robert T; Calarco, Joseph P et al. (2017) Dynamics of BAF-Polycomb complex opposition on heterochromatin in normal and oncogenic states. Nat Genet 49:213-222 |
| Braun, Simon M G; Kirkland, Jacob G; Chory, Emma J et al. (2017) Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat Commun 8:560 |
