Deciphering the contribution of enhancer transcription to enhancer function

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PROJECT SUMMARY/ABSTRACT The applicant is requesting two years of support from a Ruth L. Kirschstein National Research Service Award for Individual Predoctoral Fellowship to Promote Diversity in Health-Related Research (F31-Diversity) to examine the relationship between enhancer transcription and enhancer function. Enhancers have been shown to initiate short, non-coding transcripts in quantities rivaling promoters, yet their role on enhancer function remains controversial. My preliminary data suggests that enhancers and promoters synchronize their activity to form co- regulated domains. This result is surprising given that previous studies using total RNA measurements have indicated that enhancer eRNAs ramp up before their associated mRNAs, suggesting that enhancers get activated first and that this activation then gets transmitted to the associated promoters. Co-regulated domains explain several of the mechanisms that the textbook model of enhancer function fails to. These include that enhancer sequences are indistinguishable from promoters, that they are bound and initiate transcription by RNA polymerase II, that enhancers and promoters form hubs in the nucleus, that enhancers act in an additive or synergistic manner, and the redundancies observed when knocking out single enhancers in a multi-enhancer locus. The goal of this research is to test the hypothesis that enhancers may function by serving as simultaneously activated and mutually reinforcing transcription initiation sites within CRDs. This hypothesis implies that enhancer transcription would be essential for enhancer function. I will test this hypothesis in three specific aims: (1) determine the generality of enhancer-promoter co-regulatory domains across signaling responses, to uncover whether the formation of co-regulated domains are a general regulatory mechanism underlying signal response (2) determine the relationship between enhancer transcription and enhancer function outside of their native genomic environment, to examine the relationship between enhancer transcription and enhancer function outside of their native genomic context, and (3) determine the impact of silencing enhancer transcription on gene activation in vivo, to determine the influence that shutting down enhancer transcription ahs on enhancer function in its native genomic context. This training will allow me to (1) develop skills in the areas of research design, analysis and interpretation using next-generation sequencing and molecular biology techniques; (2) learn the fundamentals of molecular, chromatin, and enhancer biology; (3) successfully defend a dissertation; and (4) obtain a competitive post-doctoral fellowship with the long-term goal to become a successful, independently-funded scientist at a research-intensive university.