Dissecting Regulatory Mechanisms of Cardiac Regeneration Enhancers

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PROJECT SUMMARY/ABSTRACT Adult mammals possess a limited ability to regenerate cardiac tissue after an injury, such as a myocardial infarction. Following a myocardial infarction, up to a billion or more heart muscle cells die and are replaced by scar tissue that contributes to heart failure and sudden death. In contrast, adult zebrafish remarkably regenerate injured hearts with no residual scarring. Although zebrafish and mammals share homologs of genes vital for heart regeneration, their transcriptional responses to cardiac injury are distinct. However, the mechanisms governing gene expression during heart regeneration remain poorly understood. My research aims to elucidate how injury-responsive gene expression is regulated to facilitate heart regeneration. My lab identified the cardiac leptin b-linked regeneration enhancer (cLEN), which activates gene expression upon cardiac injury. Using in vivo transgenic assays, I found that multiple activation elements are required for injury-dependent cardiac enhancer activity. Surprisingly, I also found that cLEN contains a repressive element that is required for preventing enhancer activation in uninjured hearts, providing the first example of an inhibitory element within a cardiac regeneration enhancer. Based on these data, I hypothesize that cardiac regeneration enhancers are dually governed by activation and repression to direct injury-restricted expression for heart regeneration. In Aim 1, I will utilize pharmacological and genetic approaches to test the model that injury-induced MEK–ERK– AP-1 pathway signaling activates cardiac regeneration enhancers. I will also utilize ATAC-seq, ChIP-seq, and RNA-seq using FACS-sorted endocardial cells from uninjured and regenerating hearts to test whether AP-1 regulates chromatin accessibility, deposition of active histone marks, and injury-induced paracrine factor expression. In Aim 2, I will use transgenic assays to determine whether cLEN-like enhancer candidates I identified in the zebrafish, mouse, and human genomes drive regeneration-induced expression. The functionality of candidate repressive elements will be determined via mutational analyses. I will also utilize in vivo loss-of- function assays to determine whether prdm1a, a transcriptional repressor that is predicted to bind to the repressive element in cLEN, mediates heart regeneration and repression of regeneration enhancers in uninjured hearts. These studies will utilize genetic tools and the endogenous regenerative ability of the zebrafish heart to improve our understanding of transcriptional mechanisms underlying heart regeneration, build gene regulatory networks, and identify potential targets for improving heart repair.