Cardiac Transcriptional Enhancer is Repurposed During Regeneration to Activate an Anti-proliferative Program

Zebrafish have a high capacity to regenerate their hearts post-injury.Several studies have surveyed transcriptional enhancers to understand how the dynamics of gene expression are controlled during heart regeneration. We have identified a cardiac transcriptional enhancer that is activated at the site of injury called the runx1 enhancer or REN since it regulates the expression of nearby gene runx1. REN is usually active in cardiomyocytes (CMs) and epicardial tissues surrounding the cardiac valves of uninjured zebrafish hearts. However, when REN is activated in regenerating CM and epicardial tissues at the injury site, it is concurrently downregulated around the distal heart valve. Deletion of REN (?REN) results in excess Collagen around uninjured zebrafish cardiac valves. This ?REN Collagen phenotype is rescued with a runx1 deletion (?runx1), suggesting that REN and runx1 function in different genetic pathways in uninjured hearts. A different nearby gene that does change expression around valves in ?REN mutant hearts is adamts1 which encodes a metalloproteinase that degrades Collagen. Taken together, this suggests that in uninjured hearts REN regulates adamts1 independently of runx1. However, during regeneration, CM proliferation at the site of injury is enhanced in both ?REN and ?runx1 mutants, suggesting that REN is rewired to runx1 to stimulate its transcription. There are two previous descriptions for how enhancers are activated during regeneration. First, cis-regulatory sequences are reactivated from embryogenesis and second that active adult enhancers are further invigorated during regeneration. Our data point to a third and previously unappreciated mechanism for gene control during zebrafish heart regeneration. We report that an enhancer is repurposed both from one gene and cardiac domain to activate a different nearby gene in regenerating cardiac tissue. Overall design: Comparitive gene expression analysis of REN mutant and Wildtype hearts under uninjured and regenerating (7dpi) conditions using bulk RNA sequencing.