Revisiting Polycomb Repression in Appendage Regeneration

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PROJECT SUMMARY Adult zebrafish rapidly regenerate amputated fins, including complex skeletons, back to their original size and form. Lineage-restricted progenitor cells derived from injury-induced, dedifferentiated mature cells proliferate and re-differentiate to restore lost fin tissue. Therefore, understanding the control of cell state transitions between differentiated and progenitor states, with retained cell identities, is central to understanding robust appendage regeneration. Profound changes in gene expression programs drive dedifferentiation and re- differentiation state transitions. Chromatin landscapes are assumed to stabilize both progenitor and differentiated state programs and therefore regulated chromatin dynamics likely underlie state transitions. Further, chromatin mechanisms are thought to epigenetically maintain grid-like positional identities that direct the fin size-restoring amount of regenerative outgrowth. Polycomb Repressive Complex 2 (PRC2) represses gene expression by Ezh1/Ezh2-catalyzed methylation of lysine-27 of histone H3 (H3K27me). PRC2/H3K27me silences developmental regulatory genes in differentiated cells, stabilizes progenitor state programs, and maintains cell fate & positional identities, including Hox codes. We earlier linked the removal of H3K27me marks by upregulated histone demethylases to widespread gene activation associated with initiating fin regeneration. Recently, we targeted ezh1 and ezh2 to generate adult viable PRC2 mutant zebrafish. Strikingly, multiple rounds of fin regeneration occur normally despite greatly reduced global H3K27me2/3 levels accompanied by elevated, activation-associated H3K27 acetylation. Therefore, the bulk of H3K27me is not required for the regulated state transitions or maintenance of cell identities during fin regeneration. These results challenge PRC2/H3K27me3 dogma in a compelling vertebrate regeneration context. We will pursue exploratory studies to distinguish between several hypotheses explaining how fin regeneration proceeds without most of this major repressive histone modification. For Aim #1, we will profile genome-wide histone modification patterns in wildtype and PRC2-mutant regenerating fins using new CUT&Tag technology. We will use RNA-Seq to correlate the PRC2-dependent transcriptome with altered chromatin landscapes. In Aim #2, we will experimentally bypass lethality of our ezh1/ezh2 mutants by transiently expressing Ezh2 during embryonic development using mRNA injections and inducible transgenic approaches. We will characterize regeneration defects, if any, in derived null PRC2 adults towards defining key H3K27me-controlled regulatory networks of fin regeneration. Combined outcomes will provide the central premise for a larger project studying chromatin dynamics and cell transitions of organ regeneration. Broader impacts include guidance on the use of chromatin-based perturbagens to enhance regenerative medicine and as therapeutics for pediatric gliomas, other cancers, and congenital defects caused by disrupted PRC2/H3K27me.