Injury-responsive genetic elements seem to direct gene expression following injury, but whether specific elements drive regeneration in vertebrates is unclear. Now, Wang et al. describe regeneration-responsive enhancers (RREs) that drive a regeneration response programme (RRP) in killifish and zebrafish.
The authors amputated the caudal fin of killifish and zebrafish, noting that their regeneration was physiologically similar 1 day post amputation, despite the evolutionary distance between them (~230 million years). Genetically, chromatin immunoprecipitation followed by sequencing identified more putative RREs (marked by H3K27ac) in zebrafish than in killifish, and only a small portion of these RREs were linked to the same active promoter (marked by H3K4me3) in both species. In addition, RNA sequencing (RNA-seq) revealed twice as many changes in gene expression in zebrafish as in killifish, less than half of which were conserved. Thus, caudal fin regeneration is genetically more complex in zebrafish than in killifish.
However, looking at the putative RREs and upregulated genes common to both species, the authors identified 49 genes with H3K27ac-marked RREs, H3K4me3-marked promoters and elevated expression. These genes included known regulators of zebrafish regeneration and were enriched in blastema cells in both species, suggesting that a conserved RRP might be regulated by RREs. Of note, published RNA-seq data sets for mouse species that respond to injury with limited regeneration (Acomys cahirinus) or scarring (Mus musculus) showed that the expression of RRP genes following ear pinna and skin injury differed between these mice, suggesting that a RRP may determine regeneration potential in vertebrates.
To confirm whether implicated enhancers drive gene expression during regeneration, the authors cloned five of the putative enhancers into GFP constructs, observing that they drove GFP expression in the blastemal region of killifish following caudal fin amputation. Furthermore, caudal fin and heart regeneration in killifish required a minimal region of the enhancer upstream of one copy of inhba (designated K-IEN); inhba encodes activin A, an effector of vertebrate regeneration. The zebrafish orthologue of K-IEN (Z-IEN), but not the human homologue, rescued fin regeneration in K-IEN–/– killifish. Thus, evolution of the RRE could underscore the loss of regeneration in higher vertebrates.
Finally, motif enrichment analyses indicated that the consensus 12-O-tetradecanoylphorbol-13-acetate responsive element (TRE) that is bound by the AP-1 transcription factor is essential for the RRP. Mutating all the TRE motifs in K-IEN and Z-IEN abolished the expression of GFP from reporter constructs in killifish and zebrafish, respectively, following injury. Furthermore, chemical inhibition of the JNK pathway, which prevents AP-1–TRE binding, inhibited tail regeneration in killifish. Thus, AP-1 motifs seem to be essential for the activation of RREs after amputation in evolutionarily distinct fish.
“regeneration-responsive enhancers … drive a regeneration response programme (RRP) in killifish and zebrafish”
This comparative study enabled evolutionarily conserved RRPs to be distinguished from species-specific responses to regeneration and may facilitate our understanding of the broad yet uneven distribution of regeneration in vertebrates.
Wang, W. et al. Changes in regeneration-responsive enhancers shape regenerative capacities in vertebrates. Science 369, eaaz3090 (2020)
Goldman, J. A. & Poss, K. D. Gene regulatory programmes of tissue regeneration. Nat. Rev. Genet. 21, 511–525 (2020)
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Wrighton, K.H. Enhancing regeneration. Nat Rev Genet 21, 718–719 (2020). https://doi.org/10.1038/s41576-020-00290-z