HDAC Regulates Transcription at the Outset of Axolotl Tail Regeneration

Tissue regeneration is associated with complex changes in gene expression and post-translational modifications of proteins, including transcription factors and histones that comprise chromatin. We tested 173 compounds designed to target epigenetic mechanisms in an axolotl (Ambystoma mexicanum) embryo tail regeneration assay. A relatively large number of compounds (N = 31) reproducibly inhibited tail regeneration, including multiple histone deacetylase inhibitors (HDACi). In particular, romidepsin potently inhibited tail regeneration when embryos were treated continuously for 7 days. Additional experiments revealed that romidepsin acts within a very narrow, post-injury window. Romidepsin treatment for only 1 minute post-amputation inhibited regeneration through the first 7 days, however after this time, regeneration commenced with variable outgrowth of tailfin tissue and abnormal patterning. Microarray analysis showed that romidepsin altered early, transcriptional responses at 3 and 6-hour post-amputation, especially targeting genes that function in the regulation of transcription, cell differentiation, cell proliferation, pattern specification, and tissue morphogenesis. Our results show that HDAC activity is required at the time of tail amputation to regulate the initial transcriptional response to injury and regeneration. Ambystoma mexicanum embryos were first treated by an HDAC inhibitor (10um of Romidepsin or Belinostat) and amputated 2mm from tail tip. After amputation, 1 mm of the distal tail tip was collected at time of amputation (0), 3, 6, 12, 24, 48, or 72 hours post amputation (hpa). Additionally, untreated controls were also collected from animals which were not treated by an HDAC inhibitor. A total of 336 embryos were used, with each replicate representing a pool of 8 embryo tail tips.