Abstract
New patterns of gene expression are enacted and regulated during tissue regeneration. Histone deacetylases (HDACs) regulate gene expression by removing acetylated lysine residues from histones and proteins that function directly or indirectly in transcriptional regulation. Previously we showed that romidepsin, an FDA-approved HDAC inhibitor, potently blocks axolotl embryo tail regeneration by altering initial transcriptional responses to injury. Here, we report on the concentration-dependent effect of romidepsin on transcription and regeneration outcome, introducing an experimental and conceptual framework for investigating small molecule mechanisms of action. A range of romidepsin concentrations (0–10 μM) were administered from 0 to 6 or 0 to 12 h post amputation (HPA) and distal tail tip tissue was collected for gene expression analysis. Above a threshold concentration, romidepsin potently inhibited regeneration. Sigmoidal and biphasic transcription response curve modeling identified genes with inflection points aligning to the threshold concentration defining regenerative failure verses success. Regeneration inhibitory concentrations of romidepsin increased and decreased the expression of key genes. Genes that associate with oxidative stress, negative regulation of cell signaling, negative regulation of cell cycle progression, and cellular differentiation were increased, while genes that are typically up-regulated during appendage regeneration were decreased, including genes expressed by fibroblast-like progenitor cells. Using single-nuclei RNA-Seq at 6 HPA, we found that key genes were altered by romidepin in the same direction across multiple cell types. Our results implicate HDAC activity as a transcriptional mechanism that operates across cell types to regulate the alternative expression of genes that associate with regenerative success versus failure outcomes.
Highlights
Transcription differs within and between cell types and varies in response to extrinsic and intrinsic cues, such as when cells are challenged by pathogens or when cells respond to signaling molecules during development
We showed that 10 μM romidepsin, applied for 1-min post-amputation or longer, inhibits axolotl embryo tail regeneration at 6 days postamputation (DPA) (Voss et al, 2019)
In this study we evaluated a method for detailing transcriptional changes that associate with alternative regeneration outcomes
Summary
Transcription differs within and between cell types and varies in response to extrinsic and intrinsic cues, such as when cells are challenged by pathogens or when cells respond to signaling molecules during development. By quantifying transcription at concentrations that span a chemical’s critical threshold concentration, it might be possible to identify quantitative changes in key genes that determine regeneration outcome, and through subsequent experimental, computational and bioinformatic approaches, associate these quantitative changes to biological processes and properties of cell populations. We evaluate this approach using romidepsin (Ueda et al, 1994; Yang et al, 2011), an FDA approved histone deacetylase inhibitor that potently inhibits axolotl embryo tail regeneration. Application of this approach to additional chemicals offers potential to develop rich information resources that can be used to characterize and model chemical effects and gene interactions on tissue regeneration, identify promising chemical tools for regenerative biology, and identify chemical and biological mechanisms of action
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