HDAC Inhibitor Titration of Transcription and Axolotl Tail Regeneration

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.

Key Proteins for Regeneration inA. mexicanum: Transcriptomic Insights from Aged and Juvenile Limbs

“…These modules contained several proteins involved in histone modification and DNA methylation, including DNMT3A , HDAC2 , SUV39H1 , KDM3A and the Polycomb group members EED and EZH2 . Epigenetic regulation has been shown to be crucial for tissue regeneration in axolotls, pharmacological inhibition of DNA methyltransferases causes impairments in blastema formation (Aguilar & Gardiner, 2015) and histone deacetylase inhibitors inhibit regeneration (Voss et al, 2021). The Polycomb group is also known for its role in embryonic development and tissue differentiation, suggesting its likely importance in tissue regeneration for axolotls due to its evolutionary conservation (Whitcomb et al, 2007).…”

Axolotl: A resourceful vertebrate model for regeneration and beyond

“…A study employing snRNA‐seq methodology proposed a model for histone deacetylase (HDAC) mediated transcriptional regulation during tail regeneration 91 . Interestingly, this model suggests that HDAC displays a position‐specific activity between regeneration permissive and inhibitory genes.…”

“…A study employing snRNA-seq methodology proposed a model for histone deacetylase (HDAC) mediated transcriptional regulation during tail regeneration. 91 Interestingly, this model suggests that HDAC displays a positionspecific activity between regeneration permissive and inhibitory genes. Histone proteins located around enhancer and promoter of gene clusters promoting regeneration bear more acetyl groups than the ones located in intergenic and gene body regions.…”

“…A differential acetylation profile is provided by HDAC activity and is essential for proper tail regeneration. 91 While transcriptome studies identify essential genes and gene networks that extensively contribute to regeneration, the regulatory circuits controlling gene expressions post-transcriptionally are presented by miRNome projects. miRNAs are small non-coding RNAs able to control gene expression by mRNA cleavage or decay.…”

“…On the contrary, histones in the gene clusters with regeneration inhibitory functions are more acetylated from the gene body or intergenic parts compared to promoter and enhancer regions. A differential acetylation profile is provided by HDAC activity and is essential for proper tail regeneration 91 …”