Abstract
Whole-body regeneration (WBR) is the process through which a fully functional organism is regrown from a fragment of tissue. This extraordinary ability is exhibited by the colonial ascidian, Botrylloides diegensis, which can complete WBR from just 100-200 cells of the vasculature in 10 days. This requires a series of many cellular and molecular events to occur. Previous studies have found that Wnt-, Notch- and retinoic acid-signalling and histone deacetylases (HDACs) are required for successful WBR. However, the regulatory mechanisms behind this process remain unclear. This research aimed to identify key regulatory regions and transcription factors (TFs) involved in WBR in B. diegensis.
Using previously collected assay for transposase-accessible chromatin (ATAC)-seq data, enriched TF motifs were identified in differentially accessible regions (DARs) between vascular fragments at regeneration stages 0 and 2 and between stage 2 and stage 2 HDAC- inhibited (HDACi) fragments. Motifs were predominantly enriched in closed DARs in stage 0 vs. 2 data, and in open DARs in stage 2 vs. 2-HDACi data as HDACs condense chromatin, thus when inhibited more regions are open and accessible. Erg, Jund, Foxo, and Myc motifs were chosen for further analysis due to the proteins’ known functions in vasculature and stem cells. Genomic annotation revealed that the majority of these motifs were situated in promoters and in distal intergenic regions where enhancers may be located.
B. diegensis orthologues of the human ERG, JUND, FOXO, and MYC genes were identified by constructing phylogenetic trees. These TFs are expressed during WBR, as revealed in previously published RNA-seq data. Using qPCR to measure gene expression, it was found that Jund expression was significantly increased in stage 2-HDACi tissues compared to controls, but not Erg or Foxo. In situ hybridisation assays identified candidate TF expression in blood cells of stage 2 control sections as well as Jund and Myc expression in adult vasculature.
Finally, CUT&RUN was carried out to map H3K4me3 histone modifications, which are associated with active gene expression and open chromatin, to the genome in stage 2 tissues. These modifications were mapped directly upstream of the TSS where promoters are typically located. Additionally, genomic annotation revealed that H3K4me3 modifications were mostly situated in promoters and distal intergenic regions.
These findings provide a basis to understanding the role of the candidate TFs in B. diegensis WBR, as well as the presence of histone modifications and their ability to mark regulatory regions, such as promoters.