Humans have almost no regenerative capability following limb loss, resulting in decreased quality of life. In contrast, amphibians have the capability to scarlessly regenerate appendages. Following injury, complex genetic processes need to be initiated with high spatial and temporal resolution. Xenopus tropicalis are diploid frogs, whose tadpoles scarlessly regenerate properly patterned tails after amputation in approximately 72 hours, making them an ideal system to query genomic changes that occur during regeneration. An Assay for Transposase Accessible Chromatin (ATAC-seq) in Xenopus has shown that at 6 hours post amputation (hpa) differentially accessible regions of chromatin are inaccessible, and then at 24 hpa specific regions of chromatin regain accessibility. This leads to the question: how are transient changes in chromatin accessibility achieved? Studies on transient changes in gene expression during development across species have elucidated two factors of regulation: changes in epigenetic state, and nuclear lamina association. EZH2 methylates H3K27 and has a critical role in development. EZH2 has also been shown to be required for regeneration in a variety of model systems including Xenopus. H3K27Me3 is an epigenetic mark that can correspond to facultative heterochromatin, and when found in conjunction with H3K4Me3 can be a mark of ?poised? regions of chromatin, however, H3K4Me3 alone is a mark of active promoters. By studying the dynamics of methylation state and lamina association, I will gain insight into the dynamics of several different chromatin states over the course of regeneration and how these changes alter chromatin accessibility.
Aim 1 will focus on the spatiotemporal dynamics of transient changes in epigenetic state, by determining the distribution of H3K27Me3, H3K4Me3, and regions of chromatin that interact with the nuclear lamina. I will identify if regions of developmental enhancers and promoters that are transiently inactivated during the early stages of regeneration.
In Aim 2 I will query how changes in methylation state alter chromatin accessibility and gene expression during regeneration. This study will utilize cutting edge assays such as CUT&RUN and ATAC-seq together with functional analyses to probe the mechanism of transient changes in genomic programming during regeneration and how this may parallel developmental processes.
Dynamic changes in gene expression is critical for the regulation of the complex process of regeneration, however little is known about how transient repression of genes is achieved during regeneration. Increased methylation on residues such as H3K27, along with increased association with the nuclear lamina have been shown in the context of development to inactivate regions of chromatin, however it is unknown if these processes are reutilized in the context of regeneration. The proposed research uses Xenopus tropicalis tail regeneration as a system that is tractable for state-of-the-art epigenomic studies to query how transient gene repression is achieved during regeneration.
