Abstract
Purpose :
The purpose of this study is to determine how a cell becomes epigenetically reprogrammed to a myofibroblast fate associated with lens fibrotic disease. For cell reprogramming to occur, transcription factors (TF) must overcome a condensed chromatin barrier typically marked by H3K27me3. Here, we investigated how chromatin structure becomes re-established shortly after DNA replication in myofibroblast progenitor cells to identify the “window of opportunity” for the adoption of a new pro-fibrotic transcription program.
Methods :
Studies were performed in an ex-vivo mock cataract surgery wound healing/fibrosis chick embryo model to examine cell reprogramming to a myofibroblast fate, which occurs by 72hr post-injury. The chromatin assembly assay (CAA) was used to study how chromatin structure becomes re-established at the time of replication during cell reprogramming. To determine if TF binding occurs during replication, S phase entry was blocked by thymidine treatment. We examined how blocking ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) demethylase function impacts both wound healing and reprogramming to a fibrotic phenotype.
Results :
CAA revealed a delay in the accumulation of H3K27me3 on nascent DNA at 28hr post-injury, while at 48hr post-injury H3K27me3 accumulation was rapid. Thus, revealing a “window of opportunity” when chromatin is decondensed and amenable to new TF binding for cell reprogramming. The status of H3K27 is controlled by H3K27 modifying enzymes, the demethylase UTX and the methyltransferase enhancer of zeste homolog 2 (EZH2). By CAA, both UTX and EZH2 were recruited to nascent DNA at 28hr post-injury. However, blocking UTX function led to an accumulation of H3K27me3 to nascent DNA. This suggests that UTX masks EZH2 function to maintain a decondensed chromatin state for cell reprogramming. Recruitment of the pro-fibrotic TF, myocardin related transcription factor A (MRTF-A) to nascent DNA occurred during the “window of opportunity” and specifically at the time of replication. Lastly, we found that UTX demethylase function was required for MRTF-A binding to nascent DNA and transition to a fibrotic phenotype without preventing wound healing.
Conclusions :
UTX demethylase activity is required to decondense post-replicative chromatin structure to make it amenable to the adoption of a new fibrotic transcription program.
This is a 2021 ARVO Annual Meeting abstract.