Abstract
Purpose :
Our previous studies showed that the hypoxic microenvironment of nascent lens fiber cells drives the expression of genes required for the formation of the mature lens. Many of these genes are controled by the master regulator of the hypoxic response, transcription factor HIF1a, that is required for diverse fiber cell functions including elimination of non-nuclear organelles. Transcription factor function is dependent on chromatin accessibility and specific epigenetic modifications including histone modifications. Consistently, our previous studies have identified a link between genome-wide chromatin accessibility changes, DNA methylation patterns and HIF1a binding during lens fiber cell differentiation. Here, we tested the hypothesis that hypoxia drives genome-wide histone modifications to regulate gene expression for fiber cell differentiation.
Methods :
Using ex-vivo embryonic chick lens (E13) cultures we examined global histone modifications following exposure of lenses to hypoxia (1% O2). CUT & RUN was employed to map the genomic landscapes of key histone modifications associated with gene activation (H3K4me3, H3K27ac) under hypoxic conditions. Multiomic integration of the CUT n RUN data with corresponding RNA Seq data was conducted to establish the relationship between hypoxia regulation of histone modifications examined and the expression of lens fiber cell-specific genes.
Results :
Western blot analysis of histones extracted from lens fibers cells demonstrated a rapid and robust increase in multiple histone modifications that regulate chromatin accessibility and gene activation including H3K4me3, H3K27ac, H3K14ac and H3K9ac. CUT and RUN analysis of lens fiber cells following exposure to hypoxia identified increased H3K4 methylation and increased H3K27 acetylation in the promotor regions of a number of critical lens fiber genes including Maf, BNIP3L, BIRC7, Prox1 and EPHA2. These changes correlated with chromatin de-condensation previously established by ATAC sequencing.
Conclusions :
The results provide evidence that epigenetic programming through histone modifications drives lens fiber cell gene expression to achieve mature lens structure and transparency and that the lens epigenetic landscape is dependent on the hypoxic microenvironment of nascent lens fiber cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.