Hypoxia-driven histone modifications govern gene regulation for mature eye lens formation

Evidence suggests that the hypoxic environment of the lens regulates the expression of genes required for lens formation and function. Here, we tested the hypothesis that hypoxia regulates these genes through induction of specific histone modifications. Global levels of hypoxia induced histone modifications were determined in cultured day 13 chick lenses exposed to 1% oxygen. The genome-wide localizations of H3K27ac and H3K4me3 were identified by CUT&RUN analysis and mapped to within 5kb of the transcriptional start sites of genes activated or repressed by hypoxia identified by RNA Seq. The requirement for these histone modifications for hypoxia induced gene expression was determined using inhibitors of histone writers and erasers. The levels of activating modifications H3K4me3, H3K9ac, H3K14ac and H3K27ac in hypoxia treated lenses increased while levels of repressive modifications H3K9me3 and H3K27me3 remained unchanged. Hypoxia-specific H3K4me3 and/or H3K27ac regions were detected within 5 kb of the transcriptional start sites of over 900 fiber cell genes with increased expression upon hypoxia exposure and 350 genes with decreased expression. Inhibition of histone writers and erasers resulted in altered levels of key fiber cell genes upon hypoxia exposure. The results provide evidence that hypoxia induced histone modifications regulate genes required for mature lens formation and they provide a framework for understanding the role of hypoxia specific histone modifications in the regulation of genes in more complex tissues. Overall design: Whole embryonic chick lenses were incubated under normoxia (21% O2) or hypoxia (1% O2) conditions for 2H or 8H. Following incubation lenses were microdissected into epithelial and fiber cell portions and the fiber cells used for downstream analysis. CUT&RUN analysis was used to mapp genome wide histone marks resulting from exposure to hypoxia. RNAseq was used to determine gene expression changes over 8H of hypoxia treatment.