Abstract
BackgroundDuring eye lens development the embryonic vasculature regresses leaving the lens without a direct oxygen source. Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 (HIF1), in the regulation of genes required for lens fiber cell differentiation, structure and transparency. Here, we employed a multiomics approach combining CUT&RUN, RNA-seq and ATACseq analysis to establish the genomic complement of lens HIF1α binding sites, genes activated or repressed by HIF1α and the chromatin states of HIF1α-regulated genes.ResultsCUT&RUN analysis revealed 8375 HIF1α-DNA binding complexes in the chick lens genome. One thousand one hundred ninety HIF1α-DNA binding complexes were significantly clustered within chromatin accessible regions (χ2 test p < 1 × 10− 55) identified by ATACseq. Formation of the identified HIF1α-DNA complexes paralleled the activation or repression of 526 genes, 116 of which contained HIF1α binding sites within 10kB of the transcription start sites. Some of the identified HIF1α genes have previously established lens functions while others have novel functions never before examined in the lens. GO and pathway analysis of these genes implicate HIF1α in the control of a wide-variety of cellular pathways potentially critical for lens fiber cell formation, structure and function including glycolysis, cell cycle regulation, chromatin remodeling, Notch and Wnt signaling, differentiation, development, and transparency.ConclusionsThese data establish the first functional map of genomic HIF1α-DNA complexes in the eye lens. They identify HIF1α as an important regulator of a wide-variety of genes previously shown to be critical for lens formation and function and they reveal a requirement for HIF1α in the regulation of a wide-variety of genes not yet examined for lens function. They support a requirement for HIF1α in lens fiber cell formation, structure and function and they provide a basis for understanding the potential roles and requirements for HIF1α in the development, structure and function of more complex tissues.
Highlights
During eye lens development the embryonic vasculature regresses leaving the lens without a direct oxygen source
The lens resides in a hypoxic environment [4,5,6, 44], lens deletion of Hypoxia inducible factor 1 alpha (HIF1α) disrupts lens cell structure and development [17] and hypoxia and HIF1α are required for the elimination of non-nuclear organelles during mature lens cell formation through activation of the BNIP3L gene [8]
To establish the range and spectrum of lens genes regulated by HIF1α, we first sought to establish the genomic-level occupancy of HIF1α-DNA binding complexes in primary lens cells upon HIF1α activation using Cleavage Under Targets & Release Using Nuclease (CUT&RUN) (Fig. 1A-B)
Summary
During eye lens development the embryonic vasculature regresses leaving the lens without a direct oxygen source Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. Studies on interior lens oxygen levels reveal a 20-fold decrease in oxygen levels from the epithelial cellcontaining surface of the lens to the differentiating lens fiber cells [5] This oxygen gradient parallels a widevariety of hallmark events that characterize the formation of mature lens fiber cells including the elimination of cellular organelles and the expression of critical genes required for lens fiber cell transparency and function
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