Friday, April 4, 2025
4/4/2025
Hypoxia regulation of the lens - Project Summary/Abstract During ocular development, the vasculature feeding the eye lens retracts leaving the developing lens without a direct source of oxygen. This results in a 20-fold drop in oxygen levels from the undifferentiated epithelial cells of the lens surface to the mature fiber cells of the lens core. Intriguingly, the sharpest drop in lens oxygen levels occurs in the region of the lens where newly formed fiber cells initiate the rapid and robust expression of essential genes required to achieve their mature structure and transparency. These unique features of the lens lead us to hypothesize that the hypoxic microenvironment of nascent fiber cells drives transcription of essential fiber cell genes to achieve their mature structure and function through activation of novel hypoxia- dependent transcriptional regulatory and epigenetic control pathways. This hypothesis is supported by a study showing that lens exposure to artificially high oxygen levels disrupts formation of the lens organelle-free zone (OFZ) that forms from elimination of organelles during lens fiber cell maturation and by a study showing that lens-specific deletion of the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1a, (HIF1a) disrupts lens fiber cell structure and results in disintegration of the entire lens shortly after birth. These studies provide a foundation for AIM1: To establish the requirements for hypoxia, HIF1a and HIF1a- co-regulators for lens fiber cell transcription, mature lens structure and lens transparency. The feasibility of this AIM is supported by our recent CUT&RUN studies showing that HIF1a binds within -10kb of the 5’- regulatory regions of over 500 lens fiber cell genes and that HIF1a binding to these regions directly correlates with their fiber cell transcript levels. These include the mitophagy gene BNIP3L that directs the elimination of non-nuclear organelles to form the OFZ. We have now discovered that exposure of cultured chick lenses to hypoxia results in rapid and robust increases in fiber cell levels of gene activating histone modifications including H3K4me3, H3K9ac, H3K14ac and H3K27ac. These studies provide a foundation for AIM2: To identify novel hypoxia-dependent epigenetic pathways governing transcription of essential lens fiber cell genes. The feasibility of this AIM is supported by our preliminary CUT&RUN studies showing that hypoxia-exposure of cultured chick lenses induces H3K27ac and H3K4me3 modification within -10kb of the 5’-regulatory regions of a wide range of fiber cell genes and that these modifications correlate with their fiber cell expression levels. The completion of this application will establish hypoxia as a novel requirement for adult lens structure and transparency through activation of novel hypoxia-dependent transcription and epigenetic pathways regulating lens fiber cell transcription. The data will provide a foundation for establishing a role for hypoxia in formation of more complex tissues.
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