June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Multiomic analysis of HIF1a binding, regulation and gene expression combined with characterization of novel lens conditional HIF1a KO mice reveals essential requirements for hypoxia and HIF1a in mature lens formation
Author Affiliations & Notes
  • Joshua Disatham
    Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida, United States
  • Lisa A Brennan
    Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida, United States
  • Michael L Robinson
    Biology, Miami University, Oxford, Ohio, United States
  • Marc Kantorow
    Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida, United States
  • Footnotes
    Commercial Relationships   Joshua Disatham None; Lisa Brennan None; Michael Robinson None; Marc Kantorow None
  • Footnotes
    Support  NIH Grant EY029708
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 522. doi:
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      Joshua Disatham, Lisa A Brennan, Michael L Robinson, Marc Kantorow; Multiomic analysis of HIF1a binding, regulation and gene expression combined with characterization of novel lens conditional HIF1a KO mice reveals essential requirements for hypoxia and HIF1a in mature lens formation. Invest. Ophthalmol. Vis. Sci. 2023;64(8):522.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We have demonstrated that organelle-elimination during lens fiber cell maturation is dependent on the hypoxic environment of nascent fiber cells and resulting activation of transcription factor HIF1a that initiates mitophagy through BNIP3L synthesis. Here we sought to further delineate the potential genome-wide regulatory functions and requirements for HIF1a in mature lens formation.

Methods : We employed a multiomic approach involving CUT&RUN, ATAC-seq and RNA-seq to identify a wide-variety of genes and their regulatory networks requiring HIF1 for their expression and therefore function. We also used MLR10Cre to generate lens conditional KO mice and characterized the lenses for altered morphology, organelle-elimination, activation of BNIP3L and other key requirements for mature lens fiber formation. Briefly, primary embryonic chick lenses were cultured in the HIF1a-specific activator DMOG for 4 hours then CUT&RUN was employed using a HIF1a-specific antibody for targeted cleavage, release, and sequencing of HIF1a-bound DNA fragments. The data was then integrated with parallel RNA-seq to identify HIF1a-specific target genes activated or repressed upon HIF1a activation by DMOG. We also employed RNAseq of MLR10Cre HIF1a-conditional KO P1 mice lenses to functionally identify the HIF1a-dependent genes in the mouse model.

Results : CUT&RUN analysis integrated with ATACseq and RNAseq analysis of HIF1a-activated primary lens cells revealed 8,375 HIF1a-DNA binding complexes clustered within open chromatin regions that paralleled the activation or repression of 526 genes. Key gene networks identified included those functioning in glycolysis, cell cycle control, chromatin remodeling, Notch and Wnt signaling, differentiation, and transparency. Correspondingly, lens conditional HIF1a-KO mice exhibit smaller lenses, disrupted fiber cell structure, and organelle retention.

Conclusions : Collectively these data establish HIF1a as a master regulator of lens fiber cell gene expression and maturation, they further implicate hypoxia as a novel requirement for mature lens formation and transparency and they identify novel gene regulatory networks required to achieve mature lens fiber cell structure and function.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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