Abstract
Purpose :
The lens epithelial explant system represents a powerful, in vitro approach to understand how extracellular signals coordinate fiber cell differentiation. In this system, growth factors present in the vitreous humor elicit widespread transcriptomic changes resulting in proliferation and the production of proteins characteristic of both inflammation and lens fiber cell differentiation (CD). Here, we document how vitreous treatment changes chromatin accessibility (CA) through time in lens epithelial explants.
Methods :
Chromatin extracted from 8-day-old FVB/N mouse lens epithelia was subjected to ATAC-sequencing at 5 different time points: immediately after collection, after 1 day in explant culture, and after an additional explant culture period in 50% bovine vitreous/media for 1, 5 and 10 days. The ATAC data was processed to identify open chromatin regions and associated genomic features. Furthermore, the differentially accessible regions (DARs) between different time points were identified and subjected to GO term analysis. Similarly, DARs were analyzed for over-represented motifs to identify candidate transcription factors (TFs) associated with observed accessibility changes. Finally, the ATAC-seq data was integrated with RNA-seq data to identify novel relationships between CA and transcriptomic changes associated with fiber CD.
Results :
Genome-wide analysis of CA indicates a general trend toward increased accessibility of cis-regulatory elements (CREs) for vitreous treatment. GO analysis of genes associated with DARs included eye development, inflammatory response, lens development in camera-type eyes, NIK/NF-kappa B signaling and PI3K signaling. Similarly, some of the top overrepresented motifs associated with DARs in the vitreous treated samples include the transcription factors ATF3, JUN, SOX21, SOX3 and TEAD3. These motifs are consistent with a recent report (Zhao et al. Epigenetics & Chromatin 2019) of these TFs in fiber CD. Fiber cell genes Dnase2b, Cryba4, Crybb1, Crygc, Cryba3 and inflammatory response genes Mmp3, Mmp10, Mmp9, Fn1 exhibit increased accessibility in response to vitreous, consistent with their increased transcription.
Conclusions :
Vitreous treatment results in TF-mediated chromatin remodeling in CREs and within gene bodies associated with fiber CD and inflammatory response genes.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.