Genetic coding of lens fibre cell differentiation

Abstract

AbstractPurpose: The DNA genetic code operates at multiple levels, including cis‐regulatory code of promoters and enhancers regulating transcription, cis‐regulatory code of 5′‐ and 3′‐UTRs in spliced mRNAs to control their localization and stability and 3D‐organization of chromatin within the nucleus.Methods: To study chromatin landscape dynamics during lens differentiation, ATAC‐ and RNA‐seq experiments were conducted using epithelia and fibres from E14.5 and newborn (P0.5) mouse lenses. Whole genome bisulfite sequencing (WGBS) was performed to map DNA methylation and integrate the data with corresponding ATAC‐ and RNA‐seq data sets. ChIP‐seq data were generated for transcription factor CTCF and histone H3.3 in microdissected newborn lenses. ChIP‐seq data on Pax6 and RNA polymerase II landscapes in whole newborn lenses were used for comparisons. A census of the most abundant RNA‐binding proteins (RBPs) in the lens was generated using mouse proteomic data. Immunoflourescence analyses were employed to visualize selected RBPs in the mouse lens.Results: Open chromatin and DNA methylation changes were defined through identification of differentially accessible regions (DARs), unmethylated/low methylated regions (UMRs/LMRs) coupled with differential gene expression during lens differentiation. Pax6 proteins were found both in open and closed chromatin domains, including both closed and methylated regions. As expected, histone H3 variant H3.3 is present all across crystallin and other highly expressed lens genes/loci. Novel abundant RNA‐binding proteins in lens include Carhsp1, Ddx5, Ddx39b, Fxr1, Pabpc1, Rbm24, Rbm38, Ybx1 and Ybx3.Conclusions: A combination of different –omics is a powerful approach to examine molecular mechanisms governing lens differentiation. Current studies reveal additional levels of genetic code complexity and stimulate follow up mechanistic studies of the individual cellular and molecular processes of lens morphogenesis and their underlying GRNs and identification of cataract‐causing mutation in non‐coding regulatory sequences. Ongoing studies are aimed to generate Hi‐C data to probe 3D‐organization of lens chromatin using microdissected newborn lenses.