Abstract
Purpose :
Cellular differentiation is marked by temporally and spatially coordinated gene expression regulated at multiple levels, including chromatin landscape dynamics. DNA methylation represents a universal mechanism to control chromatin organization, compaction and its accessibility.Cytosine methylation of CpG dinucleotides regulates binding of methylation-sensitive DNA-binding transcription factors. Earlier studies in mouse lens examined function of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b; however, analysis of DNA methylation during lens fiber cell differentiation remains to be determined.
Methods :
Using whole genome bisulfite sequencing (WGBS), we investigated dynamics of DNA methylation changes during mouse lens fiber cell and epithelium differentiation between embryos (E14.5) and newborns (P0.5) using microdissected lenses. Embryonic stem (ES) cells and neural progenitor cells (NPCs) data were used for comparisons. To link these data with general chromatin structure and gene expression, our earlier ATAC- and RNA-seq datasets were included. Role of CpG methylation on Pax6 binding was analyzed using lens ChIP-seq data and in vitroanalyses using a set of methylated and unmethylated prototypic Pax6-binding sites.
Results :
Major differences in DNA methylation between lens, ES and NPC cells were found. For example, both Pax6and Prox1loci show low and unmethylated regions in lens cells compared to both ES and NPC cells. Within the microdissected lens cells, we identified DNA methylation and “open” chromatin changes by defining differentially methylated and/or accessible regions during mouse lens fiber cell (Path1: epi/E14.5-fib/E14.5-fib/P0.5) and epithelium (Path2: epi/E14.5-epi/P0.5) differentiation. For example, ATAC- and RNA-seq data demonstrate that reduced methylation is directly linked with increased expression of fiber cell abundant genes, including crystallins, intermediate filament proteins Bfsp1 and Bfsp2, and gap junction proteins Gja1 and Gja8. Both in vivoand in vitrodata show that CpG methylation is not detrimental for Pax6 binding to DNA.
Conclusions :
Our study has generated the first data on DNA methylation changes between two different mouse lens developmental points and linked these data with chromatin accessibility domains and gene expression. Our data support the model that between E14.5 and P0.5, DNA demethylation modulates gene expression of critical genes required for lens morphogenesis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.