Abstract
Purpose :
Chromatin is a key modulator of gene expression through its ability to control transcription by regulating access to transcription factor binding sites. To date, the potential role of chromatin in the regulation of genes expressed at specific stages of lens differentiation has not been established. Here, we employed genome-wide ATAC sequencing and RNA sequencing to establish chromatin occupancy maps of four regions of the embryonic lens representing distinct stages of lens differentiation and we correlated these with the region-specific levels of 10421 transcripts identified by RNA sequencing.
Methods :
E13 chick lenses were micro-dissected into undifferentiated central epithelium (EC), equatorial epithelium where differentiation is initiated (EQ), nascent fiber cells (FP) and mature fiber cells (FC). Chromatin binding sites were mapped at +/- 10kb within individual gene bodies and between +/- 3kb of the start of transcription for each gene analyzed and transcript levels correlated with chromatin occupancy. All samples were analyzed in biological duplicate.
Results :
3318 genes exhibited highly significant Pearson correlation (r > 0.7) between altered chromatin accessibility and region-specific differences in transcript levels. Pairwise comparison revealed the majority of chromatin-gene expression links occur during the epithelial cell to fiber cell transition (EQ vs. FP, chi square p<1e-6). >100 genes with established lens functions exhibited altered expression with corresponding changes in chromatin occupancy including those required for fiber cell structure (CP49, Filensin), transcriptional control (GATA3, Pax-6, Sox2), lens homeostasis (CX46), autophagy regulation (mTOR) and crystallins (CRYBB1, CRYBB3). Analysis of differentially accessible chromatin regions identified consensus sequences for multiple transcription factors including TEADs, FOXs, AP-1, and HIF1a.
Conclusions :
The data provide evidence that chromatin regulation is a novel mechanism for control of gene expression during lens differentiation. The data implicate chromatin in the regulation of genes important for lens differentiation, homeostasis, structure and transparency and they identify multiple transcription factor binding sites likely critical for expression of these genes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.