Abstract
Purpose :
The developing lens is an advantageous model for understanding cell fate determination, tissue-specific gene expression and general mechanisms of gene control. To further elucidate transcriptional mechanisms, understanding the three-dimensional (3D) dynamics of chromatin structure is necessary. During lens development, lens fiber cells undergo denucleation prior to their terminal differentiation to maximize the lens transparency. During this process, lens fiber cells must maintain the critical transcriptional and translational resources necessary to produce crystallin proteins at the highest levels found in nature.
Methods :
We performed comparative Hi-C analyses of newborn (P0.5) mouse lens epithelium, lens fibers and embryonic stem (ES) cells. Using ChIP-seq, we obtained lens chromatin landscape of the CCCTC-binding factor (CTCF). These data were integrated with our earlier RNA polymerase II (Pol II), ChIP-seq, and ATAC-seq data. Using the Juicer pipeline, we mapped chromatin looping, topologically associated domains (TADs), A/B compartment analysis and annotated candidate promoter/enhancer regions as well as open/closed chromatin states. Subcellular localization of chromatin insulator protein CTCF was determined by immunofluorescence in E14.5 and P0.5 lens epithelium and fiber cells.
Results :
A series of comparative analyses between ES cells, lens epithelium and lens fiber cells showed major differences between all three cell types in chromatin loop and TAD sizes, as well as A/B compartmental changes. Lens cell chromatin organization is markedly different compared to ES cells, most notably at the compartment level and at lens associated loci such as Pax6, Hif1a, and crystallin genes (mostly located in “loop deserts”). Unexpectedly, lens fiber cell chromatin showed broad CTCF peaks overlapping with Pol II at highly expressed crystallin loci. Furthermore, CTCF localization is markedly different between lens epithelium and fiber cell, showing translocation to the nucleoli in lens epithelium.
Conclusions :
For the first time, we generated a dataset to analyze nuclear organization in lens epithelium and lens fiber cells. These data give further insights into the 3D-genomic landscape and lens-specific transcriptional gene control. The current data provide a new perspective on the role of CTCF in transcriptional control of highly expressed lens-specific genes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.