Abstract
Purpose:
Retinal pigment epithelial (RPE) cells are one of the few cell types well recognized to change fate in lower vertebrates. We recently showed adult human RPE as old as 99 years differentiate down mesenchymal and neural lineages using commercially available differentiation media. Addressing whether this was due to the multipotency of the RPE cells or to a contaminating cell type, for example MSCs, in the cultures was critical. Therefore, we demonstrated that single, cloned RPE cells exhibit this ability, irrefutably demonstrating that they are multipotent cells. We therefore sought to understand the basis for this observed plasticity. Histone marks at promoter and enhancer sites can reveal both genes that are actively being transcribed - i.e. active, and genes that are poised - i.e. currently inactive, but can be activated by under appropriate conditions. Recently histone H3K27me3 mark has been shown to be enriched in regions several kilobases upstream from the promoter around enhancer regions that are poised in human embryonic stem cells (hESCs). In contrast, histone mark H3K27ac marks similar locations when the downstream gene is active. Therefore, we hypothesized that these two marks can predict gene changes from being poised to active.
Methods:
We cultured human retinal pigment epithelium into polarized layers and verified their terminal differentiation through transepithelial resistance (TER) in Ωcm2. These monolayers expressed TER of > 200Ωcm2 similar to native RPE. We also confirmed their identity immunohistochemically, by quantitative PCR. We then conducted H3K27ac and H3K27me3 Chromatin immunoprecipitation-sequencing (ChIP-seq) on adult human RPE monolayers.
Results:
We found RPE specific genes posses H3K27ac marks upstream of their promotor regions, whereas the genes we have found to increase expression during differentiation, for example RUNX2 during osteogenic conditions, possess the poised mark H3K27me3 upstream of their promotor.
Conclusions:
These results suggest the histone marks can predict not only the cell identity, but also the poised mark can predict RPE plasticity.
Keywords: 412 age-related macular degeneration •
701 retinal pigment epithelium •
721 stem cells