Abstract
Purpose: :
Genetic inactivation of many chromosomal loci in differentiated eukaryotic cells is correlated with formation of compact blocks of facultative heterochromatin. In terminally differentiated cells, chromatin that undergoes extensive condensation acquires special pattern of histone (epigenetic) modifications typical for heterochromatin. Di-/ trimethylation of histone H3 lysine 4 and acetylation of histones H3 and H4 are characteristic for actively transcribed genes, while di-/ trimethylation of histone H3 lysine 9 are manifestation of inactive state of chromosomal loci. Development of mammalian retina is characterized by progressive changes in chromatin status and accumulation of heterochromatin inside rod nuclei. The goal of this study is to explore epigenetic regulation during retina development.
Methods: :
C57BL/6j mice were from Jackson Laboratory. Animal protocols were in accordance with ARVO / IACUC guidelines. Eyeballs were collected from embryonic day 12.5 to postnatal day 28 and tissue arrays were specifically made for this study. Arrays of paraffin sections of mouse eyes were stained with epigenetic chromatin markers, H3K4me2, H3K9me3, H3K27me3, and H4K20me3 (all antibodies from Upstate, Charlottesville, VA) with DAPI counterstaining. ChIp-Seq analysis of retina chromatin was performed with same set of antibodies.
Results: :
We found that all antibodies show specific staining in cell nuclei within distinct cell types at different developmental stages. H3K4 methylation, a marker of actively transcribed genes, is very pronounced during early embryonic stages, with maximum around E18.5 in both inner and outer retina layers. In late stages it’s sharply decreased from outer retina layer, and after PN3 positive staining is observed only in inner retina. Accompanying rod photoreceptor maturation, H3K4me2 positive cells are completely lost in outer nuclear layer of retina. ChIP-Seq data shows that H3K4me2 differentially marks gene promoter regions during retina maturation. Three histone modifications associated with silent chromatin - H3K9me3, H3K27me3, and H4K20me3 - all show distinct and dynamic distribution patterns during retina development.
Keywords: retinal development • gene/expression • photoreceptors