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R. C. Rao, K. Tchedre, D. Chen; Expression of the Repressive Epigenetic Modification H3K27 in the Developing Murine Retina and Human Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1291.
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Histone H3 methylation at lysine 27 (H3K27) regulates development, stem cell pluripotency, tumorigenesis, and inflammation. Methylation at this residue is associated with heterochromatin and is directed by the histone methyltransferase, Ezh2, whose activity can be modulated by phosphorylation. However, very little is known about this repressive epigenetic modification in the retina. Here we analyze H3K27 and phospho-Ezh2 expression in the developing and adult mouse retina and human retinoblastoma cell lines.
Expression levels of epigenetic markers H3K27 and p-Ezh2 in murine and human retinal tissues were determined by immunohistochemistry, and western blotting. Sections of embryonic and adult retinae were harvested at E16, E18, P0, and adult. Three human retinoblastoma cell lines, Rb143, Rb116, and Rb107 were grown in suspension and cultured in DMEM/F-12/B27 and fixed on coverslips prior to immunocytochemical processing. Protein lysates were taken from the above tissues to be analyzed by western blotting.
There were distinctive patterns of H3K27 and p-Ezh2 expression in the developing mouse retina and human retinoblastoma cell lines. H3K27 and p-Ezh2 were largely expressed in the retinal ganglion cell (RGC) layer at all ages analyzed. Western blotting of whole retina lysates demonstrated enrichment of the H3K27 mark in adult retina when compared to the embryonic retina. While all human retinoblastoma cell lines appeared to express p-Ezh2, a subpopulation of cells appeared to express the protein at higher levels. We are currently using other proliferation-associated markers to elucidate the properties of p-Ezh-"high" retinoblastoma cells and plan to use chromatin immunoprecipitation to identify genes in retinal cancers and development associated with this epigenetic modification.
The repressive epigenetic H3K27 mark, known to be modulated by Ezh2 activity, appears to be spatially and temporally regulated in the murine retina and in human retinoblastoma cells. Elucidation of the epigenetic patterns underlying retinal development and cancer may offer insights into retinal regeneration and reveal novel drug targets in retinoblastoma.
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