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M. Assadian, K. Torres, S. L. Merbs; Genome-Wide Identification of Genes That are Differentially Methylated in the Retina Compared to the Brain. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1690.
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We have already shown that the promoter and surrounding regions of Rbp3 and Rho are differentially methylated in photoreceptors, which express both genes, compared to other retina cells (and other tissues), which do not express them. We are now taking a broad look at how DNA methylation patterns differ between the retina and other tissues. For comparison, we have chosen the brain to allow us to focus on mechanisms that mediate gene expression differences between neuronal subtypes.
Mouse brain and whole retina DNA was isolated, fragmented and enriched for methylated DNA using three different products for affinity purification of methylated DNA: (1) MethylMiner (Invitrogen), which uses the methyl-CpG binding domain of human MBD2 protein; (2) MethylCollector (Active Motif), which uses an MBD2b/MBD3L1 protein complex; and (3) a homemade MBD2-MBD protein immobilized on magnetic beads. Enrichment was confirmed using QPCR of the transcription start site regions of Rbp3 and Rho (which are relatively unmethylated in retina and methylated in brain). The samples enriched for methylated DNA are being prepared for analysis on the genome-wide mouse CHARM (Comprehensive high-throughput array for relative methylation, NimbleGen) microarrays.
Our data indicates that all 3 methods can successfully enrich for methylated DNA from the mouse brain and retina to differing degrees, depending on whether you compare methylated samples from brain and retina or whether you compare the methylated and unmethylated fractions from the same tissue. The resulting enriched material is suitable for the ongoing genome-wide microarray analyses, which should provide a far more comprehensive description of retina-specific DNA methylation patterns than is currently available. The resulting data will be discussed within the context of the emerging information on whole-genome methylation results available from other tissues.
Our comparison of genome-wide DNA methylation patterns in the murine retina and brain will provide us with an objective method to better understand the dynamic relationship between DNA methylation and biological function, particularly tissue-specific expression, and provide necessary information to allow the logical design of subsequent mechanistic experiments.
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