June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
Characterization of Genome-Wide DNA Methylation in the Mouse Retina
Author Affiliations & Notes
  • Verity Oliver
    Department of Ophthalmology, Johns Hopkins University, Baltimore, MD
  • Jun Wan
    Department of Ophthalmology, Johns Hopkins University, Baltimore, MD
  • Saurabh Agarwal
    Ludwig Institute for Cancer Research, La Jolla, CA
    Division of Biological Sciences, University of California, San Diego, La Jolla, CA
  • Donald Zack
    Department of Ophthalmology, Johns Hopkins University, Baltimore, MD
    Department of Molecular Biology and Genetics, Department of Neuroscience, and Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD
  • Jiang Qian
    Department of Ophthalmology, Johns Hopkins University, Baltimore, MD
  • Shannath Merbs
    Department of Ophthalmology, Johns Hopkins University, Baltimore, MD
  • Footnotes
    Commercial Relationships Verity Oliver, None; Jun Wan, None; Saurabh Agarwal, None; Donald Zack, Alcon (C), Merck (F), Allergan (C); Jiang Qian, None; Shannath Merbs, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 293. doi:
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    • Get Citation

      Verity Oliver, Jun Wan, Saurabh Agarwal, Donald Zack, Jiang Qian, Shannath Merbs; Characterization of Genome-Wide DNA Methylation in the Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):293.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. We have shown that retina-specific genes (Rho and Rbp3) are hypomethylated in the expressing cells of mouse retina. Little is known about DNA methylation in the regulation of other retina-specific genes. We aimed to characterize genome-wide DNA methylation patterns in the mouse retina.

Methods: We developed a novel method to characterize global DNA methylation: MBD2b/MBD3L1-enrichment of DNA followed by kinase ligation-mediated-PCR and microarray analysis (MeKL-chip). DNA was extracted from retina and brain samples of three 8 week old male C57BL/6J mice. Enrichment was performed on 250 ng DNA using the MethylCollector Ultra kit (Active Motif). Enriched and unenriched DNA was amplified using KLM-PCR. Quality control was performed pre- and post-amplification using QPCR for Rho, Rbp3 and H19 (equally methylated in retina and brain). The CHARM (comprehensive high-throughput arrays for relative methylation) 2.1M probe NimbleGen microarray platform was used to examine CpG sites throughout the genome. Tissue-specific differentially methylated regions (tDMRs) were identified. Validation of tDMRs was performed using bisulfite pyrosequencing of a second mouse cohort. The lower limits of input DNA for the MeKL-chip method were explored.

Results: 2498 tDMRs were identified between mouse retina and brain. The top 5 tDMRs were successfully validated by pyrosequencing and included genes that were more methylated in the retina (Rgs20, Hes2,Cckbr and Six3os1) and more methylated in the brain (Nfic). The top tDMR covered exon 3 of Rgs20 at an alternative transcription start site (TSS). Exon 3 is included in the brain-specific isoform and excluded from the retina-specific isoform; supporting recent evidence that intragenic DNA methylation may mediate tissue-specific expression through alternative TSS regulation. MeKL-chip enables genome-wide screening of DNA methylation using a sample containing as little as 50 ng DNA.

Conclusions: MeKL-chip is optimized for small samples and can be adapted for sequencing protocols. MeKL-chip on mouse retina has enabled novel characterization of tDMRs important for understanding retinal development and disease. MeKL-chip experiments are currently being undertaken on the rd1 mouse model to determine whether aberrant DNA methylation is implicated in the cone degeneration that follows loss of rods.

Keywords: 539 genetics • 688 retina • 533 gene/expression  

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