Comparative isoschizomer profiling of cytosine methylation: The HELP assay

  1. Batbayar Khulan1,
  2. Reid F. Thompson1,
  3. Kenny Ye2,
  4. Melissa J. Fazzari2,
  5. Masako Suzuki3,
  6. Edyta Stasiek3,
  7. Maria E. Figueroa4,
  8. Jacob L. Glass1,
  9. Quan Chen5,
  10. Cristina Montagna1,5,
  11. Eli Hatchwell6,
  12. Rebecca R. Selzer7,
  13. Todd A. Richmond7,
  14. Roland D. Green7,
  15. Ari Melnick4, and
  16. John M. Greally1,3,8
  1. 1 Molecular Genetics,
  2. 2 Epidemiology and Population Health,
  3. 3 Medicine (Hematology),
  4. 4 Developmental and Molecular Biology, and
  5. 5 Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
  6. 6 Cold Spring Harbor Laboratories, Cold Spring Harbor, New York 11797, USA;
  7. 7 NimbleGen Systems Inc., Madison, Wisconsin 53711, USA

Abstract

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.

Footnotes

  • ↵8 Corresponding author.

    ↵8 E-mail jgreally{at}aecom.yu.edu; fax (718) 824-3153.

  • [Supplemental material is available online at www.genome.org.]

  • Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.5273806

    • Received September 26, 2005.
    • Accepted May 22, 2006.
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  1. Published in Advance June 29, 2006, doi: 10.1101/gr.5273806 Genome Res. 16: 1046-1055 Copyright © 2006, Cold Spring Harbor Laboratory Press

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