Comparative isoschizomer profiling of cytosine methylation: the HELP assay.
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.
Khulan
Batbayar
B
Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Thompson
Reid F
RF
Ye
Kenny
K
Fazzari
Melissa J
MJ
Suzuki
Masako
M
Stasiek
Edyta
E
Figueroa
Maria E
ME
Glass
Jacob L
JL
Chen
Quan
Q
Montagna
Cristina
C
Hatchwell
Eli
E
Selzer
Rebecca R
RR
Richmond
Todd A
TA
Green
Roland D
RD
Melnick
Ari
A
Greally
John M
JM
D000818:Animals; D018899:CpG Islands; D003596:Cytosine / Q000378:metabolism*; D019175:DNA Methylation; D016678:Genome; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003596:Cytosine