Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques.
The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hmC.
Chromosomes and Gene Expression, MRC Human Genetics Unit at the Institute of Genetics and Molecular Medicine at the University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK, Member of MARCAR Consortium, The Centre for Individualized Medication, Linköping University Hospital, Linköping University, Linköping SE-58185, Sweden and Discovery and Investigative Safety, Preclinical Safety, Novartis Institutes for Biomedical Research, Klybeckstrasse, Basel CH-4002, Switzerland.
John P
JP
Thomson
Jennifer M
JM
Hunter
Colm E
CE
Nestor
Donncha S
DS
Dunican
Rémi
R
Terranova
Jonathan G
JG
Moggs
Richard R
RR
Meehan
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D000906:Antibodies; D002843:Chromatin / Q000378:metabolism; D018899:CpG Islands; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000276:immunology; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis; D056426:Genetic Loci; D018392:Genomic Imprinting; D007118:Immunoassay / Q000379:methods; D008099:Liver / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020411:Oligonucleotide Array Sequence Analysis; D020080:Tandem Repeat Sequences
D000906:Antibodies; D002843:Chromatin; D004268:DNA-Binding Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA