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An improved method for genome wide DNA methylation profiling correlated to transcription and genomic instability in two breast cancer cell lines.

Li J, Gao F, Li N, Li S, Yin G, Tian G, Jia S, Wang K, Zhang X, Yang H, Nielsen AL, Bolund L - BMC Genomics (2009)

Bottom Line: By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines.However a cohort of individual genomic loci with significantly different DNA methylation status between two cell lines was identified.Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Human Genetics, University of Aarhus, The Bartholin building, Aarhus C, Denmark. jianl@humgen.au.dk

ABSTRACT

Background: DNA methylation is a widely studied epigenetic mechanism known to correlate with gene repression and genomic stability. Development of sensitive methods for global detection of DNA methylation events is of particular importance.

Results: We here describe a technique, called modified methylation-specific digital karyotyping (MMSDK) based on methylation-specific digital karyotyping (MSDK) with a novel sequencing approach. Briefly, after a tandem digestion of genomic DNA with a methylation-sensitive mapping enzyme and a fragmenting enzyme, short sequence tags are obtained. These tags are amplified, followed by direct, massively parallel sequencing (Solexa 1G Genome Analyzer). This method allows high-throughput and low-cost genome-wide DNA methylation mapping. We applied this method to investigate global DNA methylation profiles for widely used breast cancer cell lines, MCF-7 and MDA-MB-231, which are representatives for luminal-like and mesenchymal-like cancer types, respectively. By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines. However a cohort of individual genomic loci with significantly different DNA methylation status between two cell lines was identified. Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells.

Conclusion: The MMSDK method will be a valuable tool to increase the current knowledge of genome wide DNA methylation profiles.

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The strategy of MMSDK. Schematic presentation of the MMSDK method. Details of the method are described in the text.
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Figure 1: The strategy of MMSDK. Schematic presentation of the MMSDK method. Details of the method are described in the text.

Mentions: MSDK [12,13] is a modification of the original digital karyotyping technique [17]. In this study, we developed MMSDK as a combination of the original MSDK and Solexa sequencing (Fig 1). The procedures prior to tag concatenation were similar to those described originally for MSDK [12,13]. Briefly, DNA was isolated from the cell lines using DNeasy® Blood & Tissue Kit (QIAGEN) following manufacturer's protocol. Genomic DNA was digested with methylation-sensitive mapping enzyme MluI (New England Biolabs). MluI has two CpG sites in its recognition sequence, ACGCGT, and therefore its recognition site is preferentially located in CpG islands (CGIs). Digested DNA was ligated to biotinylated linkers and fragmented by NlaIII (New England BioLabs) cleavage. Because MluI only cuts unmethylated regions, binding of DNA fragments to streptavidin-conjugated magnetic beads will separate the unmethylated and methylated fragments. Because of a potential risk that digested DNA without biotinylated linkers may be unspecifically bound by streptavidin-conjugated beads, we performed a parallel control experiment instead using DNA fragments without biotinylated linkers. Bound DNA was ligated to another linker (N) containing a MmeI restriction enzyme recognition site, and then digested with MmeI (New England Biolabs) that generates short sequence tags (16–17 bp, due to enzyme cut floating). Instead of the concatenation of tags and clone sequencing, tags were ligated with P7 linker and amplified by PCR with primers N and P7. To avoid the bias from PCR, amplification was stopped after 15 cycles. The sequences of the linkers and primers are available in Additional file 1.


An improved method for genome wide DNA methylation profiling correlated to transcription and genomic instability in two breast cancer cell lines.

Li J, Gao F, Li N, Li S, Yin G, Tian G, Jia S, Wang K, Zhang X, Yang H, Nielsen AL, Bolund L - BMC Genomics (2009)

The strategy of MMSDK. Schematic presentation of the MMSDK method. Details of the method are described in the text.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2696471&req=5

Figure 1: The strategy of MMSDK. Schematic presentation of the MMSDK method. Details of the method are described in the text.
Mentions: MSDK [12,13] is a modification of the original digital karyotyping technique [17]. In this study, we developed MMSDK as a combination of the original MSDK and Solexa sequencing (Fig 1). The procedures prior to tag concatenation were similar to those described originally for MSDK [12,13]. Briefly, DNA was isolated from the cell lines using DNeasy® Blood & Tissue Kit (QIAGEN) following manufacturer's protocol. Genomic DNA was digested with methylation-sensitive mapping enzyme MluI (New England Biolabs). MluI has two CpG sites in its recognition sequence, ACGCGT, and therefore its recognition site is preferentially located in CpG islands (CGIs). Digested DNA was ligated to biotinylated linkers and fragmented by NlaIII (New England BioLabs) cleavage. Because MluI only cuts unmethylated regions, binding of DNA fragments to streptavidin-conjugated magnetic beads will separate the unmethylated and methylated fragments. Because of a potential risk that digested DNA without biotinylated linkers may be unspecifically bound by streptavidin-conjugated beads, we performed a parallel control experiment instead using DNA fragments without biotinylated linkers. Bound DNA was ligated to another linker (N) containing a MmeI restriction enzyme recognition site, and then digested with MmeI (New England Biolabs) that generates short sequence tags (16–17 bp, due to enzyme cut floating). Instead of the concatenation of tags and clone sequencing, tags were ligated with P7 linker and amplified by PCR with primers N and P7. To avoid the bias from PCR, amplification was stopped after 15 cycles. The sequences of the linkers and primers are available in Additional file 1.

Bottom Line: By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines.However a cohort of individual genomic loci with significantly different DNA methylation status between two cell lines was identified.Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Human Genetics, University of Aarhus, The Bartholin building, Aarhus C, Denmark. jianl@humgen.au.dk

ABSTRACT

Background: DNA methylation is a widely studied epigenetic mechanism known to correlate with gene repression and genomic stability. Development of sensitive methods for global detection of DNA methylation events is of particular importance.

Results: We here describe a technique, called modified methylation-specific digital karyotyping (MMSDK) based on methylation-specific digital karyotyping (MSDK) with a novel sequencing approach. Briefly, after a tandem digestion of genomic DNA with a methylation-sensitive mapping enzyme and a fragmenting enzyme, short sequence tags are obtained. These tags are amplified, followed by direct, massively parallel sequencing (Solexa 1G Genome Analyzer). This method allows high-throughput and low-cost genome-wide DNA methylation mapping. We applied this method to investigate global DNA methylation profiles for widely used breast cancer cell lines, MCF-7 and MDA-MB-231, which are representatives for luminal-like and mesenchymal-like cancer types, respectively. By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines. However a cohort of individual genomic loci with significantly different DNA methylation status between two cell lines was identified. Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells.

Conclusion: The MMSDK method will be a valuable tool to increase the current knowledge of genome wide DNA methylation profiles.

Show MeSH
Related in: MedlinePlus