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Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.

Sun Z, Asmann YW, Kalari KR, Bot B, Eckel-Passow JE, Baker TR, Carr JM, Khrebtukova I, Luo S, Zhang L, Schroth GP, Perez EA, Thompson EA - PLoS ONE (2011)

Bottom Line: Gene expression in cell lines was dominated by ER-associated genes.In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines.The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota, United States of America.

ABSTRACT
We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+) and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

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ER+ and ER− cell lines exhibit differential CpG island methylation.Panel A: Unsupervised clustering of cell line data using 21,570 CpG islands, filtered for CMP methylation coverage as described in Materials and Methods. The graded colors from red, orange, yellow, to white represent correlation from high to low among samples. Panel B: A heatmap of the top 100 differentially methylated CpG islands identified using the LIMMA model. The methylation data for each CpG island were standardized by mean among the samples where red represents hypermethylation and green hypomethylation. Genes associated with these CpG islands are indicated on the right of the figure.
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pone-0017490-g003: ER+ and ER− cell lines exhibit differential CpG island methylation.Panel A: Unsupervised clustering of cell line data using 21,570 CpG islands, filtered for CMP methylation coverage as described in Materials and Methods. The graded colors from red, orange, yellow, to white represent correlation from high to low among samples. Panel B: A heatmap of the top 100 differentially methylated CpG islands identified using the LIMMA model. The methylation data for each CpG island were standardized by mean among the samples where red represents hypermethylation and green hypomethylation. Genes associated with these CpG islands are indicated on the right of the figure.

Mentions: The human genome contains 25,328 annotated CpG islands with overlapping MspI restriction sites. For purposes of analysis, we required at least 10× coverage of each dCMP residue within every CpG island, and the average of all dCMP residues within each island was calculated as described in the Materials and Methods. We analyzed 21,570 CpG islands that met these criteria in all seven breast cancer cell lines, which represents 85% of the total MspI-bounded CpG islands in the genome. We conducted unsupervised clustering using the methylation data of all 21,570 CpG islands. As shown in Figure 3A, three ER+ cell lines BT474, ZR751, and MCF7, were in the neighboring nodes of the cluster, and two ER- cell lines BT20 and MDAMB231 clustered. However, T47D (ER+) and MDAMB468 (ER-) did not cluster with ER+ and ER− samples, respectively. The data suggest that global genomic methylation status varies in a systematic manner between ER+ and ER− cell lines; however, the association between CpG island methylation and ER status is not as robust as that observed for mRNA abundance (Figure 1C).


Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.

Sun Z, Asmann YW, Kalari KR, Bot B, Eckel-Passow JE, Baker TR, Carr JM, Khrebtukova I, Luo S, Zhang L, Schroth GP, Perez EA, Thompson EA - PLoS ONE (2011)

ER+ and ER− cell lines exhibit differential CpG island methylation.Panel A: Unsupervised clustering of cell line data using 21,570 CpG islands, filtered for CMP methylation coverage as described in Materials and Methods. The graded colors from red, orange, yellow, to white represent correlation from high to low among samples. Panel B: A heatmap of the top 100 differentially methylated CpG islands identified using the LIMMA model. The methylation data for each CpG island were standardized by mean among the samples where red represents hypermethylation and green hypomethylation. Genes associated with these CpG islands are indicated on the right of the figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045451&req=5

pone-0017490-g003: ER+ and ER− cell lines exhibit differential CpG island methylation.Panel A: Unsupervised clustering of cell line data using 21,570 CpG islands, filtered for CMP methylation coverage as described in Materials and Methods. The graded colors from red, orange, yellow, to white represent correlation from high to low among samples. Panel B: A heatmap of the top 100 differentially methylated CpG islands identified using the LIMMA model. The methylation data for each CpG island were standardized by mean among the samples where red represents hypermethylation and green hypomethylation. Genes associated with these CpG islands are indicated on the right of the figure.
Mentions: The human genome contains 25,328 annotated CpG islands with overlapping MspI restriction sites. For purposes of analysis, we required at least 10× coverage of each dCMP residue within every CpG island, and the average of all dCMP residues within each island was calculated as described in the Materials and Methods. We analyzed 21,570 CpG islands that met these criteria in all seven breast cancer cell lines, which represents 85% of the total MspI-bounded CpG islands in the genome. We conducted unsupervised clustering using the methylation data of all 21,570 CpG islands. As shown in Figure 3A, three ER+ cell lines BT474, ZR751, and MCF7, were in the neighboring nodes of the cluster, and two ER- cell lines BT20 and MDAMB231 clustered. However, T47D (ER+) and MDAMB468 (ER-) did not cluster with ER+ and ER− samples, respectively. The data suggest that global genomic methylation status varies in a systematic manner between ER+ and ER− cell lines; however, the association between CpG island methylation and ER status is not as robust as that observed for mRNA abundance (Figure 1C).

Bottom Line: Gene expression in cell lines was dominated by ER-associated genes.In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines.The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota, United States of America.

ABSTRACT
We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+) and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

Show MeSH
Related in: MedlinePlus