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Epigenetics of the Placenta
Figure 6. Biological Functions of genes hypomethylated in MBC versus CVS (A) and vice versa (B) Identified Using Ingenuity
Pathways Analysis Software.
doi:10.1371/journal.pone.0014723.g006
DNA methylation in CVS based on the Infinium methylation tion and expression is nonlinear. When the genes are moderately or
data. The above trends were similarly present in the relationship highly hypomethylated (log(HpaII2/HpaII+).0.03), hypomethyla-
between DNA methylation and mRNA transcription in MBCs tion level and expression level are uncorrelated. When the genes are
(Figure 8B), though not as strong as in CVS. only weakly or not hypomethylated (log(HpaII2/HpaII+),0.03),
there is a significant negative correlation between hypomethylation
To gain further insight into the relationship between gene level and expression. In Figure 8D, the solid line shows how the
expression and methylation, we compared the CVS gene expression level relates to the hypomethylation level of the gene
expression profile with the DNA methylation profile for the 3 bodies in chromosomes 13, 18, and 21. This relationship is more
chromosomes 13, 18, and 21 based on the custom Agilent arrays. pronounced when gene bodies containing CGIs are removed from
We estimated the hypomethylation level of each MspI/HpaII site the analysis (Figure 8E). Finally, it can be seen in Figure 8F that
in the three chromosomes by determining the log ratio of the among the MspI sites inside gene bodies, the hypomethylation levels
signal of the control samples to the signal of samples digested with are higher for those inside CGIs, compared to those outside CGIs.
HpaII: log(HpaII2/HpaII+). Unlike the Infinium arrays, which
targeted only CpG sites inside the promoter regions, the custom The Relationship Between DNA Methylation Levels and
Agilent arrays targeted CpG sites at high density all over the three Gene Expression are Dependent on Promoter CpG
chromosomes. This allowed us to determine how the methylation Frequency
patterns of the different structural components of the genome were
related to the gene expression profile. Figure 9 illustrates the relationships between promoter meth-
ylation and expression in CVS samples in the context of promoter
For genes in chromosomes 13, 18, and 21, we found a positive CpG frequency. As shown in Figure 9A the genes form two
correlation of 0.194 (p value of the t test = 2.126610206) between the clusters based on their promoter CpG frequency (observed CpG/
hypomethylation level of their promoter regions and their expression expected CpG) and genes with high CpG frequency promoters
level (Figure 8C), where the promoter region was defined as 1500 tend to be more highly expressed. Figure 9B shows that the genes
upstream and 1000 downstream of the TSS. This finding agrees with also form two clusters based on their promoter CpG frequency
the previous analysis of the relationship between DNA methylation with respect to promoter methylation levels. Genes in the low CpG
and gene expression using the Illumina data. In Figure 8C, the solid frequency cluster tend to be hypermethylated, whereas the genes
line shows how the expression level relates to the hypomethylation in the high CpG frequency cluster tend to be hypomethylated.
level of the promoter regions chromosomes 13, 18, and 21. Figures 9C and D show the nonlinear relationship between
Furthermore, we found a negative correlation of 20.111 (p value expression and methylation in each of the two clusters identified in
of t test = 0.0018) between the hypomethylation level of the gene Figure 9B. When CpG frequency is #0.4 (Figure 9C), the
body and the expression level, where ‘‘gene body’’ includes both relationship between methylation and expression shows no clear
exons and introns (Figure 8D). The relation between hypomethyla-
PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e14723
