Integrative Analysis of DNA Methylation and Gene Expression Data Identifies <i>EPAS1</i> as a Key Regulator of COPD

<div><p>Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified <i>EPAS1</i> as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. <i>EPAS1</i> is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by <i>EPAS1</i> were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that <i>Epas1</i> gene expression is reduced in mice chronically exposed to cigarette smoke. As <i>EPAS1</i> downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested <i>EPAS1</i> function in human endothelial cells. <i>EPAS1</i> knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with <i>EPAS1</i> downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a ‘causal’ role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology.</p></div

The causality test of <i>trans</i> methyl-mRNA pairs.

<p><b>A</b>) and <b>B</b>) are causality test results for the causal model whereby methylation regulates <i>trans</i> gene expression (methylation → <i>cis</i> gene expression →<i>trans</i> gene expression) in control and COPD data sets, respectively. The Y-axis is the –log10 of the p-values for the Spearman correlation between and and the X-axis is –log10 of the p-values for the Spearman correlation between and . A causal relationship (methylation → <i>cis</i> gene expression →<i>trans</i> gene expression) was defined if the p-value of was <0.0001 and the p-value of was>0.01 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004898#s4" target="_blank">Methods</a> for details). A total of 30,177 and 362,095 causal pairs were inferred in control and COPD samples, respectively. <b>C</b>) and <b>D</b>) are the causality test results for the causal model whereby <i>trans</i> gene expression regulates methylation variation (<i>trans</i> gene expression→methylation → <i>cis</i> gene expression) in control and COPD data sets, respectively. The Y-axis is the –log10 of the p-values for the Spearman correlation and the X-axis is –log10 of the p-values for the Spearman correlation . A causal relationship (<i>trans</i> gene expression→methylation→ <i>cis</i> gene expression) was defined if the p-value of was <0.0001 and the p-value of was>0.01 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004898#s4" target="_blank">Methods</a> for details). A total of 1,241 and 19,173 causal pairs were inferred in control and COPD, respectively.</p

The numbers of downstream genes regulated by DNA methylation level variation follow a scale-free distribution (a linear relationship in log-log plots).

<p><b>A</b>) The numbers in control; <b>B</b>) The numbers in COPD.</p

<i>EPAS1</i> siRNA signatures in human and mouse endothelial cells overlap with multiple COPD disease severity related signature sets.

<p><i>EPAS1</i> siRNA signatures in human and mouse endothelial cells overlap with multiple COPD disease severity related signature sets.</p

Relationships between DNA methylation and gene expression.

<p><b>A</b>) <i>Cis</i> regulation was defined by the correlation of the methylation level at the promoter region of a gene with expression level of the gene. <b>B</b>) <i>Trans</i> regulation was defined by the correlation of a methylation level at the promoter region of a gene with expression level of other genes. <b>C</b>) Potential relationships between <i>cis</i> and <i>trans</i> regulations. There are two potential causal mechanisms of <i>cis</i> and <i>trans</i> connections: Model I, where the methylation level regulates <i>trans</i> gene expression via the <i>cis</i> gene expression, and Model II, where <i>Trans</i> gene expression regulates the <i>cis</i> gene via controlling its methylation level. It is also possible that <i>cis</i> and <i>trans</i> connections are independently regulated by a factor X.</p

Comparing characteristics of key regulators with 5 COPD severity related traits in LGRC.

<p><b>A</b>) Comparing lung DNA methylation profiles of key regulators with 5 COPD severity related traits by Spearman correlation. At the Fisher's exact test p-value <0.05, the DNA methylation level variations of 3 key regulators, <i>ACSF3</i>, <i>SELO</i>, and <i>EPAS1</i>, were correlated with all 5 COPD severity related traits. <b>B</b>) Comparing downstream genes of key regulators with gene signature sets for 5 COPD severity related traits by the hypergeometric test. At the Fisher’s exact test p-value<0.05, only the key regulator <i>EPAS1</i>'s downstream genes significantly overlapped with gene signature sets for all 5 COPD severity related traits.</p

Gene expression levels of <i>Epas1</i> and <i>Vegfa</i> were lower in chronic smoking mice than non-smoking age-matched mice at the time when COPD develops in different mouse models.

<p><b>A</b>) Gene expression levels of <i>Epas1</i> and <i>Vegfa</i> in C57BL/6J mice that develop COPD after 6 months chronic exposure to cigarette smoke. <b>B</b>) Gene expression levels of <i>Epas1</i> and <i>Vegfa</i> in A/J mice that develop COPD after 2 months chronic exposure to cigarette smoke. The t-test was used to compare <i>Epas1</i> or <i>Vegfa</i> expression levels in mice with or without chronic smoke exposure.</p