Epigenome-wide analysis of DNA methylation in lung tissue shows concordance with blood studies and identifies tobacco smoke-inducible enhancers

Abstract

Smoking-associated DNA hypomethylation has been observed in blood cells and linked to lung cancer risk. However, its cause and mechanistic relationship to lung cancer remain unclear. We studied the association between tobacco smoking and epigenome-wide methylation in non-tumor lung (NTL) tissue from 237 lung cancer cases in the Environment And Genetics in Lung cancer Etiology study, using the Infinium HumanMethylation450 BeadChip. We identified seven smoking-associated hypomethylated CpGs (P < 1.0 × 10-7), which were replicated in NTL data from The Cancer Genome Atlas. Five of these loci were previously reported as hypomethylated in smokers' blood, suggesting that blood-based biomarkers can reflect changes in the target tissue for these loci. Four CpGs border sequences carrying aryl hydrocarbon receptor binding sites and enhancer-specific histone modifications in primary alveolar epithelium and A549 lung adenocarcinoma cells. A549 cell exposure to cigarette smoke condensate increased these enhancer marks significantly and stimulated expression of predicted target xenobiotic response-related genes AHRR (P = 1.13 × 10-62) and CYP1B1 (P < 2.49 × 10-61). Expression of both genes was linked to smoking-related transversion mutations in lung tumors. Thus, smoking-associated hypomethylation may be a consequence of enhancer activation, revealing environmentally-induced regulatory elements implicated in lung carcinogenesis.

Figure 1

Four CpGs hypomethylated in smoker NTL are adjacent to unmethylated loci in AT2 cells and marked by regulatory histone marks in AT2 and A549 cells. Displayed from top to bottom for each CpG: the genomic location with RefSeq genes (if present; hg19 coordinates); H3K27ac (purple) & H3K4me1 (green) profiles in A549 cells and in two biological replicates of AT2 with called peaks underlined; DNase hypersensitive sites in A549; and two biological replicates of whole genome bisulfite sequencing (WGBS) with the CpG of interest magnified (blue= unmethylated; red = methylated; gray = no CpG present). A549 data were downloaded from ENCODE (45). The sequencing depth of AT2 cells is less than that of the A549 cell line owing to the difficulty in obtaining large numbers of primary human cells.

Figure 2

CSC-induced changes in enhancer marks in A459 cells. (A) Each diagram displays RefSeq genes at top (if present; hg19 coordinates) and from top to bottom, ENCODE data in A549 cells: Illumina Infinium HumanMethylation450 BeadChip CpG methylation status (blue = unmethylated; orange/purple = partially methylated; the CpGs of interest are marked by black arrowheads at bottom); DNase hypersensitive sites (DHSS) as black boxes; H3K27ac and H3K4me1 profiles with called peaks underlined and peak height scale indicated. Locations of PCR primers used for ChIP assays of H3K27ac and H3K4me1 panel B are indicated by purple and green vertical lines respectively. The region highlighted in pale yellow was cloned into a basal promoter-containing luciferase gene reporter vector (pGL4.26, panel C). The positions of AHR binding sites are indicated by gray arrowheads at the bottom. (B) ChIP assays of H3K27ac and H3K4me1 in vehicle- or CSC-treated A549 cells expressed as % input. * indicates luciferase activity statistically significantly different from vehicle (P < 0.05 paired Student's t test). Bars represent mean ± SEM of samples assayed in three or more independent trials of A549 cells exposed to CSC for 48 h. (C) Luciferase reporter assays to test for CSC-regulated enhancer activity. The region highlighted in pale yellow in panel A was cloned into the basal promoter-containing luciferase vector pLG4.26. In each case we ensured that the DNase hypersensitive site closest to the hypomethylated CpG was included. The plasmids were transfected into A549 cells that were treated with vehicle or CSC at the indicated doses 24 h after transfection. A renilla expression plasmid was cotransfected for DNA quantity normalization. Luciferase activity was measured at 24 h after CSC exposure, and is indicated relative to empty vector. All expression levels were statistically significantly elevated relative to pGL4.26. Significant induction relative to vehicle is indicated by an asterisk and the fold induction is noted for 20 µg/ml CSC. Bars represent mean ± SEM of three or more independent experiments.

Figure 3

RNA-seq investigation of 1 Mb regions flanking the putative CSC-regulated enhancers. (A) Genes induced in a 1Mb window around each CpG in A549 cells treated for 48 h or 2 weeks with 20 µg/ml CSC. RNA-seq data were obtained from three independent treatments of A549 cells with CSC. (B) RNA-seq data from TCGA NTL samples (n = 100, Supplementary Material, Table S6) was analyzed to examine whether genes identified in A were associated with smoking history. NS= non-smoker; >15 and <15 indicates smoking cessation more than or less than 15 years ago. Fold change values between NS and current smoker were 4.58 for AHRR, 2.91 for CYP1B1, -0.40 for ENTPD2 and 0.87 for NR4A3. (C) TCGA NTL lung samples were used to examine the correlation between DNA methylation and RNA expression (n = 28, Supplementary Material, Table S8).

Figure 4

Targeted sodium bisulfite pyrosequencing of cg05575921 and cg07992500 in CSC-treated A549 cells. A549 cells were exposed in quadruplicate to CSC in vitro for 48 h and 2 weeks at 0, 5, 10 or 20 µg/ml. DNA methylation was assessed by pyrosequencing of sodium bisulfite-treated DNA. All 4 plots showed a significant trend as indicated in the chart and a statistically significant reduction in DNA methylation between 0 and 20 µg/ml CSC (AHRR: P = 0.009 and 0.0177 at 48 h and 2 weeks respectively; CYP1B1: P = 0.0196 and 0.0297 at 48 h and 2 weeks, respectively (t-test, one-sided)).