Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type Specific Enhancer Activation and Gene Expression

BACKGROUND: Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SMDMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. OBJECTIVE: Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. METHOD: We integrated SM-DMRs identified by reduced representation bisullite sequencing (RRBS) of circulating CD14 + monocyte DNA collected from two independent human studies [n = 38 from Clinical Research Unit (CRU) and n = 55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4 + T cells, CD8 + T cells, CD15 + granulocytes, CD19 + B cells, and CD56 + NK cells (n = 19 females, CRU). DMRs were validated using pyrosequencing or bisullite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. RESULTS: RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AIIRR, C.5aff.55-EXOC-AS, and SASH/were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. CONCLUSIONS: Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease.