Academic journal article Environmental Health Perspectives

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

Academic journal article Environmental Health Perspectives

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

Article excerpt

Introduction

Tobacco smoke exposure is associated with a variety of human diseases including cancers of the lung, head and neck, and bladder; chronic obstructive pulmonary disease; osteoporosis; and cardiovascular disease (CDC 2010). Although tobacco smoke constituents cause DNA damage and mutation (Alexandrov et al. 2016; Pfeifer et al. 2002), many adverse outcomes are not related to DNA damage and an emerging view is that the tobacco exposure-associated epigenetic effects (Breitling 2013; Monick et al. 2012; Philibert et al. 2012) may mediate many of these adverse outcomes (Breitling et al. 2012; Breitling 2013; Knopik et al. 2012; Lee and Pausova 2013; Ostrow et al. 2013; Zeilinger et al. 2013).

DNA methylation, one of the best studied epigenetic marks, predominantly occurs at cytosine residues of cytosine phosphate guanine (CpG) dinucleotides, playing an essential role in mammalian embryonic development and gene regulation in response to developmental and environmental cues (Bird 2002; Jones 2012; Li et al. 1992; Meissner 2010; Smith and Meissner 2013). Aberrant DNA methylation can result in altered regulation of gene expression and is observed in various human diseases (Ehrlich 2009; Jones 2012; Smith and Meissner 2013). Recently, highly significant differences in DNA methylation have been observed among individuals exposed to tobacco smoke (Joehanes et al. 2016; Joubert et al. 2012; Shenker et al. 2013; Zeilinger et al. 2013). Epigenome-wide association studies (EWAS) of tobacco smoke exposure using the Illumina Human Methylation 450 BeadChip Array (450K array) on blood DNA have greatly expanded the view of smoking's impact on the genome and the relationship to disease (Fasanelli et al. 2015; Zhang et al. 2016). For example, 450K array-based EWAS have shown that smoking-associated methylation changes of coagulation factor II (thrombin) receptor-like 3 (F2RL3) at cg03636183 and aryl-hydrocarbon receptor repressor (AHRR) at cg05575921 in whole blood DNA significantly associate with smoking-related cardiovascular disease and lung cancer (Breitling et al. 2012; Fasanelli et al. 2015), suggesting a potential role for blood cells in disease etiology. However, it has been estimated that the 450K array only detects about 7% of potential differentially methylated CpGs in the genome (Ziller et al. 2013). Thus, many CpGs important in the development of smoking-related diseases may still be unknown.

Many CpGs in the AHRR gene have been associated with tobacco smoke exposure, but methylation differences have been the most significant between smokers and nonsmokers at CpG cg05575921 in studies that examined DNA from whole blood (Zeilinger et al. 2013), cord blood (Joubert et al. 2012), or other tissues/cell types (Monick et al. 2012; Reynolds et al. 2015). Recently, in the Multi-Ethnic Study of Atherosclerosis (MESA), the methylation level of cg05575921 in peripheral blood monocytes was associated with both cigarette smoking and subclinical atherosclerosis, and mediation analysis suggested that methylation of AHRR in monocytes may be intermediate in the development of preclinical, smoking-related atherosclerosis (Reynolds et al. 2015). In line with these findings, AHRR has recently been shown to be involved in pro-inflammatory signaling in human circulating monocytes (Zhang et al. 2017), a process central in the development of atherosclerosis (Moore et al. 2013). In the present work, we sought to clarify how smoking-associated AHRR methylation changes are mechanistically connected to AHRR mRNA expression, and how they might contribute to downstream effects. In addition, previous studies using the 450K array may have missed smoking-associated CpGs in AHRR or elsewhere that might be useful biomarkers or biologically important. Therefore, we tested if reduced representation bisulfite sequencing (RRBS)-based methylation analysis could identify additional CpGs associated with smoking exposure and cellular phenotypes. …

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