title: Distinct Epigenetic Effects of Tobacco Smoking in Whole Blood and among Leukocyte Subtypes.
abstract: Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking-associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p <1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) were associated with smoking duration among heavy smokers (>22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6 cell types, we confirmed cell-type specificity in the context of DMRs. We identified new CpGs associated with current smoking, pack-years, duration, and revealed unique profiles of smoking-associated DNA methylation and gene expression among immune cell types, providing potential clues to hematopoietic lineage-specific effects in disease etiology.
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Dan
authors.initials: D
authors.lastname: Su
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Xuting
authors.identifier: http://orcid.org/0000-0001-6781-8008
authors.initials: X
authors.lastname: Wang
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Michelle R
authors.identifier: http://orcid.org/0000-0001-5415-6420
authors.initials: MR
authors.lastname: Campbell
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Devin K
authors.initials: DK
authors.lastname: Porter
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Gary S
authors.initials: GS
authors.lastname: Pittman
authors.affiliation: Integrated Bioinformatics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Bennett
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Ma
authors.initials: M
authors.lastname: Wan
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Neal A
authors.identifier: http://orcid.org/0000-0003-4193-4398
authors.initials: NA
authors.lastname: Englert
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Crowl
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Ryan N
authors.identifier: http://orcid.org/0000-0002-7189-3243
authors.initials: RN
authors.lastname: Gimple
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Kelly N
authors.initials: KN
authors.lastname: Adamski
authors.affiliation: Duke University School of Medicine, Durham, NC, 27708, United States of America.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Duke University School of Medicine, Durham, NC, 27708, United States of America.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Murphy
authors.affiliation: Environmental Genomics Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, United States of America.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Bell
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001402:B-Lymphocytes / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods*; D005260:Female; D058851:GPI-Linked Proteins / Q000235:genetics; D015870:Gene Expression; D055106:Genome-Wide Association Study / Q000379:methods; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000145:classification / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008875:Middle Aged; D009000:Monocytes / Q000378:metabolism; D018179:Receptors, Thrombin / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods; D012907:Smoking; D013601:T-Lymphocytes / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D055815:Young Adult
chemical_list: D000469:Alkaline Phosphatase; D051017:Apoptosis Regulatory Proteins; D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D058851:GPI-Linked Proteins; D008565:Membrane Proteins; D018179:Receptors, Thrombin; D012097:Repressor Proteins; D014157:Transcription Factors; C492162:AHRR protein, human; C498275:GFI1 protein, human; C099815:IER3 protein, human; C548269:ALPG protein, human; C112908:protease-activated receptor 4