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Supporting Information Supporting Information Corrected September 30, 2013 Fredrickson Et Al Supporting Information Supporting Information Corrected September 30, 2013 Fredrickson et al. 10.1073/pnas.1305419110 SI Methods Analysis of Differential Gene Expression. Quantile-normalized gene Participants and Study Procedure. A total of 84 healthy adults were expression values derived from Illumina GenomeStudio software recruited from the Durham and Orange County regions of North were transformed to log2 for general linear model analyses to Carolina by community-posted flyers and e-mail advertisements produce a point estimate of the magnitude of association be- followed by telephone screening to assess eligibility criteria, in- tween each of the 34,592 assayed gene transcripts and (contin- cluding age 35 to 64 y, written and spoken English, and absence of uous z-score) measures of hedonic and eudaimonic well-being chronic illness or disability. Following written informed consent, (each adjusted for the other) after control for potential con- participants completed online assessments of hedonic and eudai- founders known to affect PBMC gene expression profiles (i.e., monic well-being [short flourishing scale, e.g., in the past week, how age, sex, race/ethnicity, BMI, alcohol consumption, smoking, often did you feel... happy? (hedonic), satisfied? (hedonic), that minor illness symptoms, and leukocyte subset distributions). Sex, your life has a sense of direction or meaning to it? (eudaimonic), race/ethnicity (white vs. nonwhite), alcohol consumption, and that you have experiences that challenge you to grow and become smoking were represented by 0/1 dummy codes. Age, BMI, and a better person? (eudaimonic), that you had something to con- minor illness symptoms were treated as continuous regressors, tribute to society? (eudaimonic); answered on a six-point fre- as were observed (log2-transformed) expression levels for tran- quency metric whereby 0 indicates never, 1 indicates once or scripts marking T lymphocyte subsets (CD3D, CD3E, CD4, twice, 2 indicates approximately once per week, 3 indicates two or and CD8A), B lymphocytes (CD19), natural killer cells (CD16/ three times per week, 4 indicates almost every day, and 5 indicates FCGR3A and CD56/NCAM1), and monocytes (CD14)(4). every day] (1, 2) and depressive symptoms [per Center for Epi- The primary “low-level” analysis model for point estimation of demiological Studies–Depression (CES-D)] (3). Participants then transcript-phenotype associations is as follows: attended a late-afternoon laboratory session in which they were assessed for weight, height, and blood pressure, and provided a = + + Log2 transcript abundance Intercept Hedonic Eudaimonic 20-mL venipuncture blood sample under resting conditions. Age, + + + = + sex, race/ethnicity, smoking history, alcohol consumption, and Age Sex White Non Alcohol + + 2-wk history of 13 minor illness symptoms (e.g., headache, upset Smoking IllnessSymptoms + + CD D + CD E + CD stomach; each rated as being experienced on a frequency scale BMI 3 3 4 + CD A + CD + FCGR A with values ranting from 0 indicating not at all to 8 indicating very 8 19 3 frequently) were assessed as potential confounders. All study + NCAM1 + CD14 + residual: procedures were approved by the institutional review board of the [S1] University of North Carolina at Chapel Hill. Analysis of Affective and Demographic Variables. Associations be- This primary low-level analysis model is used solely to provide tween affective and demographic/behavioral characteristics and point estimates of transcript–phenotype associations to serve as Short Flourishing Scale measures of hedonic and eudaimonic fi inputs into subsequent gene set expression analyses. No statistical well-being (treated as continuous variables) were quanti ed by testing is performed at the level of individual transcript–phenotype linear regression analysis for continuous variables [age, body mass associations because the goal of this study is not to discover reli- index (BMI), CES-D total scores, scores on affective and vege- able associations between the expression of individual transcripts tative symptom subscales of the CES-D, and minor illness and measured levels of hedonic or eudaimonic well-being. The symptoms, with association strength summarized by Pearson goal of this study is to test associations between eudaimonic and correlation coefficient] or by one-way ANOVA for categorical hedonic well-being and average levels of expression of specific variables (race/ethnicity, smoking history, alcohol history). Dif- sets of genes selected a priori for analysis here based on their ferences in average levels of hedonic and eudaimonic well-being previously observed involvement in the conserved transcrip- were tested by repeated-measures ANOVA, and the qualitative tional response to adversity (CTRA) (e.g., as representative predominance of eudaimonic vs. hedonic well-being was assessed proinflammatory genes, IFN-related genes, and antibody synthe- by sign test. In all analyses, residuals were checked for normal sis-related genes) as summarized in recent surveys of this re- distribution and absence of outliers, and two-tailed P values search area (5, 6). <0.05 served as the criterion for statistical significance. Analyses were conducted in SAS version 9.3. Primary Analysis: CTRA Gene Set Expression. This primary focus of this study involves testing potential differences in association of Transcriptome Profiling. Genome-wide transcriptional profiling hedonic vs. eudaimonic well-being with average expression of one was carried out on peripheral blood mononuclear cells (PBMCs) set of 53 genes selected a priori as indicators of the leukocyte isolated by Ficoll density gradient centrifugation of 20 mL CTRA gene expression profile (5, 6): (i) 19 proinflammatory anticoagulated blood samples drawn by antecubital venipuncture genes (IL1A, IL1B, IL6, IL8, TNF, PTGS1, PTGS2, FOS, FOSB, from all 80 participants who consented to provide specimens. FOSL1, FOSL2, JUN, JUNB, JUND, NFKB1, NFKB2, REL, Total RNA was extracted (RNeasy; Qiagen), tested for suitable RELA, and RELB), which are up-regulated on average in the mass (>100 ng as determined by Nanodrop ND1000) and in- CTRA; (ii) 31 genes involved in type I IFN responses (GBP1, tegrity (RNA integrity number ≥7.0; Bioanalyzer; Agilent), and IFI16, IFI27, IFI27L1-2, IFI30, IFI35, IFI44, IFI44L, IFI6, converted to fluorescent cRNA for hybridization to Illumina IFIH1, IFIT1-3, IFIT5, IFIT1L, IFITM1-3, IFITM4P, IFITM5, Human HT-12 v4 BeadArrays following the manufacturer’s IFNB1, IRF2, IRF7-8, MX1-2, OAS1-3, and OASL), which are standard protocol in the University of California, Los Angeles, down-regulated on average in the CTRA; and (iii) three genes Neuroscience Genomics Core Laboratory. Data are deposited in involved in antibody synthesis (IGJ, IGLL1, and IGLL3), which the Gene Expression Omnibus as series GSE45330. are down-regulated on average in the CTRA (5, 6). To provide Fredrickson et al. www.pnas.org/cgi/content/short/1305419110 1of9 a single summary measure of overall CTRA profile expression, recurrent emergence as gene regulatory correlates of the CTRA a weighted average association estimate was formed by multiply- profile in multiple previous studies of a diverse range of stressors ing each transcript association point estimate by an appropriate in observational studies of human adverse life circumstances signed contrast coefficient (+1proinflammatory, −1 IFN, −1an- (4, 16–20), animal models of experimentally manipulated social tibody). The resulting average gene set association measure was stress (21–23), and randomized experiments examining the effect tested for statistically significant departure from the null hypoth- of stress-buffering interventions in humans (20, 24, 25). No other esis (0 difference in log2 metric) by using a single-sample t test transcription factor hypotheses were examined in this study. with SE term estimated by nonparametric bootstrap analysis [200 Analyses were formatted as a “transcriptional asymmetry test” cycles to estimate a stable SE, as recommended (7)]. A two-tailed comparing TFBM prevalence in promoters of up-regulated P value < 0.05 served as the criterion for statistical significance. genes with that observed in promoters of down-regulated genes Following a statistically significant omnibus test of CTRA (to ensure that all analyzed transcripts were potentially expres- composite gene set association with well-being phenotypes, an- sive in the PBMC pool, and thereby exclude transcripts that cillary analyses were conducted to identify the specific CTRA could not be expressed in this cell type but are nevertheless subsets driving that overall association. These separate analyses of present in the human genome as a whole). TFBM frequencies the 19-gene proinflammatory subset, the 31-gene IFN-related were assessed by using three parametric variations in the size of subset, and the three-gene antibody-related subset paralleled the proximal promoter sequence scanned ([−300 bp, +0 bp] relative structure of the test for the overall CTRA composite, but did not to the RefSeq transcription start site, [−600, +0], and [−1,000, + require weighting as a result of the homogenous composition of 200]) and three parametric variations in the TFBM detection each subset. stringency (TRANSFAC MatInspector algorithm, mat_sim = 0.80, 0.90, 0.95). Results from each parametric scan were sum- Secondary Analysis: CTRA-Related Transcription Factors. Previous marized as a (log2-transformed) ratio of TFBM prevalence in up- research has implicated several specific transcription control
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