By HpaII (which methylates the internal Cs of the CCGG sequence) or SssI (which methylates all CpG sites) and then transfected into MCF7 cells. Data were normalized using a co-transfected renilla luciferase vector and presented as methylated promoter constructs (grey and white bars) relative to un-methylated constructs (black bars). Experiments were performed six times (SORBS) or eight times (EMX2) including four replicates in each experiment. Cells transfected with an empty pCpGL-basic vector and untransfected cells activities were journal.pone.0077579 used as background correction for firefly and renilla luciferase activity, respectively. *P < 0.05 **P < 0.005. Data are shown as mean ?SEM.as in our discovery cohort, this may have contributed to the observed inconsistencies. We also identified HOXC6 among the top ranked candidate genes with highest methylation levels in OVAT along with decreased mRNA expression, which was further supported in pure adipocytes. Homeobox genes are involved in developmental processes [39]. HOXC6 was also demonstrated by others to be differentially expressed in human SAT before and after bariatric surgery [40], between SAT andMOLECULAR METABOLISM 6 (2017) 86e100 www.molecularmetabolism.comgluteofemoral adipose tissue [13] and skeletal muscle [17]. Our findings largely support these data. Furthermore, HAND2 was higher methylated in SAT from obese subjects along with decreased expression levels in both adipose tissue biopsies and isolated adipocytes. Hand2 is involved in Notch signaling in heart development of mice [41]. Considering that NOTCH signaling also plays a major role in adipogenic differentiation [30,31], mainly through inhibiting ASC (Adipose tissue-Derived-Stem Cells)?2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Original ArticleFigure 5: Epigenome wide association study (EWAS) between DNA promoter methylation and BMI. EWAS was applied to test for a relationship (linear regression) of DNA promoter methylation per transcript with BMI. P-values for epigenome wide association analysis with BMI were calculated using an R (version 3.0.2) package called CpGassoc [58]. All analyses were adjusted for age, gender, and type 2 diabetes, separately in a) SAT wcs.1183 and b) OVAT in the samples from the Leipzig cohort, for which methylation data were available (N ?77). Different transcripts per gene showing exactly the same association results are summarized in the same dot. Bonferroni correction was used to correct for multiple testing.differentiation to adipocytes and thereby affecting the adipose tissue expansion BAY1217389 dose capacity [30], this might indicate reduced adipocyte differentiation in OVAT. In line with this, increased Notch signaling in mice blocked the expansion of white adipose tissue, ectopic fat accumulation and insulin resistance [42], which further supports the potential dysfunctional role of OVAT [1,2]. Among the successfully replicated hits was SORBS2, which was more methylated and less expressed in SAT versus OVAT in both non-obese and obese subjects. We Pepstatin web substantiated these results in isolated adipocytes. Linear regression analyses revealed a strong association ofmethylation with BMI for SORBS2 in OVAT. SORBS2 might be involved in insulin mediated translocation of GLUT4 [43] and t thereby might affect energy storage. Furthermore, we clearly show that in vitro promoter methylation of SORBS2 directly represses the tra.By HpaII (which methylates the internal Cs of the CCGG sequence) or SssI (which methylates all CpG sites) and then transfected into MCF7 cells. Data were normalized using a co-transfected renilla luciferase vector and presented as methylated promoter constructs (grey and white bars) relative to un-methylated constructs (black bars). Experiments were performed six times (SORBS) or eight times (EMX2) including four replicates in each experiment. Cells transfected with an empty pCpGL-basic vector and untransfected cells activities were journal.pone.0077579 used as background correction for firefly and renilla luciferase activity, respectively. *P < 0.05 **P < 0.005. Data are shown as mean ?SEM.as in our discovery cohort, this may have contributed to the observed inconsistencies. We also identified HOXC6 among the top ranked candidate genes with highest methylation levels in OVAT along with decreased mRNA expression, which was further supported in pure adipocytes. Homeobox genes are involved in developmental processes [39]. HOXC6 was also demonstrated by others to be differentially expressed in human SAT before and after bariatric surgery [40], between SAT andMOLECULAR METABOLISM 6 (2017) 86e100 www.molecularmetabolism.comgluteofemoral adipose tissue [13] and skeletal muscle [17]. Our findings largely support these data. Furthermore, HAND2 was higher methylated in SAT from obese subjects along with decreased expression levels in both adipose tissue biopsies and isolated adipocytes. Hand2 is involved in Notch signaling in heart development of mice [41]. Considering that NOTCH signaling also plays a major role in adipogenic differentiation [30,31], mainly through inhibiting ASC (Adipose tissue-Derived-Stem Cells)?2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Original ArticleFigure 5: Epigenome wide association study (EWAS) between DNA promoter methylation and BMI. EWAS was applied to test for a relationship (linear regression) of DNA promoter methylation per transcript with BMI. P-values for epigenome wide association analysis with BMI were calculated using an R (version 3.0.2) package called CpGassoc [58]. All analyses were adjusted for age, gender, and type 2 diabetes, separately in a) SAT wcs.1183 and b) OVAT in the samples from the Leipzig cohort, for which methylation data were available (N ?77). Different transcripts per gene showing exactly the same association results are summarized in the same dot. Bonferroni correction was used to correct for multiple testing.differentiation to adipocytes and thereby affecting the adipose tissue expansion capacity [30], this might indicate reduced adipocyte differentiation in OVAT. In line with this, increased Notch signaling in mice blocked the expansion of white adipose tissue, ectopic fat accumulation and insulin resistance [42], which further supports the potential dysfunctional role of OVAT [1,2]. Among the successfully replicated hits was SORBS2, which was more methylated and less expressed in SAT versus OVAT in both non-obese and obese subjects. We substantiated these results in isolated adipocytes. Linear regression analyses revealed a strong association ofmethylation with BMI for SORBS2 in OVAT. SORBS2 might be involved in insulin mediated translocation of GLUT4 [43] and t thereby might affect energy storage. Furthermore, we clearly show that in vitro promoter methylation of SORBS2 directly represses the tra.