Fig. 2

ANKRD26 promoter methylation in PBL of lean and obese subjects and in vitro promoter activity. a A CpG plot of the ANKRD26 gene promoter/Exon 1 (− 1000/+ 400 bp from TSS) was determined by using the EMBOSS CpGplot software. The presence of a 629 bp CpG island, straddling the ANKRD26 TSS (− 277/+ 351 bp) and two regions enriched in CpGs, respectively, at − 900/− 600 bp and at − 580/− 370 bp upstream the TSS were identified. Values in y-axis are the minimum average observed (Obs) to expected (Exp) ratio of C plus G to CpG in a set of 10 windows that are required before a CpG island is reported. The minimum length, that a CpG island has to be before it is reported, is 200 bp. A window is set by default to 100 bp. Values in the x-axis are base pairs. b Schematic representation of the ANKRD26 gene. Sub-region S1, − 991/− 693 bp; S2, − 716/− 370 bp; S3, − 349/− 48 bp; S4, − 68/+ 157 bp; S5, + 134/+ 390 bp. CpG Island, − 250/− 50. TSS, transcription start site; 5′-UTR, untranslated region. c Bisulfite sequencing methylation analysis of the ANKRD26 S2 (CpG sites, − 689, − 659, − 651, − 522, − 482, − 452, − 445, − 409, − 402 bp from the TSS) in converted genomic DNA from PBL of 14 lean and 20 obese subjects. Results are means ± SD. Statistical difference between the means of the two groups was assessed by classical OLS regression model to adjust for age. ***p < 0.001 vs. lean % CpG methylation. d Luciferase activity of in vitro CpG methylated (me) or un-methylated (unme) pCpG-ANKRD26 constructs and the pCpG empty vector was assessed as described in the “Methods” section. Firefly luciferase activity was normalized to Renilla luciferase activity. Luciferase activity is displayed in relative light units (RLU). Results are means ± SD from three independent experiments. Statistical difference between the means of the groups was assessed by one-way ANOVA. *p < 0.05 and ***p < 0.001 vs. pCpG empty vector; ^#p < 0.05 vs. pCpG-ANKRD26unme