Fig. 4: In vitro functional characterization of SOCS3 methylation.

a UCSC genome browser plot showing the SOCS3 gene (blue bar), CpGs (red lines), and DMR (orange bar) identified in the EWAS. The tracks from top to bottom indicate a CpG island (chr17: 76,354,818–76,357,038; hg19); DNase hypersensitivity clusters showing open chromatin (125 cell types from ENCODE version 3); histone tail marks H3K4Me1, H3K4Me3 and H3K27Ac indicating active regions of transcription, and transcription factor binding sites (ENCODEv3). Overlayed colored tracks for the histone tail marks represent seven cell lines from ENCODE, including GM12878: Lymphoblastoid cell line, H1-hESC human embryonic stem cell line, HSMM skeletal muscle myoblast cell line, HUVEC human umbilical vein endothelial cell line, K562 chronic myelogenous leukemia cell line, NHEK normal human epidermal keratinocyte cell line, NHLF normal human lung fibroblast cell line. b Luciferase assay experiments using CpG free Lucia vector showing enhancer activity of the SOCS3 DMR constructs (forward orientation) in human lung carcinoma (A549), human embryonic kidney (HEK293T) and human liver carcinoma (HepG2) cell lines. c Luciferase assay experiments in HepG2 cell lines showing a regulatory role of methylation at the SOCS3 DMR. Methylated (striped bars) versus unmethylated SOCS3 constructs are compared in both forward (dark green) and reverse (light green) orientations. b, c Normalized mean relative luciferase units (RLU) from three experiments are shown on the y axis and the error bars indicate standard deviation of RLU. Two-sided t tests were used for all comparisons. UCSC University of California Santa Cruz, DMR Differentially Methylated Region, EWAS Epigenome-Wide Association Study.