Figure 6: Lnc-Smad3 reduces the accessibility of the Smad3 promoter to Ash1l.
(a) CHIP analysis of the H3K4me3 and H3K27ac modifications, and Pol II occupancy around lnc-Smad3 and Smad3 loci in CD4+ T cells left unstimulated (day 0) or cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days (day 2). Four regions (capital letters A–D) across lnc-Smad3 gene locus and three regions (capital letters E–G) across Smad3 gene locus were analysed by CHIP assay. Normalized data are shown as percentage of input control. (b) HepG2 cells were transfected with a Smad3 promoter reporter construct (1 kb upstream of the transcription start site) and lnc-Smad3 expression vector (lnc-Smad3) or empty control vector (CTR), and stimulated with TGF-β. Luciferase activity was measured 48 h later. Data were normalized to renilla luciferase and presented with respect to CTR, set as 1. (c) Chromatin accessibility of the Smad3 promoter region by quantitative PCR with DNase I pretreated nucleus of CD4+ T cells transduced with a control lentivirus (Lenti-CTR) or lnc-Smad3-expressing lentivirus (Lenti-lnc-Smad3) and cultured under iTreg cell-skewing conditions (with TGF-β) for 2 days. Changed fold are concluded using 2ΔCt with respect to CD4+ T cells transduced with Lenti-CTR, set as 1. (d) CHIP analysis of the accumulation of Ash1l and H3K4me3 modification at Smad3 promoter regions in CD4+ T cells transduced and cultured as in c. Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. Error bars represent s.d. Student’s t test. *P<0.05, **P<0.01. All data are from three independent experiments (mean±s.d. of technical triplicates).
