Figure 8: IL-4 downregulates miR-130a-3p by inducing histone deacetylation.

(a) A conserved Sp1-binding element on the miR-130a promoter across species predicted by JASPAR. (b) Lysates of human macrophages with indicated treatments were prepared for the ChIP assay using anti-Sp1 Ab (n=3). (c) The nuclear extract of human macrophages with indicated treatments was prepared for EMSA(n=3). (d,e) Human macrophages were stimulated with IL-4 for indicated time and kinetics of RNA pol II (d) or AcH4 (e) on the miR-130a promoter were analysed by ChIP. The results are presented as enrichment (percentage of input DNA) of RNA pol II or AcH4 promoter occupancy(n=3). (f) Human macrophages were stimulated with IL-4 with or without pretreatment of DMSO or TSA.miR-130a expression was determined by qRT–PCR 24 h afterwards (n=3). (g) Human macrophages were stimulated with IL-4 for 24 h. The expression of the indicated HDACs was quantitated by qRT–PCR(n=3). (h) Human macrophages were stimulated with IL-4 for 48 h and the protein level of HADC2 was determined by western blot(n=3). (i) Binding of HDAC2 on the miR-130a promoter in human macrophages was analysed by ChIP(n=3). (j) Representative western blotting for HADC2 of IL-4-treated human macrophages transduced with HADC2-shRNAs(n=3). (k) Binding of AcH4 on the miR-130a promoter was analysed by ChIP (left) and the expression of miR-130a-3p (right) was analysed by qRT–PCR in cells treated as in (j) (n=3). (l) Representative western blotting for HADC2 of IL-4-treated human macrophages pretreated with STAT6 inhibitors or transduced with STAT6-shRNAs (n=3). (m) Binding of AcH4 on the miR-130a promoter was analysed by ChIP and expression of miR-130a-3p was analysed by qRT–PCR in cells treated as in (l) (n=3). (mean±s.e.m.; *P<0.05; **P<0.01 compared with control. ^#P<0.05, ^#P<0.01 compared with M(IL-4) for all the experiments above. The numbers above the blots present the intensity ratio of the indicated protein/GAPDH analysed by ImageJ).