Figure 2

EVI1 regulates MS4A3 by directly binding to a proximal element in its promoter. A) Luciferase assays with MS4A3 promoter deletion constructs. The MS4A3 5′ region, starting from -3213 relative to the transcription start site, and several 5′ deletion variants thereof were cloned into the promoterless Gaussia luciferase reporter vector, pGluc basic. Reporter plasmids and either an EVI1 expression vector (+EVI1; black bars) or empty vector as a control (-EVI1; grey bars) were transfected into U937 cells, and luciferase activity was measured from cell supernatants two days later. pGluc basic without any MS4A3 5′ sequences was used as negative control. B) Similar experiments were performed using some of the above described reporter plasmids with the HSV tk basal promoter inserted between the MS4A3 5′ regions and the luciferase gene of pGluc basic. Data in A) and B) represent means + SEMs from three independent biological replicate experiments. C) ChIP assays were performed on U937_EVI1 and U937_vec cells using two different EVI1 antibodies (AB1, sc-8707X, Santa Cruz; AB2, C50E12, Cell Signaling). Primers used for ChIP PCR amplified a region in the proximal MS4A3 promoter as indicated by the arrows in the upper panel. IgG, negative control using nonspecific IgG; no AB, negative control without antibody; +, input DNA (positive control); -, H₂O (negative) PCR control.