Role of the Sp1 binding site in c-kitpromoter activation by the SCL complex.
(A) Deletion or point mutation of a GC-box impairs c-kitpromoter activation by the SCL complex. NIH 3T3 cells were transfected with mutant c-kit reporter constructs in the absence (open bars) or presence (solid bars) of the SCL complex. Luciferase activities were normalized to the activity of CMV-βgal. Thec-kit promoter GC-box and surrounding sequences from position −122 to −70 are indicated. (B) Sp1 is the main factor that binds to the c-kit promoter GC-box in TF-1 cells. Electrophoretic mobility shift assays (EMSAs) were done with the use of 32P-labeled kit-GC-box probe and TF-1 cell nuclear extracts. Supershifting was performed with a preimmune serum (Ctl) and an anti-Sp1 antibody (lanes 1-4). Competition assays (lanes 5-8) were done with the use of a 10- and 100-fold molar excess of unlabeled kit-GC-box or kit-GC-box-mt double-stranded oligonucleotides. The plus and minus signs indicate, respectively, inclusion or omission of specific ingredients. Arrowhead points to the major Sp1 complex, while arrow points to free probe. (C) All partners of the SCL complex are required for maximal c-kit promoter activation. Expression vectors for SCL, E47, LMO2, Ldb-1, and GATA-2 were cotransfected with the kit-1146 (open bars) or kit-122 (solid bars) reporter plasmids. Where indicated (−), the corresponding vectors were omitted from the transfection mixtures. For panels A and C, results are shown as luciferase activity relative to the empty pXPIII vector and are representative of (n) independent experiments. (D) Partners of the SCL complex are expressed in B-cell precursors in which SCL induces endogenous c-kit expression. Bone marrow B-cell precursors from WT mice were purified by flow cytometry as in Figure 1C, and expression of SCL partners was assessed by RT-PCR. S16 was used as a control for the amount of cDNA.
