Dispensability of SCL transactivation and DNA binding domains forc-kit promoter activation.

(A) Mapping of SCL domains required for its function in transcription activation. The kit-1146 reporter was cotransfected in NIH-3T3 cells with complexes containing the indicated SCL point or deletion mutants (50 to 450 ng).18 Numbers correspond to amino acid residues of SCL. The bHLH indicates basic helix-loop-helix (open boxes); AD, activation domain (hatched box); F– –L→A– –A, point mutations within helix 1 of the HLH domain that disrupt dimerization with E2A; RER→AAA, point mutations within the basic domain that disrupt DNA binding; n, number of representative experiments. (B) DNA binding properties of SCL mutants. EMSA experiments were performed with the use of in vitro–synthesized ³⁵S-labeled E2A and SCL mutants and ³²P-labeled TAL1 probe.26 Where indicated, antibodies were included in the samples before addition of the labeled probes. Arrows point to the binding of E2A homodimers or SCL/E2A heterodimers, and arrowheads indicate the supershifted bands. A monoclonal antibody against c-Myc was used as a control for supershifting (lane 8), while an unprogrammed reticulocyte lysate was included as a negative control for binding (lane 9).