Figure 1

DNA binding specificity of DMRT1. (A) Summary of binding site selection. Top panel shows percentage of occurrence of each nucleotide at each position of 22 selected oligonucleotides. (At some positions percentages do not total 100, due to rounding to nearest integer.) Below the selection matrix is a comparison of the DMRT1 binding consensus to those of DSX [29] and MAB-3 [26]. (B) Test of DMRT1 DNA binding specificity. Competitive gel mobility shift assay in which labeled dsDNA probe matching DMRT1 binding consensus is competed with 40-fold excess of unlabelled dsDNA, either unaltered ("SELF") or containing the indicated sequence changes (positions align with those shown in panel A). Only the shifted DMRT1/DNA complex is shown. Darker bands indicate weaker competition. (C) Preferential binding to DNA by DMRT1 as a dimer. Gel mobility shift using increasing amounts of in vitro translated DMRT1 protein to shift a constant amount of labeled dsDNA. Positions of complexes containing monomer ("M") or dimer ("D") of DMRT1 are indicated. (D) DNA binding by DMRT1 from mouse testis. Gel mobility shift using in vitro translated DMRT1 (left) or mouse testis nuclear extract (right) to shift labeled dsDNA containing DMRT1 binding consensus. Addition of water or pre-immune rabbit serum does not affect shifted band, but addition of anti-DMRT1 immune serum [8] super-shifts the band into the gel well. (E) DMRT1 DNA recognition in cultured cells. Luciferase assay results shown for HEK293 cells transfected with expression vectors encoding only VP16, encoding DMRT1-VP16, or encoding DMRT1-VP16 with a missense mutation in the DM domain (R123A). Cells were co-transfected with a reporter plasmid containing four DMRT1 binding sites. Values shown are average of two experiments, with error bar indicating standard error of mean.