TCF recognizes repressed W-CRMs through a bipartite mechanism.

(A) A cartoon showing the Tig and Ugt36Bc loci, along with the regions that were footprinted indicating the location of the WGAWAW sites (red) and r-Helper sites (blue). (B) Example of a footprinting chromatograph showing the C-clamp-specific protection of the r-Helper in the Ugt36Bc W-CRM. The boxed region where the green peaks are higher than the blue indicates sequences protected by GST-HMG-C-clamp and not by GST-HMG. (C) Alignment of the WGAWAW and r-Helper sites identified by footprinting from the Tig and Ugt36Bc W-CRMs. The WGAWAW sites were identified by comparing footprints of GST-HMG and GST, while r-Helper sites were footprinted by GST-HMG-C-clamp and not GST-HMG. In the alignments, the footprinted sequences are underlined. The consensuses for each motif are shown, along with the classic HMG and Helper site consensuses. (D) Sequences of the probes used for EMSA, derived from two endogenous WGAWAW, r-Helper pairs. Mutations in the r-Helper and WGAWAW motifs are indicated. (E) EMSA data showing that both WGAWAW sites and r-Helper sites were required for maximal binding with GST-HMG-C-clamp protein. The reduction of binding with the Tig Hm probe was slight but reproducible. (F) EMSA showing that r-Helper sites were not required for binding by GST-HMG protein. All footprinting and EMSA experiments were performed at least three times with similar results.