TCF recognizes repressed W-CRMs through a bipartite mechanism.

<p>(<b>A</b>) A cartoon showing the <i>Tig</i> and <i>Ugt36Bc</i> loci, along with the regions that were footprinted indicating the location of the WGAWAW sites (red) and r-Helper sites (blue). (<b>B</b>) Example of a footprinting chromatograph showing the C-clamp-specific protection of the r-Helper in the <i>Ugt36Bc</i> 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. (<b>C</b>) Alignment of the WGAWAW and r-Helper sites identified by footprinting from the <i>Tig</i> and <i>Ugt36Bc</i> 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. (<b>D</b>) 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. (<b>E</b>) 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. (<b>F</b>) 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.</p>