TCF-1 binding sites are essential cis-regulatory elements for the activity of Major Peak. (A) The structural framework of constructs used in the stable transfection assays. Stars, mutated TCF sites in dmTCF construct; triangles, major peaks of TCF-1 binding as shown in Figure 6. (B) Constructs with PR1 or PR3 promoters and serial deletions of the Major Peak (in a downstream enhancer position) were made as shown schematically. The major TCF-1 binding peaks in MP are marked for reference (coordinates of construct end points provided in supplemental Table 2). Note that the CAAAG/CTTTG motifs that presumably nucleate the second TCF-1 peak are just beyond the boundary of MP-fragment 2. Graph shows normalized luciferase expression in 8 parallel cultures with each construct, stably transfected into P2C2 (red) and Raw264.7 (blue) cells after two weeks of selection. The same results were seen for these constructs in 2-3 independent experiments. (C) Either specific deletion of the two central TCF-1 binding sites or removal of the 3′ conserved region (MPF5) decreases the enhancer activity of Major Peak in P2C2 cells. dmTCF, the construct with double mutation (deletion) of TCF-1 binding peaks shown in Figure 6. MPF5, truncated construct lacking 3′ conserved region.