A repressor sequence in Tcf7l2. (A) C-terminal sequences in Tcf7l2 confer repressor function on Tcf7. C-terminal sequences containing the Ctbp binding sites (7–7L2) but not sequences containing the TLE binding domain (7L2–7 #1 and 7L2–7 #2) from Tcf7l2 fused to Tcf7 convert Tcf7 from a transcriptional activator to a repressor as measured by using the STF reporter. In some cases, two separate constructs were tested. (B) Protein sequence in addition to Ctbp binding sites contributes to Tcf7l2 repressor function. The effects of serial C-terminal deletions in a Tcf7/7L2 fusion molecule that exhibits pathway repressor activity (7–7L2) were measured in HeLa cells, using the STF reporter. Residue numbering is based on Tcf7l2 sequence. Sequence of the novel repressor domain is shown. Mean of triplicate experiments ± SD are shown. (C) Expression of Tcf7 and Tcf7l2 fusion molecules described in B. (D) Ctbp contributes to repression of STF activity. RNAi of both Ctbp 1 and 2 in HeLa cells results in increased STF activity in response to Wnt protein. The fold increase in pathway response is noted above each triplicate experiment.

TCF7L2 mutations identified in CRC abrogate Tcf7l2 pathway repressor function. (A) Predicated changes in Tcf7l2 E variant protein structure resulting from CRC mutations in the TCF7L2 gene (6, 17). NLS, nuclear localization signal; HMG box, high-mobility group DNA binding domain. (B) Effects of mutant Tcf7l2 overexpression on DLD-1 growth. Growth of DLD-1 cells overexpressing wt or mutant Tcf7l2 proteins at clonal density was measured by using the Cell-Titer Glo assay 6 days after transfection. P values are provided for each experiment. (C) Expression of Tcf7l2 WT and mutant proteins analyzed by Western blot analysis in HEK293T cells. (D) Dose-dependent effects on pathway response with overexpression of Tcf7 and Tcf7l2. Transfection of Tcf7 and Tcf7l2 (E form) DNA resulted in a respective increase and decrease of STF activity in either HeLa (with Wnt3A expression) or DLD-1 cells. (E) Effects of CRC mutations on Tcf7l2 repressor function. Overexpressed Tcf7l2 and mutant 3 but not mutants 1, 2, or 4 suppress STF activity in HeLa cells.

The function of TCF family members is cell-type-specific. (A) Tcf7l2 functions as a transcriptional repressor in many cell types. Effects of decreased Tcf7l2 function on normal Wnt pathway response were measured by using the STF reporter in HeLa and HEK293T cells expressing Wnt3A. In CRC cells, the pathway is constitutively activated as a result of respective loss- or gain-of-function mutations in APC (DLD-1) or CTNNB1 (HCT116 cells). (B) Contrasting transcriptional roles for Tcf7l2 in two different mouse cell lines. A similar experiment described in A but performed in mouse L- or NIH 3T3 cells reveals cell-type-specific functions for Tcf7l2. (C) The predominantly expressed forms of Tcf7l2 in CRC cells and L-cells as detected by Western blot analysis in HCT116 cells are respectively the E variant (containing Ctbp binding and the novel repressor sequences) and presumably the B variant (which lacks these repressor sequences). (D) Expression of Tcf7 in DLD-1 cells. Tcf7 is predominantly expressed as an isoform that comigrates on SDS/PAGE with the B form that contains a β-catenin binding domain. (E) Expression of the Tcf7 B form in different human cells lines detected by Western blot analysis.

Overview of the RNAi-based screening strategy to identify components of the Wnt/β-catenin pathway. (A) The screening strategy. HeLa cells were cotransfected with siRNA pools targeting a single gene, and various reporter and expression constructs to identify positive and negative regulators of the pathway measured by firefly luciferase (FL) activity generated from the Wnt-responsive SuperTopFlash (STF) reporter construct. Activity of the secreted control Gaussia luciferase (GL) protein reflects levels of protein exocytosis. The Renilla luciferase (RL) activity also serves as measure of general cellular functions. FL and GL activities were normalized to RL activity in the “plus Wnt screen.” Ig-Fc, control Ig protein. (B) Summary of primary screen and secondary assay results. A human siRNA library (Dharmacon) was screened in the absence or presence of Wnt-pathway activation, and interesting siRNAs were retested. Subsequent genes of interest were tested again, using nonredundant siRNAs from another human siRNA library (Qiagen). Candidate genes from the positive regulator screen were then screened in the presence of exogenous Wnt3A to eliminate those genes potentially involved in Wnt protein production. Those siRNAs likely affecting cellular response to Wnt3A were tested in mouse NIH 3T3 cells, using a mouse siRNA library (Qiagen). Hits identified from various tests (boxed) were each assigned a single point (1 pt). Format of assays are noted on the left with criteria used to identify hits described in Material and Methods.

The cellular program controlled by Tcf7l2 in CRC cells. (A) Positively acting TCF molecules and Tcf7l2 repressor control distinct target genes. Gene expression profiling identified genes regulated by positively acting TCF molecules in Ctnnb1 siRNA-treated cells (gray circle) that were also sensitive to Tcf7l2 loss (red, green circles). Both known [23; see also the Wnt homepage (www.stanford.edu/∼rnusse/wntwindow.html)] and predicted (31) TCF target genes were identified by using Ctnnb1 siRNAs (fraction of control expression shown in parenthesis for some genes; all data are shown in Table S6 and Table S7). However, the expression of these genes was mostly unchanged with loss of Tcf7l2. (B) Transcription of several known Wnt pathway components are not altered with loss of Tcf7l2 as determined by gene expression profiling. (C) Protein levels of several Wnt pathway components are not altered upon loss of Tcf7l2 as determined by Western blot analysis. Note that cell treatments in A–C were simultaneously performed by using identical conditions. (D) Identification of genes controlled by Tcf7l2 repressor function in CRC. Genes up-regulated >2-fold upon loss of Tcf7l2 (green) were identified by using expression profiling in DLD-1 cells. Of the genes within this dataset, the HGF and HECA have predicted TCF enhancer sequences in their promoter regions as identified from an in silico search for TCF target genes (31). Fold change is an average of results from duplicate microarray experiments. (E) Changes in the expression of HGF, HECA, and Tcf7l2, upon introduction of Tcf7l2 siRNAs in DLD-1 cells, were confirmed by using RT-PCR. (F) Loss of HGF or HECA abrogate the cell growth-promoting effects of Tcf7l2 RNAi. Simultaneous treatment of cells with Hgf or Heca and Tcf7l2 siRNAs abrogates the increase in DLD-1 cell growth induced by loss of Tcf7l2 alone as determined by measuring cellular ATP levels. P values for each experiment are shown. (G) RT-PCR analysis of HGF and HECA expression levels after siRNA treatment. (H) Models of normal and aberrant activation of Wnt/β-catenin pathway response in cells that harbor Tcf7l2 repressor function. Optimal induction of Wnt-mediated pathway response in such cells entails simultaneous activation of Tcf7 and inactivation of Tcf7l2. In CRC cells, oncogenic Wnt pathway activity is initiated in most cases by homozygous loss of APC function. Additional loss of TCF7L2 function likely results in greater tumorigenicity.