PMC

Two modes of TCF7L2-mediated transcriptional repression of GATA3 target genes. (a) GATA3 tethers TCF7L2 to the genome and both factors cooperate to repress target genes. (b) GATA3 tethers TCF7L2 to the genome with TCF7L2 antagonizing GATA3-mediated transcriptional activation.

Association of other motifs with TCF7L2 binding sites. (a,b) TCF7L2 binding sites unique to HepG2 cells (a) or MCF7 cells (b) were analyzed for the indicated motifs; the position of each motif is plotted relative to the center of the TCF7L2 binding site.

Cell type-specific binding of TCF7L2. (a,b) The ChIP-seq binding patterns of TCF7L2 are compared in six cell lines, demonstrating both common peaks (a) and cell type-specific binding (b). (c) The ChIP-seq binding patterns of TCF7L2 near and within the SH3BP4 locus is shown for three cell lines. The number of tags reflecting the ChIP enrichments are plotted on the y-axis; the chromosomal coordinates (hg19) shown are: (a) chr19:7,701,591-7,718,750; (b) chr1:112,997,195-113,019,766; and (c) chr2:235,767,270-235,974,731.

ChIP-seq analysis of TCF7L2 in six different human cell lines. Shown is the distribution of TCF7L2 binding within ±3 kb windows around distinct genomic regions (n = 3,000) bound by TCF7L2 in a given cell type. ChIP-seq tags for each cell line were each aligned with respect to the center of the combined top 500 peaks from each dataset and clustered by genomic position.

Association of TCF7L2 and HNF4α in HepG2 cells. (a) HNF4α and FOXA2 ChIP-seq data were downloaded from the UCSC genome browser, and peaks were called and overlapped with the HepG2 cell type-specific TCF7L2 peaks. (b) Peaks bound only by HNF4α, only by TCF7L2, or by both factors were analyzed for the presence of HNF4α and TCF7L2 motifs. (c) For the set of 7,576 peaks bound by all three factors, the location of the HNF4α and FOXA2 peaks were plotted relative to the center of the TCF7L2 peak. (d) A comparison of TCF7L2, HNF4α, and FOXA2 binding patterns near the GREB1 locus is shown. The hg19 genomic coordinates are chr2:11,636,208-11,708,654. The number of tags reflecting the ChIP enrichments is plotted on the y-axis.

TCF7L2 binding sites are distal and enriched for active enhancer histone marks. (a) Shown for the TCF7L2 binding sites in the six cell types and for the 1,864 peaks commonly bound in all six cells is the percentage of TCF7L2 binding sites in different genomic regions (hg19) relative to the nearest transcription start site (TSS). (b) The percentage of active enhancer regions containing a TCF7L2 binding site; active enhancers were defined by taking the regions that have an overlap of H3K4me1 and H3K27ac ChIP-seq peaks for the given cell line. (c) Heatmaps of the ChIP-Seq tags for H3K27ac and H3K4me1 at TCF7L2-bound regions (±3 kb windows around the center of all TCF7L2 peaks) for each cell line were generated by k-means cluster analysis. (d) The average RNA polymerase II and histone modification profiles of MCF7 cells are shown for the ±3 kb windows around the center of TCF7L2 peaks identified as proximal to RefSeq genes (upper graph) or distal to RefSeq genes (lower graph).

Transcriptional regulation of TCF7L2 and GATA3 target genes. (a) The nearest gene to each TCF7L2 binding site and the nearest gene to each GATA3 binding site was identified and the two lists were compared to identify 3,614 genes that are potentially regulated by both GATA3 and TCF7L2. (b) The expression of the 3,614 GATA3+TCF7L2 bound genes was analyzed in control cells, cells treated with siRNAs to TCF7L2, and cells treated with siRNAs to GATA3; the number of genes whose expression increases or decreases is shown. (c) A scatterplot of expression data from RNA-seq experiments. Each point corresponds to one NCBI Reference Sequence (RefSeq) transcript with fragments per kilobase of gene per million reads (FPKM) values for control and siGATA3 or control and siTCF7L2 knockdown samples shown on a log10 scale. The dashed line represents no change in gene expression between the two samples. Differentially expressed genes whose function corresponds to Gene Ontology categories of breast cancer, cell differentiation, and response to hormone stimulus are highlighted.

Association of TCF7L2 and GATA3 in MCF7 cells. (a) GATA3 ChIP-seq in MCF7 cells was performed, and peaks were called and then overlapped with the MCF7 cell type-specific TCF7L2 peaks; a comparison of TCF7L2 and GATA3 binding patterns near the CDT1 locus is shown. The hg19 genomic coordinates are chr16:88,861,964-88,880,233. (b) Peaks bound only by GATA3, only by TCF7L2, or by both factors were analyzed for the presence of GATA3 and TCF7L2 motifs. The GATA3 motif is found in sites bound by GATA3 only and in sites bound by both factors, whereas the TCF7L2 motif is found only in the sites bound only by TCF7L2 and not in the sites bound by both factors. (c) Depletion of GATA3 results in loss of TCF7L2 occupancy at sites bound by TCF7L2 and GATA3 sites but not at sites only bound by TCF7L2. MCF7 cells were transfected with siRNAs specific for TCF7L2 or GATA3 or control siRNAs. ChIP-qPCR assays were performed using antibodies specific for TCF7L2 (left panel) or GATA3 (right panel) using primers specific for peaks bound only by GATA3, only by TCF7L2, or by both factors. Shown are ChIP-qPCR results performed in triplicate and plotted with the standard error of two independent experiments. (d) Co-immunoprecipitation of endogenous GATA3 and FLAG-tagged TCF7L2 constructs from MCF7 cells. The left panel analyzes whole-cell extracts (WCE) and FLAG immunoprecipitation (FLAG IP) eluates from MCF7 cells transfected with the indicated FLAG-tagged plasmids; the membrane was incubated with both anti-FLAG and anti-GATA3 antibodies. Note that the GATA3 signal in input WCE extracts is quite weak and can generally only be visualized after concentration by immunoprecipitation. The right panel is a separate blot prepared in the same way (using the GATA antibody for immunoprecipitation), but does not include the WCE extracts. V, vector control; E, full length TCF7L2; EΔ, TCF7L2 lacking the amino terminus; B, TCF7L2 isoform lacking the carboxyl terminus; BΔ, TCF7L2 isoform lacking the amino and carboxyl termini.