Figure 6

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.