Figure 5. ZEB1 functionally interacts with YAP.
(a,b) Schematic representation of the promoter region of human CTGF (a) and CYR61 (b) genes on chromosomes 6q23.2 and 1p22.3, respectively. The potential ZEB1 (E-boxes) and YAP/ TEAD (MCAT) binding sites, the regions amplified after chromatin immunoprecipitation (ChIP) (blue arrows represent primer pairs) are depicted. Numbers indicate positions in bp on chromosomal DNA relative to the transcription start site (+1). Used luciferase (luc2) reporter constructs with and without E-boxes analysed in MCF7 cells transiently transfected with ZEB1, YAP or empty vector control are shown; n=3. qPCR analysis after ChIPs for endogenously expressed ZEB1 and YAP in MDA-MB231 cells shows direct binding of both factors, the promoters of CTGF and CYR61. HPRT1 is used as negative control; n=3. (c) qPCR analyses of anti-ZEB1/anti-YAP sequential ChIP (re-ChIP) of HEK293T cells overexpressing ZEB1 alone or in combination with V5-tagged YAP at the indicated gene loci (initial ChIP against ZEB1 and re-ChIP against V5). We confirmed that ZEB1 and YAP can simultaneously bind to the same region of the CTGF gene but not at the CDH1 and MIR200C loci, two genes repressed by ZEB1 and at control HPRT1; n=2. (d) CTGF-promoter reporter assay upon overexpression of ZEB1 and YAP expression constructs (YAP=wild type, del TA=without transactivation domain, del TB=without TEAD binding domain) in MCF7; n=3. (e) CTGF-promoter reporter constructs with sequential mutations in the three TEAD bindings sites (red crosses). Reporter assays in MCF7 cells showing that TEAD binding sites are important for functional interaction of ZEB1 and YAP; n=3. (f) Reporter constructs with four tandem repeats of wild-type or mutated (mut) TEAD binding sites upstream of a minimal promoter analysed in MCF7 cells showing that functional interaction of ZEB1 and YAP is transferred through TEAD binding sites n=3. For a–f, mean±s.e.m. For reporter assays, firefly luciferase activity was normalized to co-transfected renilla luciferase; *P=0.01–0.05, **P=0.001–0.01, ***P=0.001–0.0001, ****P<0.0001, NS, not significant, two-way analysis of variance with Bonferroni multiple comparisons test. ****P<0.0001, unpaired two-tailed Student's t-test. For ChIP assays, *P=0.01–0.05, nonparametric Mann–Whitney U-test.