Fig. 5

Zeb1 directly regulates miR-1199-5p expression. a Zeb1 controls the promoter activity of the miR-1199 gene. NMuMG/E9 cells were transfected with a miR-1199 promoter Firefly luciferase reporter construct, a Renilla luciferase reporter construct as well as with (left) a siRNA against Zeb1 (siZeb1) or a negative control siRNA (siCtr) or (right) a Zeb1 expression construct (6xMyc-tag-Zeb1) or a negative control construct (6xMyc-tag). Cells transfected with siRNAs were cultured for 4 days with TGFβ, while cells transfected with 6xMyc or 6xMyc-tagged Zeb1 were cultured in the absence of TGFβ. Relative luminescence (Firefly/Renilla) was calculated and normalized to the control Firefly luciferase reporter (pGL4; mean fold changes±s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05, **P < 0.01). b Zeb1 controls miR-1199-5p transcript levels. NMuMG/E9 cells were transfected with siRNAs (left) and Zeb1 constructs (right) as described in a and further cultured in the absence or presence of TGFβ. MiR-1199-5p transcript levels were examined by RT-PCR analysis (mean fold changes±s.e.m.; left: n = 5; right: n = 3). c Schematic presentation of the genomic localization of the murine miR-1199 gene and its promoter region. Red: mmu-miR-1199; grey: 2210011C24Rik gene; green: E-boxes (CANNTG, N = G or C); blue: promoter fragments examined by ChIP-qPCR analysis. d Zeb1 directly binds to the miR-1199 promoter. Chromatin of Py2T cells cultured in the absence (green) and presence of TGFβ (red) was subjected to chromatin immunoprecipitation with antibodies against Zeb1 followed by RT-PCR analysis using primers amplifying different regions of the miR-1199 promoter illustrated in c. An intergenic region was used as negative control. Data were normalized to control IgG and are presented as mean fold enrichment above background±s.e.m. (n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05, **P < 0.01, ****P < 0.0001). e Analysis of E-box-binding motifs in the miR-1199 promoter as potential Zeb1-binding sites. NMuMG/E9 cells were transfected and cultured as described in a (right). Relative luminescence (Firefly/Renilla) was calculated as described in a (mean fold changes±s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05, ***P < 0.001)