Fig. 4

JUNB binds to and regulates the expression of TGFB2 gene. A Schematic representation of the TGFB2 gene. The position of the identified intragenic AP-1 site (B) and that of the AP-1 site located 114 kb downstream the TSS (C) are indicated. Both of them are bound by JUNB in U2OS cells. Position A, located +22 kb from the TSS, corresponds to a region not bound by JUNB and devoid of any AP-1 binding site and was used as a negative control for unspecific binding. B ChIP-qPCR analysis of the enrichment of JUNB binding to site +31 kb and +114 kb of the TGFB2 locus in inducible UTA6 cells expressing pCDNA3 (UTA6-Control) and UTA6 cells overexpressing JUNB (UTA6-JUNB) cells. Cells were grown in the absence of tetracycline, and ChIP was performed using JUNB antibody or IgG antibody as control. qPCR was carried out on the three TGFB2 regions A, B, and C. Statistical analyses were performed using two-way ANOVA with Tukey’s multiple comparisons test (**p < 0.01, ***p < 0.001, ****p < 0.0001). C UTA6-Control or UTA6-JUNB were synchronized in mitosis by using a double block with thymidine and nocodazole. Cells were subjected to a thymidine block prior to the nocodazole block, and Tet was removed from the cell culture at the same time as thymidine is removed and nocodazole added to ensure substantial induction of the Tet-regulated JUNB, which requires several hours [11]. Mitotic cells were collected, released into the cell cycle, and DNA content was analyzed by flow cytometry of propidium iodide (PI)-stained cells at the indicated times (in h) as shown in the histograms. A representative experiment out of two is shown. D TGFB2 mRNA levels during G1/S cell cycle progression in UTA6-control and UTA6-JUNB cell under the same conditions as described in C. TGFB2 mRNA levels were analyzed by RT-qPCR. Results are the mean of two independent experiments. Statistical analyses were performed with two-way ANOVA with Tukey’s multiple comparisons test (*p<0.05, **p<0.01). E JUNB overexpression decreases TGFB2 and increases CCNE1 protein levels during cycle progression. Protein extracts of the mitotic synchronized cells in C were analyzed by immunoblot using anti-JUNB, CCNE1, and TGFB2 antibodies. HSP90 and β-actin were used as loading control. F ChIP-qPCR analysis of the enrichment of JUNB binding to the site located 31 and 114 kb downstream of the TGFB2 TSS in UTA6-control and UTA6-JUNB synchronized cells. Cells were synchronized as described in C. Mitotic cells were collected, released into the cell cycle, and collected after 3 and 9 h for ChIP-qPCR analysis. ChIP was performed using a JUNB antibody or IgG antibody as control. qPCR was carried out on the three regions of TGFB2 locus depicted in A. Results are the mean of two independent experiments. Statistical analyses were performed by two-way ANOVA with Tukey’s multiple comparison test (*p<0.05, **p < 0.01, and ****p < 0.0001). G Addition of exogenous TGFB2 ligand impaired cell cycle progression in UTA6-Control and UTA6-JUNB cells. Inducible UTA6-Control and UTA6-JUNB cells were synchronized in mitosis as in E. Mitotic cells were collected, released into the cell cycle, and concomitantly treated with 8 ng/ml of exogenous TGFB2 ligand for up to 15 h. Histograms of a representative experiment out of two are shown