Fig. 1

Transcriptional repression of TGFB2 by Fra-1. In all experiments, MDA-MB-231 cells were transfected with either siCTL (control) or siFra-1 for 72 h. A TGFB2 gene. The 8 exons of TGFB2 are indicated by black boxes. The amplicons used for assaying nascent RNAs by run-on or TGFB2 pre-mRNA are located in the first and second introns and are indicated in red and green, respectively, whereas the one used for assaying TGFB2 mRNA overlaps exons 3 and 4 and is shown in blue. B TGFB2 mRNA abundance upon RNAi-mediated depletion of Fra-1. Fra-1 amounts were assayed by immunoblotting, taking glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an invariant loading control (left panel). TGFB2 mRNA was assayed by RT-qPCR in 4 independent experiments (right panel). The S26 mRNA was taken as an internal standard and signals were normalized to the siCTL condition arbitrarily set to 1. C Assay of TGFB2 nascent RNAs upon Fra-1 depletion. Run-on assays were performed in 3 independent experiments. Quantifications were performed using GAPDH as an invariant standard and normalized to the siCTL condition arbitrarily set to 1. D Assay of TGFB2 pre-mRNA upon Fra-1 depletion. TGFB2 pre-mRNA contained in total cell RNA was assayed by RT-qPCR in 7 independent experiments GAPDH mRNA was used as an invariant standard and data were normalized to the siCTL condition arbitrarily set to 1. E Assay of H3K36me3 at the beginning of TGFB2 after Fra-1 depletion. ChIP-qPCRs were carried out as indicated in Materials and Methods. The positions of the amplicons used are indicated with respect to the TGFB2 TSS, which is indicated by an arrow. The values are the mean of 5 independent experiments and were normalized to that of amplicon + 2,9 under control condition, which was arbitrarily set to 1. The sequences, or the commercial references, of the siRNAs used are provided in Additional file 7: Table S1A whereas the sequences of the oligonucleotides used in RT-PCR assays are given in Additional file 7: Table S1B