Fig. 4

Effects of Fra-1 knockdown on the activity and accessibility of TGFB2 enhancers. A Fra-1-dependent regulation of eRNA production at most Fra-1-bound TGFB2 enhancers. MDA-MB-231 cells were subjected to RNAi-mediated knock-down of Fra-1 for 72 h and eRNA levels were quantified by RT-qPCR at all TGFB2 enhancers. The sequences of the PCR primers are given in Additional file 7: Table S1C. Left panel: immunoblotting analysis of Fra-1 down-regulation upon siFra-1 versus siCTL transfection. GAPDH was used as an invariant control. Right panel: quantifications of eRNA levels. The data correspond to 7 independent experiments using S26 mRNA as an internal standard. They were normalized to siCTL condition arbitrarily set to 1 for each amplicon. B Chromatin accessibility at all Fra-1-bound enhancers in MDA-MB-231 cells. MDA-MB-231 cells were subjected to RNAi-mediated knockdown of Fra-1 for 72 h before ATAC-seq analyses were carried out. The metaprofiles correspond to the merge of 3 independent experiments using cells transfected with siCTL as a control. A threshold (i.e. the ratio of signal intensity in siFra-1- versus siCTL conditions) of ± 1.5 was used to classify the 4129 Fra-1-bound enhancers in three categories (-1.5 < FC <  + 1.5, FC ≤ − 1.5 and FC ≥ 1.5). The number of enhancers per category is indicated on the figure. C Chromatin accessibility assessed by ATAC-seq in the TGFB2 TAD. ATAC-seq- and NG Capture-C data were aligned along the whole TGFB2 locus using the IGV browser. D Chromatin accessibility assessed by FAIRE-qPCR at the Fra-1-bound TGFB2 enhancers and at the TGFB2 promoter. The data are the mean of 8 independent experiments. The sequences of the qPCR primers are given in Additional file 7: Table S1D. Left panel: FAIRE-qPCR assay at two control positions, one shown to be more accessible (left) and one less accessible (right) upon RNAi knockdown of Fra-1 in ATAC-seq experiments conducted in MDA-MB-231 cells. Right panel: FAIRE-qPCR experiments at the TGFB2 locus 72 h post-transfection of siCTL or siFra-1. Position + 481 kb is devoid of any ATAC-seq signal and was used as a negative control. The GAPDH promoter was used as an internal control to normalize the results to compensate for differences among samples according to Rodriguez-Gil [63]