Citation
- Authors: Bejjani F. et al.
- Year: 2021
- Journal: Nucleic Acids Res 49 2488-2508
- Applications: in vitro / siRNA / INTERFERin
- Cell type: MDA-MB-231
Description: Human breast adenocarcinoma cells
Known as: MDAMB231
Method
For RNAi-mediated Fra-1 and/or Fra-2 depletion, we used pools of 3 siRNA directed to each protein (siFra-1 and siFra-2, respectively) and low siRNA concentrations to minimize potential off-target effects without altering on-target ones.
For simple Fra-1 or Fra-2 depletion, 4.5 nM (i.e. 1.5 nM of each siRNA constituting the pools) siFra-1 or siFra-2 were used in 72 h-long experiments.
For depletion of both proteins together, 9 nM (4.5 nM of siFra-1 + 4.5 nM of siFra-2) of siFra-1+siFra-2 were used.
siRNA transfections were conducted using INTERFERin (Polyplus) according to the supplier's specifications.
For transcriptomic analysis (see Supplementary Data S1A), we included two control conditions: cells transfected with a control siRNA (siCTL, 4.5 nM for single depletion, and 9 nM for double knockdown) and cells placed in the presence of the transfection agent without siCTL (NT) to take into consideration possible off-targets effects of the control siRNA.
Abstract
The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.