The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

Plau codes for the urokinase-type plasminogen activator (uPA), critical in cancer metastasis. While the mechanisms driving its overexpression in tumorigenic processes are unknown, it is regulated by the AP-1 transcriptional complex in diverse situations. The AP-1 component Fra-1 being overexpressed in aggressive breast cancers, we have addressed its role in the overexpression of Plau in the highly metastatic breast cancer model cell line MDA-MB231 using ChIP, pharmacological and RNAi approaches. Plau transcription appears controlled by 2 AP-1 enhancers located -1.9 (ABR-1.9) and -4.1 kb (ABR-4.1) upstream of the transcription start site (TSS) of the uPA-coding mRNA, Plau-001, that bind Fra-1. Surprisingly, RNA Pol II is not recruited only at the Plau-001 TSS but also upstream in the ABR-1.9 and ABR-4.1 region. Most Pol II molecules transcribe short and unstable RNAs while tracking down toward the TSS, where there are converted into Plau-001 mRNA-productive species. Moreover, a minority of Pol II molecules transcribes a low abundance mRNA of unknown function called Plau-004 from the ABR-1.9 domain, whose expression is tempered by Fra-1. Thus, we unveil a heretofore-unsuspected transcriptional complexity at Plau in a reference metastatic breast cancer cell line with pleiotropic effects for Fra-1, providing novel information on AP-1 transcriptional action.

The c-Fos proto-oncogenic transcription factor defines a multigene family controlling many processes both at the cell and the whole organism level. To bind to its target AP-1/12-O-tetradecanoylphorbol-13-acetate-responsive element or cAMP-responsive element DNA sequences in gene promoters and exert its transcriptional part, c-Fos must heterodimerize with other bZip proteins, its best studied partners being the Jun proteins (c-Jun, JunB, and JunD). c-Fos expression is regulated at many transcriptional and post-transcriptional levels, yet little is known on how its localization is dynamically regulated in the cell. Here we have investigated its intranuclear mobility using fluorescence recovery after photobleaching, genetic, and biochemical approaches. Whereas monomeric c-Fos is highly mobile and distributed evenly with nucleolar exclusion in the nucleus, heterodimerization with c-Jun entails intranuclear redistribution and dramatic reduction in mobility of c-Fos caused by predominant association with the nuclear matrix independently of any binding to AP-1/12-O-tetradecanoylphorbol-13-acetate-responsive element or cAMP-responsive element sequences. In contrast to c-Jun, dimerization with JunB does not detectably affect c-Fos mobility. However, dimerization with JunB affects intranuclear distribution with significant differences in the localization of c-Fos.c-Jun and c-Fos.JunB dimers. Moreover, c-Jun and JunB exert comparable effects on another Fos family member, Fra-1. Thus, we report a novel regulation, i.e. differentially regulated intranuclear mobility and distribution of Fos proteins by their Jun partners, and suggest the existence of intranuclear storage sites for latent c-Fos.c-Jun AP-1 complexes. This may affect the numerous physiopathological functions these transcription factors control.

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.

The architectural chromatin protein HMGA1 and the transcription factor Fra-1 are both overexpressed in aggressive triple-negative breast cancers (TNBC), where they both favor epithelial-to-mesenchymal transition, invasion, and metastasis. We therefore explored the possibility that Fra-1 might be involved in enhanced transcription of the HMGA1 gene in TNBCs by exploiting cancer transcriptome datasets and resorting to functional studies combining RNA interference, mRNA and transcriptional run-on assays, chromatin immunoprecipitation, and chromosome conformation capture approaches in TNBC model cell lines. Our bioinformatic analysis indicated that Fra-1 and HMGA1 expressions positively correlate in primary samples of patients with TNBC. Our functional studies showed that Fra-1 regulates HMGA1 mRNA expression at the transcriptional level via binding to enhancer elements located in the last two introns of the gene. Although Fra-1 binding is required for p300/CBP recruitment at the enhancer domain, this recruitment did not appear essential for Fra-1–stimulated HMGA1 gene expression. Strikingly, Fra-1 binding is required for efficient recruitment of RNA Polymerase II at the HMGA1 promoter. This is permitted owing to chromatin interactions bringing about the intragenic Fra-1–binding enhancers and the gene promoter region. Fra-1 is, however, not instrumental for chromatin loop formation at the HMGA1 locus but rather exerts its transcriptional activity by exploiting chromatin interactions preexisting to its binding. Implications: We demonstrate that Fra-1 bound to an intragenic enhancer region is required for RNA Pol II recruitement at the HMGA1 promoter. Thereby, we provide novel insights into the mechanisms whereby Fra-1 exerts its prooncogenic transcriptional actions in the TNBC pathologic context.

Background: How transcription factors (TFs) down-regulate gene expression remains ill-understood, especially when they bind to multiple enhancers contacting the same gene promoter. In particular, it is not known whether they exert similar or significantly different molecular effects at these enhancers. Results: To address this issue, we used a particularly well-suited study model consisting of the down-regulation of the TGFB2 gene by the TF Fra-1 in Fra-1-overexpressing cancer cells, as Fra-1 binds to multiple enhancers interacting with the TGFB2 promoter. We show that Fra-1 does not repress TGFB2 transcription via reducing RNA Pol II recruitment at the gene promoter but by decreasing the formation of its transcription-initiating form. This is associated with complex long-range chromatin interactions implicating multiple molecularly and functionally heterogeneous Fra-1-bound transcriptional enhancers distal to the TGFB2 transcriptional start site. In particular, the latter display differential requirements upon the presence and the activity of the lysine acetyltransferase p300/CBP. Furthermore, the final transcriptional output of the TGFB2 gene seems to depend on a balance between the positive and negative effects of Fra-1 at these enhancers. Conclusion: Our work unveils complex molecular mechanisms underlying the repressive actions of Fra-1 on TGFB2 gene expression. This has consequences for our general understanding of the functioning of the ubiquitous transcriptional complex AP-1, of which Fra-1 is the most documented component for prooncogenic activities. In addition, it raises the general question of the heterogeneity of the molecular functions of TFs binding to different enhancers regulating the same gene.