OPPOSING FUNCTIONS OF ROS AND SUMO TO CONTROL AP-1 TRANSCRIPTION COMPLEX ACTIVITY – ROSUAP

The AP-1 transcription complex is a collection of ubiquitously expressed dimeric transcription factors, principally made up of dimers of the Fos and Jun multigene families. It regulates many genes and, thereby, occupies a central physiological position. Its composition is dynamic and the various dimers exert different effects depending on the situation. Signaling converging to AP-1-constituting proteins is largely documented. However, the mechanisms whereby AP-1 transcriptional activity is molecularly regulated at the level of target promoters has surprisingly still received little attention. This concerns induction of AP-1-dependent transcription and, more critically, AP-1 activity termination. Gaining knowledge on these molecular events is essential as activity of most AP-1 dimers must be limited to strict physiological windows of time and controlled in intensity to avoid deleterious effects. Sumo-1, -2 and -3 (referred as Sumo here) are members of the ubiquitin family of peptidic post-translational modifiers. They are conjugated to acceptor lysines on their substrates. As for Ub and the other Ub-like proteins, the cycles of Sumo conjugation/deconjugation have recently appeared as important as those of phosphorylation/dephosphorylation to control the activity of many target proteins implicated in key cellular functions (transcription, replication, DNA repair, intracellular routing, cell cycle, etc…). In most cases, sumoylation of transcription factors results in transcriptional repression. However, the molecular mechanisms underlying this inhibition are far from being understood. Importantly here, we have formerly described transcriptional repression of AP-1 upon sumoylation of its c-Fos and/or c-Jun components, repression most probably occuring via sumoylation of a small fraction of Fos/Jun dimers bound to target promoters. Reactive oxygen species (ROS) are endogenously produced by aerobic cells as a consequence of oxygen metabolism. Exclusively considered as toxic molecules for long, they are now recognized as "second messengers" essential for proper regulation of intracellular cell signaling. They are produced endogenously in all cells by cell membrane-associated NAPDH oxidase complexes. However, little is known on how ROS play their physiological functions of second messengers, especially for activation of certain transcription factors including AP-1. Importantly, one of us has recently demonstrated that ROS can inhibit sumoylation through inactivation of sumoylation enzymes. Noteworthy, c-Fos and c-Jun are selectively and rapidly desumoylated in the presence of physiological concentrations of ROS or after low oxidative stress. As redox signaling is known to activate AP-1 in a number of situations, this ROS-induced desumoylation is likely to be important for target gene activation and more globally for cellular response. Based on our observations (repression of c-Fos:c-Jun dimers by sumoylation and inhibition of sumoylation by ROS) our project aims at elucidating (i) the intimate mechanisms of Fos/Jun AP-1 transcriptional repression by sumoylation at the level of target gene promoters (i.e. how the dynamics of chromatin modifications and configuration is affected), (ii) the finely tuned interplay between protein modification by Sumo and ROS signaling to control the activity of AP-1 on specific genes and (iii) the ROS-Sumo-AP-1 connection in two well-characterized models where ROS signaling plays important roles: one, macrophagic differentiation, is physiologic and the other, tumorigenesis, is pathologic. To achieve our goals, we will combine cutting-edge biochemical, cellular and molecular biology, cell-imaging, proteomic and transcriptomic approaches. Elucidating the molecular effects of sumoylation is a hot topic in the intracellular signaling field and ROS as physiological second messengers is still an emerging concept. Studying the antagonistic influences of Sumo and ROS on AP-1 is of a high added value issue as (i) AP-1 is one of the main transcriptional integrators of extracellular cues for cell to adapt to their environment and (ii) the knowledge gained throughout this project may be generic, as Sumo and ROS both target other transcription factors. We feel we are in a strong position to achieve our goal because, not only our project is built on some of our most recent observations, but also because of (i) our long-standing expertise of AP-1 and protein modification by peptidic modifiers and (ii) we can make use of biochemical and immunological tools that are not available to most laboratories. Strengthening the interest of our project, many pathologies are associated with, or caused by, dysfunction of AP-1, Sumo and ROS pathways. Examples include cancer and neurodegenerative-, inflammatory- and immune illnesses. Therefore, they constitute potential targets for new therapies either individually or in combination, due to the connections we have recently demonstrated between them.