The class IIa HDACs prevent degradation of RBFOX2 by Chaperone-Mediated Autophagy

By modulating the acetylation level of histones, histone deacetylases (HDACs) are enzymes playing a key role in the control of gene expression. In addition to histones, HDACs also have non-histone substrates, which may relate to potential roles for HDACs outside of gene regulation. Because HDACs are key regulators of major cellular processes such as cell division, apoptosis and differentiation, it is not surprising that these enzymes have emerged as attractive therapeutic targets for cancer. While the in vivo antitumoral activities of several small molecules HDACs inhibitors have generated a lot of hope, these molecules often showed dramatic side-effects. It is suspected that these side effects could be related to unknown functions of HDACs. The goal of this project is to identify novel, unsuspected functions of HDACs that will help developing more efficient and specific HDAC-based antitumoral therapies.
To identify novel functions of HDACs, we used a high-throughput yeast two hybrid (Y2H) approach. This led to the first comprehensive interactomic map of class IIa HDACs that includes 84 protein partners. Among new HDACs partners, we identified several RNA-binding proteins (RBPs) involved in mRNA processing. In this work, we focused on one, the alternative splicing regulator RBFOX2, and investigated its regulation by class IIa HDACs. Through various approaches, we verified that HDAC7 interacts with RBFOX2. In addition, we found that silencing of HDAC7 correlates with a decrease in stability of RBFOX2. Because we identified a potential CMA-specific KFERQ motif in RBFOX2, we tested the possibility that RBFOX2 might be degraded through Chaperone-Mediated Autophagy (CMA). Supporting this hypothesis, we found that RBFOX2 interacts with the co-chaperone HSC70. RBFOX2 levels are sensitive to CMA inducers, including serum starvation and 6AN. Interestingly, we found that a lysine residue in the KFERQ motif of RBFOX2 is acetylated, suggesting that HDAC7 might control RBFOX2 degradation through reversible acetylation. Indeed, degradation of RBFOX2 following HDAC7 silencing was reverted with an inhibitor of autophagy, bafilomycin A1. Analysis of RNA splicing pattern in cells depleted for HDAC7 showed that absence of HDAC7 is associated with 159 alternative splicing events. These events mostly include exon skipping that is known to be the major splicing event in which RBFOX2 is involved. In addition, we observed a highly statistically overlap between splicing events associated with RBFOX2 and HDAC7 depletion.