An Acetylation/Deacetylation-SUMOylation Switch through a Phylogenetically Conserved ψKXEP Motif in the Tumor Suppressor HIC1 Regulates Transcriptional Repression Activity

Abstract

Tumor suppressor HIC1 (hypermethylated in cancer 1) is a gene that is essential for mammalian development, epigenetically silenced in many human tumors, and involved in a complex pathway regulating P53 tumor suppression activity. HIC1 encodes a sequence-specific transcriptional repressor containing five Krüppel-like C₂H₂ zinc fingers and an N-terminal BTB/POZ repression domain. Here, we show that endogenous HIC1 is SUMOylated in vivo on a phylogenetically conserved lysine, K314, located in the central region which is a second repression domain. K314R mutation does not influence HIC1 subnuclear localization but significantly reduces its transcriptional repression potential, as does the mutation of the other conserved residue in the ψKXE consensus, E316A, or the overexpression of the deSUMOylase SSP3/SENP2. Furthermore, HIC1 is acetylated in vitro by P300/CBP. Strikingly, the K314R mutant is less acetylated than wild-type HIC1, suggesting that this lysine is a target for both SUMOylation and acetylation. We further show that HIC1 transcriptional repression activity is positively controlled by two types of deacetylases, SIRT1 and HDAC4, which increase the deacetylation and SUMOylation, respectively, of K314. Knockdown of endogenous SIRT1 by the transfection of short interfering RNA causes a significant loss of HIC1 SUMOylation. Thus, this dual-deacetylase complex induces either a phosphorylation-dependent acetylation-SUMOylation switch through a ψKXEXXSP motif, as previously shown for MEF2, or a phosphorylation-independent switch through a ψKXEP motif, as shown here for HIC1, since P317A mutation severely impairs HIC1 acetylation. Finally, our results demonstrate that HIC1 is a target of the class III deacetylase SIRT1 and identify a new posttranslational modification step in the P53-HIC1-SIRT1 regulatory loop.

We thank M. Crossley, R. Hay, T. Kouzarides, S. H. Lin and D. Wotton for their generous gifts of reagents. We are indebted to Bénédicte Grasland and Aurélie Bauduin for the construction and the initial analyses of the Gal4-HIC1 and HIC1-Gal4 chimeras. We thank Brian Rood and Alexis Verger for comments and critical reading of the manuscript and Gil Privé for helpful discussions on the BTB/POZ structure.

This work was supported by funds from the CNRS, the Pasteur Institute, the Ligue Nationale contre le Cancer (Comité Interrégional du Septentrion), the EEC “Rubicon,” and the Association pour la Recherche contre le Cancer (to J.S. and D.L.). N. Stankovic-Valentin was supported by a fellowship from the Ministère de la Recherche et de la Technologie and by the Association pour la Recherche contre le Cancer.