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 C 2 H 2 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 KXE 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 dualdeacetylase complex induces either a phosphorylation-dependent acetylation-SUMOylation switch through a KXEXXSP motif, as previously shown for MEF2, or a phosphorylation-independent switch through a KXEP 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.

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity-based search with the suicide inhibitor haemagglutinin (HA)-SUMOvinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin-specific protease-like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l-an essential but distant zebrafish homologue of USPL1-also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low-abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.Keywords: SUMO protease; ubiquitin-specific protease family; USPL1; zebrafish C13orf22l; Cajal body EMBO reports (2012) 13, 930-938.

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc-chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a de-ubiquinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome, Associated-molecule-with-the-SH3-Domain-of-STAM (AMSH), which regulates ubiquitin-dependent sorting of cell-surface receptors, and Brcc36, a K63-specific deubiquitnase of BRCC36-containing isopeptidase complex (BRISC) and BRCA1-BRCA2-containing complex (BRCC). We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

The tumor suppressor gene HIC1 encodes a transcriptional repressor involved in regulatory loops modulating P53-dependent and E2F1-dependent cell survival, growth control, and stress responses. Despite its importance, few HIC1 corepressors and target genes have been characterized thus far. Using a yeast two-hybrid approach, we identify MTA1, a subunit of the NuRD complex, as a new HIC1 corepressor. This interaction is regulated by two competitive posttranslational modifications of HIC1 at lysine 314, promotion by SUMOylation, and inhibition by acetylation. Consistent with the role of HIC1 in growth control, we demonstrate that HIC1/MTA1 complexes bind on two new target genes, Cyclin D1 and p57KIP2 in quiescent but not in growing WI38 cells. In addition, HIC1/MTA1 and HIC1/CtBP complexes differentially bind on two mutually exclusive HIC1 binding sites (HiRE) on the SIRT1 promoter. SIRT1 transcriptional activation induced by short-term serum starvation coincides with loss of occupancy of the distal sites by HIC1/MTA1 and HIC1/CtBP. Upon longer starvation, both complexes are found but on a newly identified proximal HiRE that is evolutionarily conserved and specifically enriched with repressive histone marks. Our results decipher a mechanistic link between two competitive posttranslational modifications of HIC1 and corepressor recruitment to specific genes, leading to growth control.HIC1 (Hypermethylated in Cancer 1), a tumor suppressor gene frequently deleted or epigenetically silenced in human cancers, encodes a transcriptional repressor (7,20,65). A regulatory feedback loop between HIC1 and P53 has been deciphered. HIC1 is a direct target gene of P53 (5, 24, 65). HIC1 directly represses the transcription of SIRT1, a NAD ϩ -dependent class III histone deacetylase (HDAC) that deacetylates and inactivates P53, thereby modulating P53-dependent DNA damage responses (8). We have shown that SIRT1 also deacetylates HIC1. In contrast to P53, this deacetylation activates HIC1 by strengthening its transcriptional repression potential (59).Aside from P53, SIRT1 and HIC1 have also been implicated in a feedback regulatory loop with E2F1. E2F1 is a crucial activator of SIRT1 transcription in response to DNA damage, but SIRT1 binds and deacetylates E2F1 that inhibits E2F1-mediated gene activation (30, 66). In addition, E2F1 directly activates HIC1 (27) and HIC1 directly represses the E2F1 promoter in quiescent but not in G 1 human fibroblasts, which contributes to the growth suppression induced by serum deprivation (71). Thus, HIC1 is placed at the intersection of complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival, growth control, and stress responses (15).HIC1 encodes a sequence-specific transcriptional repressor with five Krüppel-like C 2 H 2 zinc fingers mediating DNA binding to a HIC1 responsive element (HiRE), (C/G)NG(C/ G)GGGCA(C/A)CC (48). To date, SIRT1, ATOH1 (a proneuronal transcription factor) (4), E2F1, CXCR7 (a receptor for the chemokine CXCL12) (62), and ephrin-A1 (a cell surface liga...

While the semi or total synthesis of ubiquitin or polyubiquitin conjugates have attracted a lot of attention the last decade, the preparation of small-ubiquitin like modifier (SUMO) conjugates is much less developed. We describe hereinafter some important molecular features to consider when preparing SUMO-2/3 conjugates by chemical synthesis using the native chemical ligation and extended methods. In particular, we clarify the role of the conserved cysteine residue on SUMO-2/3 domain stability and properties. Our data reveal that SUMO-2 and 3 proteins behave differently to the CysAla modification with SUMO-2 being less impacted than SUMO-3 likely due to a stabilizing interaction occurring in SUMO-2 between its tail and the SUMO core domain. While the CysAla modification has no effect on the enzyme-catalyzed conjugation, it shows a deleterious effect on the enzyme-catalyzed deconjugation process, especially with the SUMO-3 conjugate. Whereas it is often stated that SUMO-2 and SUMO-3 are structurally and functionally indistinguishable, here we show that these proteins have specific structural and biochemical properties. This information is important to consider when designing and preparing SUMO-2/3 conjugates, and should help making progress in the understanding of the specific role of SUMO-2 and/or SUMO-3 modifications on protein structure and function.

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The covalent attachment of the cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) to hundreds of substrate proteins leads to their rapid degradation by the 26 S proteasome independently of ubiquitylation. Here, we identify another function of FAT10, showing that it interferes with the activation of SUMO1/2/3 in vitro and down-regulates SUMO conjugation and the SUMO-dependent formation of promyelocytic leukemia protein (PML) bodies in cells. Mechanistically, we show that FAT10 directly binds to and impedes the activity of the heterodimeric SUMO E1 activating enzyme AOS1/UBA2 by competing very efficiently with SUMO for activation and thioester formation. Nevertheless, activation of FAT10 by AOS1/UBA2 does not lead to covalent conjugation of FAT10 with substrate proteins which relies on its cognate E1 enzyme UBA6. Hence, we report that one ubiquitin-like modifier (FAT10) inhibits the conjugation and function of another ubiquitin-like modifier (SUMO) by impairing its activation.

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