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Background: Type 2 diabetes (T2D) is an independent risk factor for the development of nonalcoholic steatohepatitis (NASH. However, there is a need for non-invasive tests (NIT) to identify at-risk NASH patients requiring intense lifestyle or clinical intervention. Aim: The aim of this study was to access clinical performance metrics of NIS4™ either alone or in combination with other NITs in patients characterized as nondiabetic, pre-diabetic and T2D and with either at-risk of NASH (NAS≥4 and F≥2) or advanced fibrosis (F≥3). Methods: Patients in the RESOLVE-IT DIAG cohort presented with one or more risk factors for NAFLD and had corresponding NIT data: NIS4™ (miR-34a-5p, α2M, YKL-40, HbA1c), Fibrosis-4 (FIB-4), NAFLD activity score (NFS), and FibroScan™ (FS). NIT data were used to simulate different testing strategies to identify patients with at-risk NASH or advanced fibrosis. Results: The 467 patients were divided into 3 subgroups with 200 (43%) non-T2D, 67 (14%) pre-diabetic and 200 (43%) T2D with a prevalence for at-risk NASH ranging 42-71%, 58-78% and 66-84%, respectively. The corresponding prevalence for advanced fibrosis in the same groups was 18-27%, 34-40%, 42-49%, respectively. Generally, the performance was impacted by variability in sample size and prevalence which mainly affected PPV and NPV. Under these circumstance NIS4™ together with FS or FIB-4 provided a strong overall balanced performance (Acc: 66-82%, Sen: 50-80%, Spe: 58-94%). Conclusion: In this cohort, the sequential use of NIS4™ together with another blood-based test FIB-4 or an imaging-based FibroScan™ test provided an overall more balanced performance for identification of at-risk NASH or advanced fibrosis for nondiabetic and T2D patients. Therefore, NIS4™ has the potential to be a valuable clinical tool for also identifying diabetic patients at higher risk of negative outcomes. Disclosure C. Rosenquist: Employee; Self; GENFIT. Y. Hajji: None. J. Magnanensi: Employee; Self; GENFIT. N. Stankovic-valentin: Employee; Self; GENFIT, Stock/Shareholder; Self; GENFIT. S. Hosmane: Stock/Shareholder; Self; GENFIT, Stock/Shareholder; Spouse/Partner; Vertex Pharmaceuticals Incorporated. A. J. Sanyal: Advisory Panel; Self; Novo Nordisk, Consultant; Self; Akero Therapeutics, Inc., Alnylam Pharmaceuticals, Inc., Amgen Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Bristol-Myers Squibb Company, Genentech, Inc., Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc., Inventiva Pharma, Lilly Diabetes, Lipocine Inc., Merck & Co., Inc., NGM Biopharmaceuticals, Novartis Pharmaceuticals Corporation, Pfizer Inc., Regeneron Genetics Center, Siemens Corporation, Other Relationship; Self; AstraZeneca, UpToDate, Stock/Shareholder; Self; GENFIT, HemoShear Therapeutics, LLC, Indalo Therapeutics, Inc., Tiziana Life Sciences plc.

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., FAT10 is an ubiquitin-like modifier that targets proteins to proteasomal degradation. Here, the authors show that FAT10 also regulates SUMO activation in vitro and in cells, providing evidence for functional crosstalk between two ubiquitin-like modifiers.

Hypoxia occurs in pathological conditions, such as cancer, as a result of the imbalance between oxygen supply and consumption by proliferating cells. HIFs are critical molecular mediators of the physiological response to hypoxia but also regulate multiple steps of carcinogenesis including tumor progression and metastasis. Recent data support that sumoylation, the covalent attachment of the Small Ubiquitin-related MOdifier (SUMO) to proteins, is involved in the activation of the hypoxic response and the ensuing signaling cascade. To gain insights into differences of the SUMO1 and SUMO2/3 proteome of HeLa cells under normoxia and cells grown for 48 h under hypoxic conditions, we employed endogenous SUMO-immunoprecipitation in combination with quantitative mass spectrometry (SILAC). The group of proteins whose abundance was increased both in the total proteome and in the SUMO IPs from hypoxic conditions was enriched in enzymes linked to the hypoxic response. In contrast, proteins whose SUMOylation status changed without concommitant change in abundance were predominantly transcriptions factors or transcription regulators. Particularly interesting was transcription factor TFAP2a (Activating enhancer binding Protein 2 alpha), whose sumoylation decreased upon hypoxia. TFAP2a is known to interact with HIF-1 and we provide evidence that deSUMOylation of TFAP2a enhances the transcriptional activity of HIF-1 under hypoxic conditions. Overall, these results support the notion that SUMO-regulated signaling pathways contribute at many distinct levels to the cellular response to low oxygen.

Molecular switches are essential modules in signaling networks and transcriptional reprogramming. Here, we describe a role for small ubiquitin‐related modifier SUMO as a molecular switch in epidermal growth factor receptor (EGFR) signaling. Using quantitative mass spectrometry, we compare the endogenous SUMO proteomes of HeLa cells before and after EGF stimulation. Thereby, we identify a small group of transcriptional coregulators including IRF2BP1, IRF2BP2, and IRF2BPL as novel players in EGFR signaling. Comparison of cells expressing wild type or SUMOylation‐deficient IRF2BP1 indicates that transient deSUMOylation of IRF2BP proteins is important for appropriate expression of immediate early genes including dual specificity phosphatase 1 (DUSP1, MKP‐1) and the transcription factor ATF3. We find that IRF2BP1 is a repressor, whose transient deSUMOylation on the DUSP1 promoter allows—and whose timely reSUMOylation restricts—DUSP1 transcription. Our work thus provides a paradigm how comparative SUMO proteome analyses serve to reveal novel regulators in signal transduction and transcription., Transcriptional repressors of the IRF2BP family and SUMO play a critical role in EGFR signaling. Transient de‐SUMOylation of IRF2BP proteins in response to EGF contributes to immediate early gene transcription.

Elafibranor and GLP-1 receptor agonists synergize to attenuate hepatic inflammation and fibrosis in a rodent model of obesity, insulin resistance and NASH. Liver steatosis contributes to the development of hepatic insulin resistance and is present in the majority of T2DM patients, who are 50 or older. Therefore, many NAFLD patients with diabetes are currently treated with the antidiabetics, including GLP-1 receptor agonists (GLP-1RA). Elafibranor (ELA), a phase 3 candidate for the treatment of NASH, ameliorates insulin sensitivity and lowers HbA1c in patients with NASH-associated T2DM. The aim of our study was to evaluate the pharmacological benefit of combining GLP-1RA and ELA in the context of NASH-associated insulin resistance. GLP-1RA administration induced body weight loss, reduced insulinemia, HOMA-IR and liver steatosis in obese mice with NASH and insulin resistance, but its action on liver fibrosis was negligible. ELA co-administration allowed for GLP-1RA dose reduction and resolved all NASH features. Food intake was also normalized at reduced GLP-1RA doses. Plasma CRP concentration was reduced by 56% in mice that received the combination treatment. Transcriptomic analyses revealed that the synergism between ELA and GLP-1RA in the liver was limited to the modulation of the inflammatory pathways with no effect on metabolic genes. Since ELA and GLP-1RA also synergize in vitro to reduce TNFa secretion in LPS-activated THP1 macrophages, we postulate that both drugs have a direct action on inflammatory cells in the liver. Further analyses to characterize ELA and GLP-1RA effects on innate and adaptive immune responses will be presented. Conclusion: ELA and GLP-1RA synergize to reduce hepatic inflammation and fibrosis in a model of NASH and obesity. These results suggest a potential therapeutic interest of combining ELA and GLP-1RA for the treatment of T2DM in NASH patients. Disclosure V. Legry: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. E. Descamps: Employee; Self; GENFIT. Employee; Spouse/Partner; GENFIT. Stock/Shareholder; Spouse/Partner; GENFIT. Stock/Shareholder; Self; GENFIT. S. Debaecker: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. N. Stankovic-Valentin: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. P. Parroche: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. N. Degallaix: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. P. Poulain: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT. B. Staels: Consultant; Self; Genfit. R. Walczak: Employee; Self; GENFIT. Stock/Shareholder; Self; GENFIT.

Reversible post-translational modifications (PTMs) ensure rapid signal transmission from sensors to effectors. Reversible modification of proteins by the small proteins Ubiquitin and SUMO are involved in virtually all cellular processes and can modify thousands of proteins. Ubiquitination or SUMOylation is the reversible attachment of these modifiers to lysine residues of a target via isopeptide bond formation. These modifications require ATP and an enzymatic cascade composed of three classes of proteins: E1 activating enzymes, E2 conjugating enzymes and E3 ligases. The reversibility of the modification is ensured by specific isopeptidases. E1 and E2 enzymes, some E3 ligases and most isopeptidases have catalytic cysteine residues, which make them potentially susceptible for oxidation. Indeed, an increasing number of examples reveal regulation of ubiquitination and SUMOylation by reactive oxygen species, both in the context of redox signaling and in severe oxidative stress. Importantly, ubiquitination and SUMOylation play essential roles in the regulation of ROS homeostasis, participating in the control of ROS production and clearance. In this review, we will discuss the interplay between ROS homeostasis, Ubiquitin and SUMO pathways and the implications for the oxidative stress response and cell signaling.

SummaryMolecular switches are essential modules in signaling networks and transcriptional reprogramming. Here, we describe a role for small ubiquitin-related-modifier SUMO as a molecular switch in epidermal growth factor receptor (EGFR) signaling. Using quantitative mass spectrometry, we compared the endogenous SUMO-proteomes of Hela cells before and after EGF-stimulation. Thereby, we identified a small group of transcriptional co-regulators including IRF2BP1, IRF2BP2 and IRF2BPL as novel players in EGFR signaling. Comparison of cells expressing wildtype or SUMOylation deficient IRF2BP1 indicated that transient deSUMOylation of IRF2BP1 is important for appropriate expression of immediate early genes including Dual specificity phosphatase 1 (DUSP1, MKP-1), an important feedback regulator of EGFR signaling. We find that IRF2BP1 is a SUMO-dependent repressor, whose transient deSUMOylation on the DUSP1 promotor allows - and whose timely reSUMOylation restricts - DUSP1 expression. Our work thus provides a paradigm how comparative SUMO proteome analyses serve to reveal novel regulators in signal transduction and transcription.

International audience; While the semi or total synthesis of ubiquitin or polyubiquitin conjugates has attracted a lot of attention the past 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 from 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 in making progress in the understanding of the specific role of SUMO-2 and/or SUMO-3 modifications on protein structure and function.

Pyrophosphate (PPi), a byproduct of macromolecule biosynthesis is maintained at low levels by soluble inorganic pyrophosphatases (sPPase) found in all eukaryotes. In plants, H+-pumping pyrophosphatases (H+-PPase) convert the substantial energy present in PPi into an electrochemical gradient. We show here, that both cold- and heat stress sensitivity of fugu5 mutants lacking the major H+-PPase isoform AVP1 is caused by reduced SUMOylation. In addition, we show that increased PPi concentrations interfere with SUMOylation in yeast and we provide evidence that SUMO activating E1-enzymes are inhibited by micromolar concentrations of PPi in a non-competitive manner. Taken together, our results do not only provide a mechanistic explanation for the beneficial effects of AVP1 overexpression in plants but they also highlight PPi as an important integrator of metabolism and stress tolerance in eukaryotes.

To sense and defend against oxidative stress, cells depend on signal transduction cascades involving redox‐sensitive proteins. We previously identified SUMO (small ubiquitin‐related modifier) enzymes as downstream effectors of reactive oxygen species (ROS). Hydrogen peroxide transiently inactivates SUMO E1 and E2 enzymes by inducing a disulfide bond between their catalytic cysteines. How important their oxidation is in light of many other redox‐regulated proteins has however been unclear. To selectively disrupt this redox switch, we identified a catalytically fully active SUMO E2 enzyme variant (Ubc9 D100A) with strongly reduced propensity to maintain a disulfide with the E1 enzyme in vitro and in cells. Replacement of Ubc9 by this variant impairs cell survival both under acute and mild chronic oxidative stresses. Intriguingly, Ubc9 D100A cells fail to maintain activity of the ATM–Chk2 DNA damage response pathway that is induced by hydrogen peroxide. In line with this, these cells are also more sensitive to the ROS‐producing chemotherapeutic drugs etoposide/Vp16 and Ara‐C. These findings reveal that SUMO E1~E2 oxidation is an essential redox switch in oxidative stress. ![][1] E1 and E2 enzymes in the SUMO conjugation pathway become reversibly disulfide‐linked between their active site cysteines upon oxidative stress. A Ubc9 mutant selectively deficient for this mechanism allows to study the cellular roles of redox‐dependent global SUMOylation shutdown. The EMBO Journal (2016) 35: 1312–1329 [1]: /embed/graphic-1.gif

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.

HIC1 (hypermethylated in cancer) is a tumour suppressor gene located in 17p13.3, a region frequently hypermethylated or deleted in many types of prevalent human tumour. HIC1 is also a candidate for a contiguous-gene syndrome, the Miller-Dieker syndrome, a severe form of lissencephaly accompanied by developmental anomalies. HIC1 encodes a BTB/POZ-zinc finger transcriptional repressor. HIC1 represses transcription via two autonomous repression domains, an N-terminal BTB/POZ and a central region, by trichostatin A-insensitive and trichostatin A-sensitive mechanisms, respectively. The HIC1 central region recruits the corepressor CtBP (C-terminal binding protein) through a conserved GLDLSKK motif, a variant of the consensus C-terminal binding protein interaction domain PxDLSxK/R. Here, we show that HIC1 interacts with both CtBP1 and CtBP2 and that this interaction is stimulated by agents increasing NADH levels. Furthermore, point mutation of two CtBP2 residues forming part of the structure of the recognition cleft for a PxDLS motif also ablates the interaction with a GxDLS motif. Conversely, in perfect agreement with the structural data and the universal conservation of this residue in all C-terminal binding protein-interacting motifs, mutation of the central leucine residue (leucine 225 in HIC1) abolishes the interaction between HIC1 and CtBP1 or CtBP2. As expected from the corepressor activity of CtBP, this mutation also impairs the HIC1-mediated transcriptional repression. These results thus demonstrate a strong conservation in the binding of C-terminal binding protein-interacting domains despite great variability in their amino acid sequences. Finally, this L225A point mutation could also provide useful knock-in animal models to study the role of the HIC1-CtBP interaction in tumorigenesis and in development.

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located at chromosome 17p13.3, a region frequently hypermethylated or deleted in human tumors and in a contiguous-gene syndrome, the Miller-Dieker syndrome. HIC1 is a transcriptional repressor containing five Kruppel-like C(2)H(2) zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Although some of the HIC1 transcriptional repression mechanisms have been recently deciphered, target genes are still to be discovered. In this study, we determined the consensus binding sequence for HIC1 and investigated its DNA binding properties. Using a selection and amplification of binding sites technique, we identified the sequence 5'-(C)/(G)NG(C)/(G)GGGCA(C)/(A) CC-3' as an optimal binding site. In silico and functional analyses fully validated this consensus and highlighted a GGCA core motif bound by zinc fingers 3 and 4. The BTB/POZ domain inhibits the binding of HIC1 to a single site but mediates cooperative binding to a probe containing five concatemerized binding sites, a property shared by other BTB/POZ proteins. Finally, full-length HIC1 proteins transiently expressed in RK13 cells and more importantly, endogenous HIC1 proteins from the DAOY medulloblastoma cell line, repress the transcription of a reporter gene through their direct binding to these sites, as confirmed by chromatin immunoprecipitation experiments. The definition of the HIC1-specific DNA binding sequence as well as the requirement for multiple sites for optimal binding of the full-length protein are mandatory prerequisites for the identification and analyses of bona fide HIC1 target genes.

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)-SUMO-vinylmethylester 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.

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.

To measure rates of sumoylation and isopeptidase cleavage in vitro, we developed an enzyme assay that is based on fluorescence resonance energy transfer (FRET). FRET is a process by which the excited state energy of a fluorescent donor molecule is transferred to an acceptor molecule. Efficient energy transfer requires very close proximity, and can therefore be used as a read-out for covalent and non-covalent protein interactions. The assay described here uses bacterially expressed and purified YFP-SUMO-1 and CFP-RanGAP1 as model substrates that are covalently coupled in the presence of recombinant SUMO E1 and E2 enzymes and ATP. Reactions of 25 microl volume, set up in 384-wells plates, give sufficient signal for analysis. Consequently, this assay requires very low amounts of recombinant proteins and allows measurement of time courses in high-throughput format.

To measure rates of sumoylation and isopeptidase cleavage in vitro, we developed an enzyme assay that is based on fluorescence resonance energy transfer (FRET). FRET is a process by which the excited state energy of a fluorescent donor molecule is transferred to an acceptor molecule. Efficient energy transfer requires very close proximity, and can therefore be used as a read-out for covalent and non-covalent protein interactions. The assay described here uses bacterially expressed and purified YFP-SUMO-1 and CFP-RanGAP1 as model substrates that are covalently coupled in the presence of recombinant SUMO E1 and E2 enzymes and ATP. Reactions of 25 microl volume, set up in 384-wells plates, give sufficient signal for analysis. Consequently, this assay requires very low amounts of recombinant proteins and allows measurement of time courses in high-throughput format.

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 psiKXE 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 psiKXEXXSP motif, as previously shown for MEF2, or a phosphorylation-independent switch through a psiKXEP 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.

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located at chromosome 17p13.3, a region frequently hypermethylated or deleted in human tumors and in a contiguous-gene syndrome, the Miller-Dieker syndrome. HIC1 is a transcriptional repressor containing five Krüppel-like C(2)H(2) zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Although some of the HIC1 transcriptional repression mechanisms have been recently deciphered, target genes are still to be discovered. In this study, we determined the consensus binding sequence for HIC1 and investigated its DNA binding properties. Using a selection and amplification of binding sites technique, we identified the sequence 5'-(C)/(G)NG(C)/(G)GGGCA(C)/(A) CC-3' as an optimal binding site. In silico and functional analyses fully validated this consensus and highlighted a GGCA core motif bound by zinc fingers 3 and 4. The BTB/POZ domain inhibits the binding of HIC1 to a single site but mediates cooperative binding to a probe containing five concatemerized binding sites, a property shared by other BTB/POZ proteins. Finally, full-length HIC1 proteins transiently expressed in RK13 cells and more importantly, endogenous HIC1 proteins from the DAOY medulloblastoma cell line, repress the transcription of a reporter gene through their direct binding to these sites, as confirmed by chromatin immunoprecipitation experiments. The definition of the HIC1-specific DNA binding sequence as well as the requirement for multiple sites for optimal binding of the full-length protein are mandatory prerequisites for the identification and analyses of bona fide HIC1 target genes.

Hypermethylated in Cancer 1 (HIC1) is a human tumor suppressor gene located at chromosome 17p13.3 which is frequently hypermethylated and transcriptionally silent in many types of tumors. In addition, its location in the Miller-Dieker syndrome's (MDS) deletion region, its embryonic expression pattern in mice and the phenotype of the HIC1-deficient mice have provided strong evidence for its implication in this contiguous-gene syndrome. HIC1 encodes a five C2H2-type zinc finger transcriptional repressor belonging to the BTB/POZ family. We have isolated the true zebrafish orthologue of human HIC1 since it has a comparable intron-exon structure and since its predicted gene product, ZfHIC1 displays much higher sequence similarities in its overall sequence (737 residues) with human HIC1 (714 residues) than the 454 residues encoded by the only zebrafish HIC1 sequence (AF111712) described so far, which has been renamed ZfHIC1alpha. Notably, the C-terminal end and one zinc finger in the DNA-binding domain are missing in ZfHIC1alpha. As a consequence, ZfHIC1 proteins bind the human HIC1 consensus DNA-binding sequence in vitro, whereas ZfHIC1alpha cannot. Analyses of the expression pattern of ZfHIC1 and of its paralogue ZfHRG22 (HIC1 related gene on chromosome 22) show that they share expression domains with their respective orthologous vertebrate genes. ZfHRG22 is prominently expressed in the brain and in neural tissues. Interestingly, the predominant expression of ZfHIC1 in the mesenchyme of the head, around the nose and the eye and in the branchial arches is possibly consistent with some of the abnormalities seen in the HIC1-deficient mice and provides another clue for the implication of HIC1 in MDS.