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HIC1 (Hypermethylated in Cancer 1), a tumor suppressor gene inactivated through promoter hypermethylation in numerous tumors and leukemia is involved in complex feedback regulatory loops with P53 and SIRT1 and codes for a transcriptional repressor (Wales et al. Nat Med 1995 ; 1:570). Definitive clues to the tumor suppressor function of HIC1 have come from animal models developed in Steve Baylin’s laboratory. Heterozygous Hic1+/- mice develop a late-onset and gender-specific spectrum of spontaneous tumors (Chen et al. Nat Genet 2003; 33:197). In addition, using Hic1 and p53 double heterozygous knockout mice, it has been shown that Hic1 cooperates with p53 in determining cancer progression and spectrum (Chen et al. Cancer Cell 2004; 6:387). Another double heterozygote cross between the medulloblastoma forming Ptch1+/- model and Hic1+/- resulted in an unchanged latency of tumor onset but a fourfold increase in tumor incidence compared to the Ptch1+/- mouse (Briggs et al. Genes Dev 2008; 22:770). These studies also identified the gene coding the pro-neuronal transcription factor ATOH1 which is essential for normal cerebellum development as another HIC1 direct target gene (Briggs et al. Genes Dev 2008; 22:770). Finally, a circular regulatory loop has been proposed for HIC1, SIRT1 and p53 during the apoptotic response to DNA damages. HIC1 directly represses the transcription of SIRT1 through a SIRT1/HIC1 (Chen et al. Cell 2005; 123:437) or a CtBP/HIC1 (Zhang et al. PNAS 2007; 104:829) repressor complex. SIRT1 is a class III NAD+ dependant deacetylase which deacetylates many protein targets, including p53. Thus, SIRT1 negatively modulates P53 DNA-binding properties and hence transactivation of its direct target genes, including HIC1. HIC1 directly regulates SIRT1 transcription to modulate the P53-dependant apoptotic DNA damage response (Chen et al. Cell 2005; 123:437).
For the last fifteen years, we have performed functional studies on HIC1 and defined the HIC1 protein as a sequence specific transcriptional repressor containing three main functional domains: (i) the N-terminal BTB/POZ (which stands for Broad complex, Tramtrack and Bric à brac/Poxviruses and Zinc finger) domain of about 120 amino-acid is a dimerization domain known to play direct or indirect (through conformational effects) roles in protein-protein interactions. This domain is also an autonomous transcriptional repression domain; (ii) the C-terminal end contains five Krüppel-like C2H2 zinc fingers which bind a specific DNA sequence, termed the HiRE (HIC1 responsive element) that we have defined (Pinte et al. 2004 J Biol Chem 2004; 279:38313) and a C-terminal tail that displays no obvious functional domain but has been phylogenetically conserved (Bertrand et al. 2004 BBA 2004; 1678:57); (iii) a central region which is a second autonomous transcriptional repression domain (Deltour et al. Mol Cell Biol 2002; 22:4890) (Stankovic-Valentin et al. FEBS J 2006; 273:2879 - Stankovic-Valentin et al. Mol Cell Biol 2007; 27:2661).
The BTB/POZ domain is a highly conserved and widely distributed structural motif found mainly in transcription factors. BTB/POZ domains are protein-protein interaction domains and mediate homo-oligomerization, hetero-oligomerization as well as interactions with non-BTB/POZ proteins. We have shown that he HIC1 BTB/POZ domain is also an autonomous transcriptional repression domains but is insensitive to trichostatin A (TSA), a specific inhibitor of class I and class II HDACs (Deltour et al. PNAS 1999; 96:14831 – Deltour et al. BBRC 2001; 287:427). It has been shown that HIC1 forms a transcriptional repression complex with the class III HDAC, SIRT1 and that this complex directly binds the SIRT1 promoter to repress its transcription (Chen et al. Cell 2005; 123:437).
We have recently deciphered a potential repression mechanism for this BTB/POZ domain which interacts with the human Polycomb-like proteins (hPCL), PHF1 and hPCL3/PHF19. These hPCL proteins are cofactors of the Polycomb PRC2 repression complex. Indeed, we have shown that the recruitment of EZH2 and the deposition of the repressive H3K27me3 repressive epigenetic mark in human fibroblasts on ATOH1 are dependent of HIC1. Furthermore, we demonstrated that in vivo, ATOH1 repression by HIC1 is associated with Polycomb activity during mouse cerebella development (Boulay et al. J Biol Chem 2012b; 287:10509).
This work has been published in Boulay et al. Biochem J 2011; 434:333 – Boulay et al. J Biol Chem 2012b; 287:10509
Our results thus provided the first example of a human transcription factor able to recruit PRC2 to some target promoters through interaction with hPCL proteins. Interestingly, a flurry of very recent papers demonstrated that hPCL proteins recognition of H3K6me3 promotes the intrusion of PRC2 complexes into active chromatin regions to promote gene silencing through optimal H3K27me3 deposition.
The HIC1 central region is also an autonomous transcriptional repression domain but sensitive to TSA (Deltour et al. Mol Cell Biol 2002; 22:4890). This region has not been subjected to a strong selection pressure except for 4 peptidic motifs perfectly conserved from human to zebrafish (Bertrand et al. BBA 2004; 1678:57). One of them, GLDLSKK, is highly related to the canonical motif PxDLSxK/R found in proteins interacting with the co-repressor CtBP (C-terminal Binding Protein). We have demonstrated that HIC1 interacts with the two related CtBP1 and CtBP2 corepressors through this conserved GLDLSKK motif thus extending the CtBP binding site (Deltour et al. Mol Cell Biol 2002; 22:4890 – Stankovic-Valentin et al. FEBS J 2006; 273:2879). Thus, the HIC1 central region appears to be a second repression domain exhibiting both CtBP-dependent and CtBP-independent repression mechanisms, both of which are sensitive to TSA.
The second conserved motif is an YRWM/VK314xEP motif (Stankovic-Valentin et al. Mol Cell Biol 2007; 27:2661) which contains a SUMOylation consensus site,KxE. SUMOylation is a reversible post-translational modification in which a member of the Small Ubiquitin-like Modifier (SUMO) family of proteins is covalently conjugated to lysine residues in target proteins. Unlike ubiquitination which generally marks proteins for rapid degradation, SUMOylation of nuclear proteins has very diverse effects on transcriptional activity ranging from regulation of DNA-binding activity, subcellular localization and assembly of multiprotein complexes. In the case of HIC1, SUMOylation of K314 does not affect its subnuclear localization and its interaction with CtBP, HDAC4 and SIRT1 but does positively regulate the transcriptional repression potential (Stankovic-Valentin et al. Mol Cell Biol 2007; 27:2661).
Lysine residues can be targeted by several post-translational modifications including SUMOylation, acetylation, ubiquitination or methylation. The KxEP motif in HIC1 with the Proline residue conserved from human to zebrafish is related to the G/SKxxP consensus motif for acetylation by CBP/P300. Indeed, we have shown that HIC1 is acetylated on various Lysine residues including K314. Thus, the KxEP motif is an acetylation/SUMOylation switch motif. The cross-talk between these two competitive post-translational modifications of Lysine residues is orchestrated by a new complex associating two distinct types of deacetylases, HDAC4 and SIRT1. Even though the precise mechanisms are not fully understood, these results identify HIC1 as a new target of the class III deacetylase SIRT1. Our results thus provide the first mechanistic clues to the HIC1/SIRT1 interaction. SIRT1 is not involved in the repression mediated by the isolated BTB/POZ domain but by deacetylating HIC1, SIRT1 favors its SUMOylation and thus the establishment of an optimal transcriptional repression. In addition, they bring new insights into the HIC1, P53 and SIRT1 regulatory loop. This loop relies not only on direct transcriptional effects since P53 activates the HIC1 promoter and HIC1 represses the SIRT1 promoter but also on post-translational effects. Thus, the HIC1 central region is essential for the transcriptional repression potential of HIC1. But, corepressors and complexes interacting with this region, notably those whose recruitment is regulated by the SUMOylation/Acetylation switch have still to be characterized.
This task was achieved during the recent years since we have demonstrated that HIC1 interacts with MTA1, a subunit of the NuRD repression complex and that this interaction is regulated by the two competitive post-translational modifications of HIC1 at K314, promotion by SUMOylation and inhibition by Acetylation
This work has been published in Van Rechem et al. Mol Cell Biol 2010; 30:4045
Previous studies also suggested that HIC1 could be a central actor in the DNA damage response (DDR), a process largely under the control of SUMOylation. Indeed, HIC1 is a direct target of P53 and HIC1 regulates the p53-dependant apoptotic DNA-damage response through the direct transcriptional repression of SIRT1 which deacetylates and inactivates P53 (Chen et al., Cell, 2005, 123:437). Recently, we have shown that down-regulation of endogenous HIC1 by siRNA in normal human fibroblasts treated with etoposide, a DSBs inducer, delays DNA repair as shown by the persistence of H2AX foci and comet assays. Conversely, ectopic expression of wild-type HIC1 but not of non-SUMOylatable mutants reduced the number of H2AX foci supporting a role of HIC1 in the regulation of DNA repair in a SUMO-dependent manner. Indeed, HIC1 SUMOylation is increased by DSBs inducers in an ATM-dependent way since we have shown that the ATM inhibitor Ku-55933 abolishes the SUMOylation increase of HIC1. This increase of HIC1 SUMOylation favors the interaction with endogenous MTA1 in etoposide-treated human fibroblasts and hence the recruitment of NuRD complexes on HIC1 target genes which remain to be identified (Dehennaut et al. J Biol Chem 2013; 288:10254).
Thus, we have demonstrated that HIC1 is involved in the DNA Damage Response and regulates DNA repair in a SUMO-dependent way.
This work has been published in Dehennaut et al. J Biol Chem 2013; 288:10254
The C-terminal end contains a cluster of four conserved C2H2 zinc fingers (ZF 2-5) which binds the sequence 5’-C/GNGC/GGGGCAC/ACC-3’ that we have identified as an optimal HIC1 binding site (HiRE for HIC1-responsive element) and validated by mutational analyses a GGCA core motif bound by Zinc fingers 3 and 4 (Pinte et al. J Biol Chem 2004; 279:38313). The first bona fide HIC1 direct target gene, SIRT1, contains a cluster of two HiRE in the same orientation located at the 5’ end of the promoter (Chen et al. Cell 2005; 123:437).
To identify direct target genes of HIC1, we and other performed microarray gene expression profiling analyses of total RNAs from HIC1-deficient transformed cells (U2OS, MCF7 or PC3 cells) infected by adenoviruses or retroviruses -expressing HIC1 or GFP as control. In our studies in collaboration with Dr Brian ROOD (CNMC, Washington), we have used RNAs extracted during a time course infection of U20S cells (from 8 to 26 Hours), the rationale behind being that the earliest repressed genes are more likely to be the direct targets of HIC1. Indeed, we identified a total of 94 genes whose expression was down-regulated at least 3-fold. Some of these genes are involved in proliferation, apoptosis and/or cell cycle control with no obvious “clustering”.
Following our first studies focused on CXCR7/RDC1, an orphan G protein coupled receptor (GPCR) shown to be a second receptor, in addition to CXCR4, for the chemokine SDF-1 (stromal cell-derived factor 1)/CXCL12 (Van Rechem et al. J Biol Chem 2009; 284:20927), we have now validated through promoter luciferase activity, and chromatin immunoprecipitation (ChIP) and sequential ChIP experiments two other membrane bound receptors as direct HIC1 target genes endogenously in normal WI-38 fibroblasts and upon HIC1 re-expression in breast cancer cells : the Tyrosine kinase receptor EphA2 whose overexpression in cancers correlates with poor prognosis and increased metastatic properties (Foveau et al. J Biol Chem 2012; 287:5366) and the GPCR receptor ADRB2 whose activation by the stress-released hormones adrenaline/noradrenaline stimulates tumor growth and invasion (Boulay et al. J Biol Chem 2012a; 287:5379). These results are in good agreement with our observation that overexpression of HIC1 trough retroviral expression in MDA-MB231 cells inhibits their anchorage-dependent and anchorage –independent growth as well as their migration and invasion properties.
In addition, ephrinA1, the gene coding for the cell-bound ligand of the Tyrosine kinase receptor EphA2 is also a direct target gene of HIC1 in MCF-7 breast cancer cells (Zhang et al., Oncogene 2010; 29:2467). These results are in good agreement with the mutually exclusive Expression of EphA2 and ephrinA1 observed in a panel of 28 breast tumor cell lines including MCF-7 and MDA-MB231. Indeed EphA2 is expressed in cells with mesenchymal characteristics such as MDA-MB231 whereas ephrinA1 is expressed in cells which have retained epithelial markers. An inverse correlation has been recently described in metastatic ductal carcinoma samples whereas normal breast and in situ ductal carcinomas expressed both EphA2 and ephrinA1. In conclusion, HIC1 directly regulates the whole EphA2/ephrinA1 signaling pathways in normal mammary cells. The early genetic alteration of HIC1 coupled with additional genetic or epigenetic mutational events might then yield distinct populations of transformed cells expressing either the tyrosine kinase receptor or its ligand. This could also ultimately contribute to intra-tumoral heterogeneity.
This work has been published in Van Rechem et al. J Biol Chem 2009; 284:20927 – Foveau et al. J Biol Chem 2012; 287:5366 – Boulay et al. J Biol Chem 2012a; 287:5379
In conclusion, during the last five years, we have identified several HIC1 target genes, defined the PRC2 Polycomb complex as a new co-repressor of HIC1 and characterized several post-translational modifications regulating its interaction with the NuRD complex, especially during the DNA damage response.
As for the tumor suppressor gene function of HIC1 in general, our work has revealed two new aspects which are still under investigation:
- The regulation of signaling pathways involved in cell migration and invasion properties.
- The identification of HIC1 as a new player in the DNA Damage Response.