Institut Curie / CNRS UMR 3244 / UPMC Dynamics of Genetic Information: Fundamental Basis and Cancer

Institut Curie / CNRS UMR 3244 / UPMC Dynamics of Genetic Information: Fundamental Basis and Cancer Unit director: Michelle Debatisse, PhD Teams in this unit aim at elucidating the regulation of mechanisms crucial to the maintenance of genome integrity, like DNA replication, repair and recombination, and cell cycle checkpoints. Experimental models include yeast, mammalian and human cell lines, mutant mouse models and tumour samples. A large variety of methods is being used such as genetic, molecular and cytogenetic techniques, fluorescence in situ hybridisation and DNA combing', DNA microarrays for transcriptome analysis and ChIP-chip mapping of chromatinassociated proteins at sites of DNA lesions.

Michelle Debatisse, PhD Functional organization and plasticity of mammalian genomes Tel : +33 1 56 24 66 72 Fax : +33 1 56 24 66 74 Email : michelle.debatisse@curie.fr DNA replication, genome instability, replication dynamics, dynamic molecular combing, checkpoint, fragile sites Our team is interested in the process of cellular DNA replication and how it relates to genome instability. Our work aims at understanding how, during DNA replication, the density of initiation events is regulated in mammalian cells, at determining which proteins of the complexes recruited on initiation sites respond to these regulatory processes, at defining the role of the intra-s checkpoint in this control and at analyzing the replication dynamics at common fragile sites. performing experiments to analyze further the underlying regulatory processes. Figure 1 : Fibers with labeled newly synthesized DNA, IdU (blue) and CldU (green) and hybridized with probes specific to the AMPD2 region (red) identify a specific initiation site. Figure 2: Visualization of chromatin loops (grey halo) surounding the nuclear matrix (black) by the nuclear halo technique. The cells have been grown in conditions leading to high (left) or low (right) replication speed. The position and frequency of initiation regions and the identification of pausing sites along the chromosomes of mammalian cells is still poorly understood, mostly because the study of mammalian replication dynamics relies on rather difficult techniques. Recently, an efficient method for the analysis of replication initiation was set up, which combines the dynamic molecular combing technology to pulse labeling of the cells by two thymidine analogues, IdU and CldU. This makes it possible to visualize neo-synthesized DNA on individual DNA molecules upon specific immunodetection of each analogue. Double labeling permits the determination of the direction and the rate of fork progression, and unambiguous identification of initiation versus termination events. An additional step of FISH (fluorescent in situ hybridization) allows analysis of specific loci (Fig. 1). We have applied this technique to the study of the AMP deaminase 2 (AMPD2) domain in Chinese hamster cells. We have demonstrated that the efficiency and spacing of initiation sites are flexible and that inter-origin distances directly rely on fork speed. Indeed, variations in replication speed ranging from 0.4 to 1.5 kb/min are almost instantaneously compensated by modifications of the number of active origins, so that S-phase duration remains constant. In all conditions, initiation events appear confined to short ATrich sequences previously identified as matrix attachment regions (MAR), but a functional relationship between matrix anchorage and origin selection was missing. We have recently shown that the most efficient origin of the AMPD2 domain displays the strongest matrix affinity as compared to other potential initiation sites (Fig. 2). Altogether, our data suggest the existence of two new levels of origin programming. First, replication speed determines the spacing of initiation events. Second, origin hierarchy reflects their relative affinity for the nuclear matrix. We are now Courbet S et al. (2008) Replication fork movement sets chromatin loop size and origin choice in mammalian cells. Nature, 455, 557-560 Quignon F et al. (2007) Sustained mitotic block elicits DNA breaks: one-step alteration of ploidy and chromosome integrity in mammalian cells. Oncogene, 26, 165-172 Haber JE and Debatisse M (2006) Gene amplification: yeast takes a turn. Cell, 125, 1237-1240 Debatisse M et al. (2006) Common fragile sites nested at the interfaces of early and late-replicating chromosome bands: cis acting components of the G2/M checkpoint? Cell Cycle, 5, 578-581 El Achkar E et al. (2005) Premature condensation induces breaks at the interface of early and late replicating chromosome bands bearing common fragile sites. Proc. Natl. Acad. Sci. U. S. A., 102, 18069-18074

Arturo Londoño-Vallejo, MD-PhD "Telomeres and cancer" Institut Curie / CNRS UMR 3244/ UPMC Tel : +33 1 56 24 66 11 Fax : +33 1 56 24 66 17 Email : Arturo.Londono@curie.fr Telomeres, replication, telomerase RNP biogenesis, genome instability, microrna, chromatin, epigenetics, cancer, anti-cancer drugs Tumorigenesis is a multistep process in which chromosomal instability seem to play an important role. We have shown that telomere instability due to excessive shortening is the main driving force of chromosome aberrations during the early stages of cell transformation. However, telomere instability appears to be insufficient to convert a pre-malignant cell into a malignant one, but it is likely that this instability contributes to more subtle aspects of tumor development. We have recently explored the impact of such instability on microrna and global gene expression. Our results suggest that cells traversing a period of telomere instability develop adaptive responses impinging on their capacity to survive, to become immortal and to form tumors. We are now studying the role of epigenetic mechanisms on these adaptive responses as well as on the tumorigenic potential of transformed cells. Understanding how pre-malignant cells become malignant will help to identify potential targets for drug development. We also study the effects of anti-cancer drugs on telomere metabolism. Because telomeres are essential for a cell to divide, and despite of the many basic issues that still remain to be addressed, telomeres have become a remarkable target for the development of anticancer drugs. Indeed, several compounds, including important chemotherapeutic agents currently used in clinics, have been shown to induce telomere shortening and senescence in many types of cancer cells in vitro but the specific effects on telomere replication remain largely unexplored. We therefore aim at evaluating the effects of such drugs (in particular, telomestatin, cisplatin, camptothecin and etoposide) on telomere replication, in the presence of normal and altered levels of telomeric proteins. This information is likely to help in the definition of more rational therapeutic strategies. Finally, we study the role of factors directly impinging on telomere length homeostasis, both through telomerase dependent and independent mechanisms. These factors are potentially important in defining stem cell capability, in fertility and in the development of aging related pathologies. Figure 1 : Quantitative CO-FISH analyses of tumor cells treated sith cis-platin during a single S-phase. The intensities of the fluorescent signals on each of the parental telomeric strands are measured and their distribution is represented in the histograms. Cis-platin induces massive losses on both strands at certain telomers (arrows), which also bear the signs of postreplicative recombination (T-SCE). This results in a bimodal distribution of telomere lengths. Arnoult N et al (2010) Replication timing of human telomeres is chromosome-arm specific, influenced by subtelomeric structures and connected to nuclear localization. PLoS Genet. 2010 Apr 22;6(4):e1000920. Arnoult N et al (2009) Human Pot1 is required for efficient replication of the telomeric C-strand in the absence of WRN. Genes and Dev. Dec 15, 23 (24) Draskovic I et al (2009) Probing PML body function reveals spatiotemporal requirements for telomere recombination. 2009. PNAS 106(37):15726-31. Epub 2009 Aug 26. Marie-Egyptienne D et al (2009). Cell cycle defects in mouse telomerase RNA-deficient cells expressing a template-mutated mouse telomerase RNA. Cancer letters 275(2):266-76. Epub 2008 Dec 3. Arnoult N et al (2008) Studying telomere replication by Q-CO FISH: the effect of telomestatin, a potent G-quadrupex ligand. Cytogenet Genome Res 122:229-36. Epub 2009 Jan 30.

Bernard Malfoy,PhD Chromosome Alterations and Cancer Tel : +33 1 56 24 66 85 Fax : +33 1 56 24 66 74 Email : bernard.malfoy@curie.fr Chromosome stability, radiation-induced tumours, sarcoma, glioma, amplification, Our research is focused on two different biological questions Looking for a molecular signature of the ionising radiation, in radiation-induced tumors. We are interested in identifying specific alterations that could discriminate between radiation-induced and sporadic tumors. To ascertain the causal relationship, we analyze radiation-induced sarcomas (RIS) that develop in the field of irradiation in patients treated by radiotherapy for a primary cancer. This occurs at 0,03 to 0,1% of the treated cases. We observe in these RIS a high rate of small deletions in TP53 gene, as compared with mutations in sporadic tumors. Patients carrying a germ-line mutation in the retinoblastoma gene (RB1) are predisposed not only to retinoblastoma (a tumour of the retina) but also to radiation-induced sarcomas, suggesting that RB1 inactivation may be a pre-requisite for the development of RIS. RB1 is not a target of ionising radiation, since we observe no somatic mutation in these tumors. RB1 is, nevertheless, inactivated by a mechanism involving genetic instability. In retinoblastoma, TP53 is rarely inactivated by mutation; the suppression of its function is due to activation of regulatory pathways subsequent to the loss RB1. We have shown that the same tumorigenesis pathways are used in retinal and mesenchymal cells, but in RIS, inactivation of TP53 is a consequence of irradiation, and not of the RB1 loss. We now intend to determine a gene expression signature of ionising radiations, using of global transcription and genome analyses. Elucidating extra-chromosomal amplification mechanisms in gliomas. Our aim is to elucidate the mechanism, by which oncogenes are amplified in the tumoral cells. Amplified DNA fragments can appear as intrachromosomic structures (homogeneously staining regions) or form small circular autonomously replicating extrachromosomes called dmin, for «double minute» (Figure 1). We first investigated the molecular structure of dmins containing the epidermal growth factor receptor (EGFR) gene in a series of gliomas. The founding extrachromo somal DNA molecule was generated by a simple event that circularizes a chromosome fragment overlapping EGFR. The corresponding chromosomal loci were not rearranged, which strongly suggests that a post-replicative event was responsible for the formation of each of these initial amplicons. At contrast, in a glioma containing four extrachromosomally amplified loci, complex extrachromosomal DNA molecules were formed by the fusion of several syntenic or non-syntenic DNA fragments. Fragments ranged from a few base pairs to megabase pairs. Scars of the amplification process remained at the original locus in the form of deletions or chromosome rearrangements. Chromosome fragmentation, due to replication stress, could explain this complex situation. In contrast, at 1q32.1, the initial extrachromosomal DNA molecule resulted from the circularization of a single fragment associated with an intrachromosomal deletion including, but larger than, the amplified sequence. The nature of the sequences involved in these rearrangements suggests that a V(D)J-like illegitimate recombination contributes to its formation Figure 1 : The formation of dmins Cohybridisation of chromosomes with a probe containing the EGFR gene (red) and a probe painting chromosome 7 in green (pale blue aspect) shows that three copies of chromosome 7 are present, as usual recurrently found in glioma cells. Each one contains the EGFR locus at its expected location (arrowheads), and numerous extra-chromosomal dmins, containing the EGFR gene, can also be seen (arrows). Gibaud A et al. (2010) Extrachromosomal amplification mechanisms in a glioma with amplified sequences from multiple chromosome loci. Hum Mol Genet 19(7):1276-85. Gonin-Laurent N. et al. (2007) RB1 and TP53 pathways in radiation-induced sarcomas. Oncogene. 26, 6106-6112. Gonin-Laurent N. et al.(2006) Specific TP53 mutation pattern in radiation-induced sarcomas.carcinogenesis. 27, 1266-1272 Chevillard S. et al. (2006) Deregulated pathways in a human lymphoblastoid cell line after low doses gamma irradiation. Cancer Genomics & Proteomics. 3, 295-310. Vogt, N. et al. (2004). Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas. Proc. Natl. Acad. Sci. U S A. 101, 11368-11373.

Antonin Morillon, PhD Non coding RNA, epigenetic and genome fluidity Institut Curie/CNRS UMR 3244 / UPMC Tel : +33 1 56 24 65 72 Fax : +33 1 56 24 66 74 Email : antonin.morillon@curie.fr epigenetic, ncrna, chromatin, yeast, genome fluidity Our research interest is focused on regulatory non coding (nc)rnas. In high eukaryotes, regulatory ncrnas have been shown to regulate gene expression, chromatin domains and genome stability. There are a growing number of evidence suggesting that they play central roles on cancer formation and cellular differentiation. Regulatory ncrna can be classified in two categories depending on their size. Short interfering (si)rnas, also known to be part of the RNA interference pathway, have been extensively studied and control gene expression and chromosome segregation. Large ncrnas participate also in gene silencing and are key players in cell differentiation and development but, in contrast to sirnas, their mode(s) of action remain poorly characterized. Our lab was one of the first to describe large ncrnamediated epigenetic regulation in the budding yeast that control transposon proliferation and gene expression, providing powerful genetic and large scale tools to uncover their regulatory mechanisms in this classic model organism. Since the beginning of our research project in 2005, we obtained two main results showing the existence of a transacting ncrna controlling the Ty1 transposon in yeast (Figure 1). In addition, we provided evidence that cryptic transcription mediates the deposition of histone marks controlling inducible genes (Figure 2) Our work shows that yeast is indeed an excellent organism to study regulatory ncrna that are involved in chromatin regulation. Interestingly, the processing and the mode of action of the yeast large ncrna implicate pathways important for genome integrity and cell development in mammalian cells, suggesting that their mechanisms might be conserved among the eukaryotic kingdom. Our future aims are to extensively identify all the regulatory ncrna in yeast and to further characterize their associated proteins to understand the mechanisms controlling histone modifications both at the euchromatin and heterochromatin domains. Our ongoing work will set up the fundamental basis for future studies in higher eukaryotes, especially during differentiation and cancer. Berretta J et al. (2009) A cryptic unstable transcript mediates transcriptional trans-silencing of the Ty1 retrotransposon in S.cerevisiae. Genes and Dev, 22:615-626 Pinskaya M et al. (2009) Nucleosome remodeling and transcriptional repression are distinct functions of Isw1 in Saccharomyces cerevisiae. Mol Cell Biol, 29:2419-2430 Pinskaya M et al. (2009) H3K4 di and tri-methylation deposited by cryptic transcription attenuate promoter activity. EMBO J, 28:1697-1707 Berretta J et al. (2009) Pervasive transcription a novel regulatory level in eukaryotes? EMBO Rep, 10:973-982 Pinskaya M et al. (2009) H3K4 di methylation: a novel mark for transcriptional fidelity? Epigenetics, 4:302-306

Alain Nicolas,PhD Recombination and Genetic Instability 75248 Paris Cedex 05 Tel : +33 1 56 24 65 20 Fax : +33 1 56 24 66 44 Email : alain.nicolas@curie.fr Genome integrity, Replication, Repair, Recombination, G-Quadruplex, Meiosis, Double- Strand Breaks (DSBs) Our group aims at identifying and characterizing the biological processes that maintain genomic integrity and ensure the faithful transmission of genetic information during reproduction, as well as endogenous and exogenous events that enhance genome instability. We are focusing our work on two biological situations in which DNA double-strand breaks (DSBs) form in the budding yeast Saccharomyces cerevisiae. First, we study the exchange of genetic material by recombinationwhich occurs between sister chromosomes during yeast meiosis. Using chromatin immunoprecipitation and microarrays(chip chip), we were able to show that each yeast chromosome has a unique map of meiotic DSBs with alternating 'hot' and 'cold' domains where recombination occurs more or less frequently, and correlate with the binding sites of the Mre11 protein, as well as enriched region of histone H3-K4 trimethylation, independently of transcriptional levels (Figure 1). variety of length variants due to expansion and contraction of the repeat units. We have also found that the mitotic stability of CEB1 depends on the activity of the Pif1 helicase. In vitro and in vivo analyses showed that CEB1 repeats formed stable G- quadruplex (G4) secondary structures and that the Pif1 protein unwinds these structures efficiently. This was further confirmed by using the PhenDCs G4-quadruplexes ligands developed by the group of M-P. Teulade-Fichou (Institut Curie, Orsay); These molecules specifically destabilized CEB1 in wild-type treated cells and yielded CEB1 rearrangements similar to that in pif1 cells. The steps leading to CEB1 rearrangements are illustrated in Figure 2. Figure 2: Genetic control and mechanisms of CEB1 rearrangements Figure 1: Genome-wide 'ChIP-chip' map of the DNA double-strand breaks induced by Spo11 in the wild type (SET1) and histone H3- K4 methyl transferase mutant (set1 ) strains. Also, we have developed a Gal4-Spo11 fusion protein, which allows us to modify the usual DNA cleavage sites along the chromosomes and envisage to extent our studies of meiotic homologous recombination to other species and screen mutations in candidate genes for human infertility syndromes. Second, we study the instability of human tandem repeated DNA sequences (minisatellites) inserted in the yeast genome In the S. cerevisiae genome, as in the human genome, tandem repeated minisatellite DNA sequences are unstable during meiosis when they may undergo expansion and/or contraction of the number of tandem repeats. To investigate the mechanism(s) underlying tandem-repeat instability, we introduced two human minisatellite CEB1 alleles into the S. cerevisiae genome. We found that deletion of the RAD27 gene, which is involved in DNA replication and repair, causes a high level of instability of the CEM1-1.8 allele in cells growing vegetatively, indicating that replication defects destabilise these repeated sequences. It gives rise to a large Piazza et al., (2010). Genetic instability triggered by G- quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae. Nucleic Acids Research. doi :10.1093/nar/gkq136. Székvölgyi & Nicolas A. (2010) From meiosis to postmeiotic events : Meiotic recombination is obligatory but flexible. FEBS Journal, 277, 571-589. Borde V. et al., (2009). Histone H3 Lysine 4 trimethylation marks meiotic recombination initiation sites. EMBO J., 28, 99-111. Ribeyre, C. et al., (2009) The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming sequences in vivo. PloS Genetics 5, e1000475 Murakami H. & Nicolas A. (2009). Locally, meiotic doublestrand breaks targeted by Gal4BD-Spo11 occur at discrete sites with sequence preference. Mol. Cell. Biol., 29, 3500-3516. Patent available for licensing : 1

Franck Toledo, PhD Genetics of tumor suppression 75248 Paris CEDEX 05 Tel : +33 1 56 24 66 71 Fax : +33 1 42 34 66 74 Email : Franck.Toledo@curie.fr Mouse models, preclinical mouse models, p53 regulation, MDM2, p53 activity, MDM4, anticancer strategies The transcription factor p53 is altered in most tumours. In more than half of human cancers, the p53 gene is mutated and, in the other half, the p53 protein is frequently inactivated by, for example, overexpression of its specific inhibitors MDM2 and MDM4. A better understanding of the pathways that regulate p53 could lead to development of new and broadly applicable anti-cancer strategies. Our group is using mouse models to gain a better understanding of the regulation of p53. Much of what we know about the regulation of p53 results from biochemical studies and analyses relying on transfection of expression plasmids into cells in culture. In recent years, studies of several mouse models carrying targeted p53 mutations have found significant differences between the data in vivo and those obtained by earlier in vitro approaches. For example, we found that mutation of threonine and proline residues in p53's proline rich domain (PRD), which were thought to be essential for regulation of the protein, did not significantly affect the transactivation or tumor suppressor function of p53 in the mouse - a finding that may explain the sequence variability of the PRD in evolution. We also generated the mutant mouse p53δp, which expresses a p53 that lacks the proline-rich domain, and has provided tremendous insight into p53 regulation. Studies of this mutant showed that MDM2 and MDM4 have distinct and complementary roles in p53 regulation: MDM2 mainly regulates p53 stability, whereas MDM4 regulates its activity (Fig. 1). Our group is now generating new p53 mutant mice to pursue the analysis of this protein's regulation. These studies will be facilitated by the use of a new targeting strategy we recently designed, called RMCE-ASAP (recombinase-mediated cassette exchange adapted for targeting in somatic cells to accelerate phenotyping, Fig.2). Figure 2: Rationale for a RMCE-ASAP. The targeting of specific mutations (green or orange boxes) relies on the combined use of heterologous inverted LoxP sites (blue and purple arrowheads) and a positive/negative selection cassette (red box). Cre/Lox recombination ensures highly efficient targeting, and the screening of recombinants clones relies on the selection cassette. Targeting of mutations can be performed in embryonic stem (ES) cells to generate a mutant mouse (path A), as well as in mouse embryonic fibroblasts (MEFs) to accelerate the phenotypic analysis of introduced mutations (path B). Figure 1 : A model for co-operative control of p53 by Mdm2 and Mdm4 : Mdm2 promotes degradation of p53, whereas Mdm4 inhibits the transcriptional activity of p53. In addition, we have shown that MDM4 is a promising target for anti-cancer strategies, and that the combined use of MDM2 and MDM4 antagonists may reactivate p53 in some cancers. Indeed, in recent years, several mouse models have been used to establish the clinical potential of reactivating p53 in tumours. We estimate that such an approach might be used to treat between two and three million new cancers each year. These studies demonstrate just how much information can be gained from studying p53 regulation in vivo, as well as the potential of such approaches for developing effective therapies. F. Toledo & B. Bardot (2009) Cancer : Three birds with one stone. Nature, 460, 466-467. F. Toledo et al. (2007) Mouse mutants reveal that putative protein interaction sites in the p53 proline-rich domain are dispensable for tumor suppression. Mol. Cell. Biol., 27, 1425-1432 F. Toledo et al. (2006) A mouse p53 mutant lacking the proline rich domain rescues Mdm4 deficiency and provides insight into the Mdm2-Mdm4-p53 regulatory network. Cancer Cell, 9, 273-285. F. Toledo et al. (2006) RMCE-ASAP : a gene targeting method for ES and somatic cells to accelerate phenotype analyses. Nucleic Acids Res., 34, e92 F. Toledo & G.M. Wahl (2006) Regulating the p53 pathway : in vitro hypotheses, in vivo veritas. Nature Rev. Cancer, 6, 909-923.