Publications de l’équipe
Année de publication : 2015
Functional genomics analysis reveals a MYC signature associated with a poor clinical prognosis in liposarcomas.
The American journal of pathology : 717-28 : DOI : 10.1016/j.ajpath.2014.11.024 En savoir plusRésumé
Liposarcomas, which are malignant fatty tumors, are the second most common soft-tissue sarcomas. Several histologically defined liposarcoma subtypes exist, yet little is known about the molecular pathology that drives the diversity in these tumors. We used functional genomics to classify a panel of diverse liposarcoma cell lines based on hierarchical clustering of their gene expression profiles, indicating that liposarcoma gene expression profiles and histologic classification are not directly correlated. Boolean probability approaches based on cancer-associated properties identified differential expression in multiple genes, including MYC, as potentially affecting liposarcoma signaling networks and cancer outcome. We confirmed our method with a large panel of lipomatous tumors, revealing that MYC protein expression is correlated with patient survival. These data encourage increased reliance on genomic features in conjunction with histologic features for liposarcoma clinical characterization and lay the groundwork for using Boolean-based probabilities to identify prognostic biomarkers for clinical outcome in tumor patients.
ReplierHuman regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.
Nucleic acids research : 1834-47 : DOI : 10.1093/nar/gku1402 En savoir plusRésumé
Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS.
ReplierAnnée de publication : 2014
RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.
EMBO reports : 221-31 : DOI : 10.15252/embr.201439458 En savoir plusRésumé
Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.
ReplierThe basic N-terminal domain of TRF2 limits recombination endonuclease action at human telomeres.
Cell cycle (Georgetown, Tex.) : 2469-74 : DOI : 10.4161/cc.29422 En savoir plusRésumé
The stability of mammalian telomeres depends upon TRF2, which prevents inappropriate repair and checkpoint activation. By using a plasmid integration assay in yeasts carrying humanized telomeres, we demonstrated that TRF2 possesses the intrinsic property to both stimulate initial homologous recombination events and to prevent their resolution via its basic N-terminal domain. In human cells, we further showed that this TRF2 domain prevents telomere shortening mediated by the resolvase-associated protein SLX4 as well as GEN1 and MUS81, 2 different types of endonucleases with resolvase activities. We propose that various types of resolvase activities are kept in check by the basic N-terminal domain of TRF2 in order to favor an accurate repair of the stalled forks that occur during telomere replication.
ReplierFISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool.
Lab on a chip : 811-22 : DOI : 10.1039/c4lc01059k En savoir plusRésumé
Microfluidic systems bear promise to provide new powerful tools for the molecular characterization of cancer cells, in particular for the routine detection of multiple cancer biomarkers using a minute amount of the sample. However, taking miniaturized cell-based assays into the clinics requires the implementation and validation of complex biological protocols on chip, as well as the development of disposable microdevices produced at a low cost. Based on a recently developed microfluidic chip made of Cyclic Olefin Copolymer for cell immobilization with minimal dead volume and controlled shear stress, we developed a protocol performed entirely in the liquid phase, allowing the immobilization and fixation of cells and their quantitative characterization by fluorescence in situ hybridization. We demonstrated first in cell lines and then in two clinical case studies the potential of this method to perform quantitative copy number measurement and clinical scoring of the amplification of the ERBB2 gene, a decisive biomarker for the prescription of HER2+ related targeted therapies. This validation was performed in a blind protocol in two clinical case studies, in reference to the gold standard and clinically used method based on glass slides. We obtained a comparable reproducibility and a minor difference in apparent amplification, which can be corrected by internal calibration. The method thus reaches the standard of robustness needed for clinical use. The protocol can be fully automated, and its consumption of samples and DNA probes is reduced as compared to glass slide protocols by a factor of at least 10. The total duration of the assay is divided by two.
ReplierA timeless but timely connection between replication and recombination.
Cell : 697-8 : DOI : 10.1016/j.cell.2014.07.029 En savoir plusRésumé
Initiation of meiotic recombination by DNA double-strand break formation is temporally coordinated with replication. Murakami and Keeney show that this coordination requires recruitment of the Dbf4-dependent kinase to the replication fork by the conserved TIM-TIPIN complex. The same mechanism may regulate other important replication-associated processes.
ReplierCRIPTO overexpression promotes mesenchymal differentiation in prostate carcinoma cells through parallel regulation of AKT and FGFR activities.
Oncotarget : 11994-2008 En savoir plusRésumé
Members of the EGF-CFC (Cripto, FRL-1, Cryptic) protein family are increasingly recognized as key mediators of cell movement and cell differentiation during vertebrate embryogenesis. The founding member of this protein family, CRIPTO, is overexpressed in various human carcinomas. Yet, the biological role of CRIPTO in this setting remains unclear. Here, we find CRIPTO expression as especially high in a subgroup of primary prostate carcinomas with poorer outcome, wherein resides cancer cell clones with mesenchymal traits. Experimental studies in PCa models showed that one notable function of CRIPTO expression in prostate carcinoma cells may be to augment PI3K/AKT and FGFR1 signaling, which promotes epithelial-mesenchymal transition and sustains a mesenchymal state. In the observed signaling events, FGFR1 appears to function parallel to AKT, and the two pathways act cooperatively to enhance migratory, invasive and transformation properties specifically in the CRIPTO overexpressing cells. Collectively, these findings suggest a novel molecular network, involving CRIPTO, AKT, and FGFR signaling, in favor of the emergence of mesenchymal-like cancer cells during the development of aggressive prostate tumors.
ReplierPHD and TFIIS-Like domains of the Bye1 transcription factor determine its multivalent genomic distribution.
PloS one : e102464 : DOI : 10.1371/journal.pone.0102464 En savoir plusRésumé
The BYpass of Ess1 (Bye1) protein is a putative S. cerevisiae transcription factor homologous to the human cancer-associated PHF3/DIDO family of proteins. Bye1 contains a Plant Homeodomain (PHD) and a TFIIS-like domain. The Bye1 PHD finger interacts with tri-methylated lysine 4 of histone H3 (H3K4me3) while the TFIIS-like domain binds to RNA polymerase (Pol) II. Here, we investigated the contribution of these structural features to Bye1 recruitment to chromatin as well as its function in transcriptional regulation. Genome-wide analysis of Bye1 distribution revealed at least two distinct modes of association with actively transcribed genes: within the core of Pol II- and Pol III-transcribed genes concomitant with the presence of the TFIIS transcription factor and, additionally, with promoters of a subset of Pol II-transcribed genes. Specific loss of H3K4me3 abolishes Bye1 association to gene promoters, but doesn’t affect its binding within gene bodies. Genetic interactions suggested an essential role of Bye1 in cell fitness under stress conditions compensating the absence of TFIIS. Furthermore, BYE1 deletion resulted in the attenuation of GAL genes expression upon galactose-mediated induction indicating its positive role in transcription regulation. Together, these findings point to a bimodal role of Bye1 in regulation of Pol II transcription. It is recruited via its PHD domain to H3K4 tri-methylated promoters at early steps of transcription. Once Pol II is engaged into elongation, Bye1 binds directly to the transcriptional machinery, modulating its progression along the gene.
ReplierTelomere recombination and the ALT pathway: a therapeutic perspective for cancer.
Current pharmaceutical design : 6466-71 En savoir plusRésumé
Telomeres are essential for cell proliferation and tumor cell immortalization requires the presence of a telomere maintenance mechanism. Thus, interfering with this mechanism constitutes a potential means to impede cell proliferation and tumor progression. Many cancer cells rely on telomerase activity to ensure indefinite proliferation capacity and developing therapeutic approaches that target telomerase has attracted much attention in the last couple of decades. Nevertheless, a non-negligible proportion of tumors utilize telomerase- independent, alternative mechanisms to lengthen telomeres (ALT). Here we briefly discuss both our current understanding of ALT mechanisms and the potential to develop a therapeutic approach targeting ALT.
ReplierRecombinogenic telomeres in diploid Sorex granarius (Soricidae, Eulipotyphla) fibroblast cells.
Molecular and cellular biology : 2786-99 : DOI : 10.1128/MCB.01697-13 En savoir plusRésumé
The telomere structure in the Iberian shrew Sorex granarius is characterized by unique, striking features, with short arms of acrocentric chromosomes carrying extremely long telomeres (up to 300 kb) with interspersed ribosomal DNA (rDNA) repeat blocks. In this work, we investigated the telomere physiology of S. granarius fibroblast cells and found that telomere repeats are transcribed on both strands and that there is no telomere-dependent senescence mechanism. Although telomerase activity is detectable throughout cell culture and appears to act on both short and long telomeres, we also discovered that signatures of a recombinogenic activity are omnipresent, including telomere-sister chromatid exchanges, formation of alternative lengthening of telomeres (ALT)-associated PML-like bodies, production of telomere circles, and a high frequency of telomeres carrying marks of a DNA damage response. Our results suggest that recombination participates in the maintenance of the very long telomeres in normal S. granarius fibroblasts. We discuss the possible interplay between the interspersed telomere and rDNA repeats in the stabilization of the very long telomeres in this organism.
ReplierA preclinical mouse model of glioma with an alternative mechanism of telomere maintenance (ALT).
International journal of cancer : 1546-58 : DOI : 10.1002/ijc.29171 En savoir plusRésumé
Glioblastoma multiforme is the most aggressive primary tumor of the central nervous system. Glioma stem cells (GSCs), a small population of tumor cells with stem-like properties, are supposedly responsible for glioblastoma multiforme relapse after current therapies. In approximately thirty percent of glioblastoma multiforme tumors, telomeres are not maintained by telomerase but through an alternative mechanism, termed alternative lengthening of telomere (ALT), suggesting potential interest in developing specific therapeutic strategies. However, no preclinical model of ALT glioma was available until the isolation of TG20 cells from a human ALT glioma. Herein, we show that TG20 cells exhibit a high level of telomeric recombination but a stable karyotype, indicating that their telomeres retain their protective function against chromosomal instability. TG20 cells possess all of the characteristic features of GSCs: the expression of neural stem cell markers, the generation of intracerebral tumors in NOD-SCID-IL2Rγ (NSG) mice as well as in nude mice, and the ability to sustain serial intracerebral transplantations without expressing telomerase, demonstrating the stability of the ALT phenotype in vivo. Furthermore, we also demonstrate that 360B, a G-quadruplex ligand of the pyridine derivative series that impairs telomere replication and mitotic progression in cancer cells, prevents the development of TG20 tumors. Together, our results show that intracerebral grafts of TG20 cells in immunodeficient mice constitute an efficient preclinical model of ALT glioblastoma multiforme and that G-quadruplex ligands are a potential therapy for this specific type of tumor.
ReplierMdm4 loss in mice expressing a p53 hypomorph alters tumor spectrum without improving survival.
Oncogene En savoir plusRésumé
The p53 pathway is inactivated in most human cancers, and its reactivation in tumors appears as a promising therapeutic strategy. Overexpression of Mdm4, a p53 negative regulator, occurs in a significant fraction of human cancers. Mouse models were used to evaluate the therapeutic potential of strategies against Mdm4, and encouraging results were obtained for tumor cells in which Mdm4 overexpression prevents wild-type p53 to exert its tumor suppressive functions. However, missense mutations in the p53 gene occur in about half of human cancers, and 15% of such mutations lead to the expression of a mutant protein that retains partial activity. In this report, we used mouse models to address the therapeutic potential of strategies against Mdm4 in tumors expressing an hypomorphic p53 mutant. We found that, in an Rb(+/-) background promoting pituitary and thyroid tumors, decreased Mdm4 levels improved the survival of mice expressing wild-type p53, but not that of mice expressing p53(ΔP), a p53 hypomorph lacking the proline-rich domain. Importantly, however, most Rb(+/-) p53(ΔP/ΔP) mice developped pituitary adenomas, but these tumors were rare in Rb(+/-) p53(ΔP/ΔP) Mdm4(-/-) animals, because Mdm4 loss led to increased p21 levels, a suppressor of pituitary tumor growth. On the contrary, Rb(+/-) p53(ΔP/ΔP) and Rb(+/-) p53(ΔP/ΔP) Mdm4(-/-) mice developped anaplastic thyroid carcinomas at equal frequencies. Importantly, wild-type p53 represses the Plk1 gene, which encodes a promising therapeutic target in anaplastic thyroid carcinomas, and this repression is improved when Mdm4 levels are decreased. On the opposite, p53(ΔP) is a mediocre transcriptional repressor that is not improved by Mdm4 loss. In sum, depending on the tumor type, strategies against Mdm4 that work in cells expressing wild-type p53 may not work in cells expressing an hypomorphic p53. Furthermore, p53-mediated transcriptional repression should be considered when evaluating strategies to reactivate p53 in tumors.
ReplierAlternative Lengthening of Telomeres is characterized by reduced compaction of telomeric chromatin.
Nucleic acids research : 4391-405 : DOI : 10.1093/nar/gku114 En savoir plusRésumé
Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric RNA species. These results offer new insight on telomeric chromatin properties in ALT cells and support the hypothesis that telomeric chromatin decondensation is important for ALT pathway.
ReplierPRC2-independent chromatin compaction and transcriptional repression in cancer.
Oncogene : 741-51 : DOI : 10.1038/onc.2013.604 En savoir plusRésumé
The silencing of large chromosomal regions by epigenetic mechanisms has been reported to occur frequently in cancer. Epigenetic marks, such as histone methylation and acetylation, are altered at these loci. However, the mechanisms of formation of such aberrant gene clusters remain largely unknown. Here, we show that, in cancer cells, the epigenetic remodeling of chromatin into hypoacetylated domains covered with histone H3K27 trimethylation is paralleled by changes in higher-order chromatin structures. Using fluorescence in situ hybridization, we demonstrate that regional epigenetic silencing corresponds to the establishment of compact chromatin domains. We show that gene repression is tightly correlated to the state of chromatin compaction and not to the levels of H3K27me3-its removal through the knockdown of EZH2 does not induce significant gene expression nor chromatin decompaction. Moreover, transcription can occur with intact high-H3K27me3 levels; treatment with histone deacetylase inhibitors can relieve chromatin compaction and gene repression, without altering H3K27me3 levels. Our findings imply that compaction and subsequent repression of large chromatin domains are not direct consequences of PRC2 deregulation in cancer cells. By challenging the role of EZH2 in aberrant gene silencing in cancer, these findings have therapeutical implications, notably for the choice of epigenetic drugs for tumors with multiple regional epigenetic alterations.
ReplierAnnée de publication : 2013
[RTEL1 (regulator of telomere elongation helicase 1), a DNA helicase essential for genome stability].
Médecine sciences : M/S : 1138-44 : DOI : 10.1051/medsci/20132912018 En savoir plusRésumé
RTEL1 (regulator of telomere length helicase 1) is a DNA helicase that has been identified more than 10 years ago. Many works since, mainly in the nematode Caenorhabditis elegans and the mouse, have highlighted its role in chromosomal stability, maintenance of telomere length, and DNA repair. Recently, four laboratories have characterized RTEL1 mutations in patients with dyskeratosis congenita (DC) and Hoyeraal-Hreidarsson (HH) syndrome, a rare and severe variant of DC. We here summarize the current knowledge on RTEL1 and discuss the possible other functions that RTEL1 could play.
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