Publications by authors named "Annick Harel-Bellan"

47 Publications

MasterPATH: network analysis of functional genomics screening data.

BMC Genomics 2020 Sep 14;21(1):632. Epub 2020 Sep 14.

Institut des Hautes Etudes Scientifiques, Le Bois-Marie 35 rte de Chartres, 91440, Bures-sur-Yvette, France.

Background: Functional genomics employs several experimental approaches to investigate gene functions. High-throughput techniques, such as loss-of-function screening and transcriptome profiling, allow to identify lists of genes potentially involved in biological processes of interest (so called hit list). Several computational methods exist to analyze and interpret such lists, the most widespread of which aim either at investigating of significantly enriched biological processes, or at extracting significantly represented subnetworks.

Results: Here we propose a novel network analysis method and corresponding computational software that employs the shortest path approach and centrality measure to discover members of molecular pathways leading to the studied phenotype, based on functional genomics screening data. The method works on integrated interactomes that consist of both directed and undirected networks - HIPPIE, SIGNOR, SignaLink, TFactS, KEGG, TransmiR, miRTarBase. The method finds nodes and short simple paths with significant high centrality in subnetworks induced by the hit genes and by so-called final implementers - the genes that are involved in molecular events responsible for final phenotypic realization of the biological processes of interest. We present the application of the method to the data from miRNA loss-of-function screen and transcriptome profiling of terminal human muscle differentiation process and to the gene loss-of-function screen exploring the genes that regulates human oxidative DNA damage recognition. The analysis highlighted the possible role of several known myogenesis regulatory miRNAs (miR-1, miR-125b, miR-216a) and their targets (AR, NR3C1, ARRB1, ITSN1, VAV3, TDGF1), as well as linked two major regulatory molecules of skeletal myogenesis, MYOD and SMAD3, to their previously known muscle-related targets (TGFB1, CDC42, CTCF) and also to a number of proteins such as C-KIT that have not been previously studied in the context of muscle differentiation. The analysis also showed the role of the interaction between H3 and SETDB1 proteins for oxidative DNA damage recognition.

Conclusion: The current work provides a systematic methodology to discover members of molecular pathways in integrated networks using functional genomics screening data. It also offers a valuable instrument to explain the appearance of a set of genes, previously not associated with the process of interest, in the hit list of each particular functional genomics screening.
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http://dx.doi.org/10.1186/s12864-020-07047-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491077PMC
September 2020

Identification of microRNAs in skeletal muscle associated with lung cancer cachexia.

J Cachexia Sarcopenia Muscle 2020 04 11;11(2):452-463. Epub 2019 Dec 11.

Department of Respiratory Medicine, NUTRIM, Maastricht University Medical Center+, Maastricht, The Netherlands.

Background: Cachexia, highly prevalent in patients with non-small cell lung cancer (NSCLC), impairs quality of life and is associated with reduced tolerance and responsiveness to cancer therapy and decreased survival. MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in post-transcriptional gene regulation. Changes in intramuscular levels of miRNAs have been implicated in muscle wasting conditions. Here, we aimed to identify miRNAs that are differentially expressed in skeletal muscle of cachectic lung cancer patients to increase our understanding of cachexia and to allow us to probe their potential as therapeutic targets.

Methods: A total of 754 unique miRNAs were profiled and analysed in vastus lateralis muscle biopsies of newly diagnosed treatment-naïve NSCLC patients with cachexia (n = 8) and age-matched and sex-matched healthy controls (n = 8). miRNA expression analysis was performed using a TaqMan MicroRNA Array. In silico network analysis was performed on all significant differentially expressed miRNAs. Differential expression of the top-ranked miRNAs was confirmed using reverse transcription-quantitative real-time PCR in an extended group (n = 48) consisting of NSCLC patients with (n = 15) and without cachexia (n = 11) and healthy controls (n = 22). Finally, these miRNAs were subjected to univariate and multivariate Cox proportional hazard analysis using overall survival and treatment-induced toxicity data obtained during the follow-up of this group of patients.

Results: We identified 28 significant differentially expressed miRNAs, of which five miRNAs were up-regulated and 23 were down-regulated. In silico miRNA-target prediction analysis showed 158 functional gene targets, and pathway analysis identified 22 pathways related to the degenerative or regenerative processes of muscle tissue. Subsequently, the expression of six top-ranked miRNAs was measured in muscle biopsies of the entire patient group. Five miRNAs were detectable with reverse transcription-quantitative real-time PCR analysis, and their altered expression (expressed as fold change, FC) was confirmed in muscle of cachectic NSCLC patients compared with healthy control subjects: miR-424-5p (FC = 4.5), miR-424-3p (FC = 12), miR-450a-5p (FC = 8.6), miR-144-5p (FC = 0.59), and miR-451a (FC = 0.57). In non-cachectic NSCLC patients, only miR-424-3p was significantly increased (FC = 5.6) compared with control. Although the statistical support was not sufficient to imply these miRNAs as individual predictors of overall survival or treatment-induced toxicity, when combined in multivariate analysis, miR-450-5p and miR-451a resulted in a significant stratification between short-term and long-term survival.

Conclusions: We identified differentially expressed miRNAs putatively involved in lung cancer cachexia. These findings call for further studies to investigate the causality of these miRNAs in muscle atrophy and the mechanisms underlying their differential expression in lung cancer cachexia.
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http://dx.doi.org/10.1002/jcsm.12512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113505PMC
April 2020

Mathematical modeling reveals the factors involved in the phenomena of cancer stem cells stabilization.

PLoS One 2019 11;14(11):e0224787. Epub 2019 Nov 11.

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris-Sud, University Paris-Saclay, Gif-sur-Yvette, France.

Cancer Stem Cells (CSC), a subset of cancer cells resembling normal stem cells with self-renewal and asymmetric division capabilities, are present at various but low proportions in many tumors and are thought to be responsible for tumor relapses following conventional cancer therapies. In vitro, most intriguingly, isolated CSCs rapidly regenerate the original population of stem and non-stem cells (non-CSCs) as shown by various investigators. This phenomenon still remains to be explained. We propose a mathematical model of cancer cell population dynamics, based on the main parameters of cell population growth, including the proliferation rates, the rates of cell death and the frequency of symmetric and asymmetric cell divisions both in CSCs and non-CSCs sub-populations, and taking into account the stabilization phenomenon. The analysis of the model allows determination of time-varying corridors of probabilities for different cell fates, given the particular dynamics of cancer cells populations; and determination of a cell-cell communication factors influencing these time-varying probabilities of cell behavior (division, transition) scenarios. Though the results of the model have to be experimentally confirmed, we can anticipate the development of several fundamental and practical applications based on the theoretical results of the model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224787PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844488PMC
March 2020

Basic, simple and extendable kinetic model of protein synthesis.

Math Biosci Eng 2019 07;16(6):6602-6622

Lobachevsky University, Nizhny Novgorod, Russia.

Protein synthesis is one of the most fundamental biological processes. Despite existence of multiple mathematical models of translation, surprisingly, there is no basic and simple chemical kinetic model of this process, derived directly from the detailed kinetic scheme. One of the reasons for this is that the translation process is characterized by indefinite number of states, because of the structure of the polysome. We bypass this difficulty by applying lumping of multiple states of translated mRNA into few dynamical variables and by introducing a variable describing the pool of translating ribosomes. The simplest model can be solved analytically. The simplest model can be extended, if necessary, to take into account various phenomena such as the limited amount of ribosomal units or regulation of translation by microRNA. The introduced model is more suitable to describe the protein synthesis in eukaryotes but it can be extended to prokaryotes. The model can be used as a building block for more complex models of cellular processes. We demonstrate the utility of the model in two examples. First, we determine the critical parameters of the synthesis of a single protein for the case when the ribosomal units are abundant. Second, we demonstrate intrinsic bi-stability in the dynamics of the ribosomal protein turnover and predict that a minimal number of ribosomes should pre-exists in a living cell to sustain its protein synthesis machinery, even in the absence of proliferation.
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http://dx.doi.org/10.3934/mbe.2019329DOI Listing
July 2019

miR-600 Acts as a Bimodal Switch that Regulates Breast Cancer Stem Cell Fate through WNT Signaling.

Cell Rep 2017 02;18(9):2256-2268

Molecular Oncology "Equipe labellisée Ligue Contre le Cancer," Aix-Marseille Université, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, 13273 Marseille, France. Electronic address:

Breast cancer stem cells (bCSCs) have been implicated in tumor progression and therapeutic resistance; however, the molecular mechanisms that define this state are unclear. We have performed two microRNA (miRNA) gain- and loss-of-function screens to identify miRNAs that regulate the choice between bCSC self-renewal and differentiation. We find that micro-RNA (miR)-600 silencing results in bCSC expansion, while its overexpression reduces bCSC self-renewal, leading to decreased in vivo tumorigenicity. miR-600 targets stearoyl desaturase 1 (SCD1), an enzyme required to produce active, lipid-modified WNT proteins. In the absence of miR-600, WNT signaling is active and promotes self-renewal, whereas overexpression of miR-600 inhibits the production of active WNT and promotes bCSC differentiation. In a series of 120 breast tumors, we found that a low level of miR-600 is correlated with active WNT signaling and a poor prognosis. These findings highlight a miR-600-centered signaling network that governs bCSC-fate decisions and influences tumor progression.
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http://dx.doi.org/10.1016/j.celrep.2017.02.016DOI Listing
February 2017

A Broad Set of Chromatin Factors Influences Splicing.

PLoS Genet 2016 Sep 23;12(9):e1006318. Epub 2016 Sep 23.

Institut Pasteur, Unit of Epigenetic regulation, Paris, France.

Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035054PMC
http://dx.doi.org/10.1371/journal.pgen.1006318DOI Listing
September 2016

The WEE1 regulators CPEB1 and miR-15b switch from inhibitor to activators at G2/M.

Cell Cycle 2016 ;15(5):667-77

a Université Paris Sud, Laboratoire Epigénétique et Cancer, Formation de Recherche en Evolution 3377 , Gif-Sur-Yvette , France.

MicroRNAs (miRNAs) in the AGO-containing RISC complex control messenger RNA (mRNA) translation by binding to mRNA 3' untranslated region (3'UTR). The relationship between miRNAs and other regulatory factors that also bind to mRNA 3'UTR, such as CPEB1 (cytoplasmic polyadenylation element-binding protein), remains elusive. We found that both CPEB1 and miR-15b control the expression of WEE1, a key mammalian cell cycle regulator. Together, they repress WEE1 protein expression during G1 and S-phase. Interestingly, the 2 factors lose their inhibitory activity at the G2/M transition, at the time of the cell cycle when WEE1 expression is maximal, and, moreover, rather activate WEE1 translation in a synergistic manner. Our data show that translational regulation by RISC and CPEB1 is essential in cell cycle control and, most importantly, is coordinated, and can be switched from inhibition to activation during the cell cycle.
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http://dx.doi.org/10.1080/15384101.2016.1147631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845936PMC
December 2016

UV-dependent phosphorylation of COP9/signalosome in UV-induced apoptosis.

Oncol Rep 2016 May 11;35(5):3101-5. Epub 2016 Mar 11.

Institute for Integrative Biology of the Cell (I2BC), IBITECS, CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette Cedex, France.

The COP9/signalosome (CSN) multi-protein complex regulates the activity of cullin-RING ubiquitin ligases (CRLs), including the DDB2 and CSA CRL4 ligases (CRL4DDB2 and CRL4CSA), which are involved in the repair of UV-induced DNA damages. In the present study, we demonstrated that the protein kinase ATM, a key component of the DNA damage response (DDR), phosphorylates CSN1 and CSN7a, two subunits of the CSN complex, in a UV-dependent manner. The phosphorylation of CSN1 on serine 474 was detected as early as 3 h after UV-exposure, peaked at 8 h and persisted until 48 h post-UV irradiation. Such a time course suggests a role in late DDR rather than in DNA repair. Consistently, overexpression of a phosphorylation-resistant S474A CSN1 mutant reduced UV-induced apoptosis. Thus, CSN1 appears to play a role not only in DNA repair but also in UV-induced apoptosis.
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http://dx.doi.org/10.3892/or.2016.4671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918815PMC
May 2016

Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation.

Biochim Biophys Acta 2016 Feb 1;1863(2):263-70. Epub 2015 Dec 1.

Institute for Integrative Biology of the Cell (I2BC), IBiTecs, CEA, CNRS, Univ Paris Saclay, Gif-sur-Yvette F-91198, France. Electronic address:

CNOT6L is a deadenylase subunit belonging to the CCR4-NOT complex, a major deadenylase complex in eukaryotes involved at multiple levels in regulation of gene expression. While CNOT6L is expressed in skeletal muscle cells, its specific functions in this tissue are still largely unknown. Our previous work highlighted the functional of CNOT6L in skeletal muscle cell differentiation. To further explore how CNOT6L regulates myogenesis, we used here gene expression analysis to identify CNOT6L mRNA targets in human myoblasts. Among these novel targets, IL-8 (interleukin 8) mRNA was the most upregulated in CNOT6L knock-down (KD) cells. Biochemical approaches and poly (A) tail length assays showed that IL-8 mRNA is a direct target of CNOT6L, and further investigations by loss- and gain-of-function assays pointed out that IL-8 is an important effector of myogenesis. Therefore, we have characterized CNOT6L-IL-8 as a new signaling axis that regulates myogenesis.
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http://dx.doi.org/10.1016/j.bbamcr.2015.11.018DOI Listing
February 2016

miRNA expression in control and FSHD fetal human muscle biopsies.

PLoS One 2015 18;10(2):e0116853. Epub 2015 Feb 18.

Sorbonne Universités, UPMC Univ Paris 06, Center of Research in Myology UM76, Paris, France; INSERM UMRS974, Paris, France; CNRS FRE 3617, F-75013, Paris, France; Institut de Myologie, F-75013, Paris, France.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder and is one of the most common forms of muscular dystrophy. We have recently shown that some hallmarks of FSHD are already expressed in fetal FSHD biopsies, thus opening a new field of investigation for mechanisms leading to FSHD. As microRNAs (miRNAs) play an important role in myogenesis and muscle disorders, in this study we compared miRNAs expression levels during normal and FSHD muscle development.

Methods: Muscle biopsies were obtained from quadriceps of both healthy control and FSHD1 fetuses with ages ranging from 14 to 33 weeks of development. miRNA expression profiles were analyzed using TaqMan Human MicroRNA Arrays.

Results: During human skeletal muscle development, in control muscle biopsies we observed changes for 4 miRNAs potentially involved in secondary muscle fiber formation and 5 miRNAs potentially involved in fiber maturation. When we compared the miRNA profiles obtained from control and FSHD biopsies, we did not observe any differences in the muscle specific miRNAs. However, we identified 8 miRNAs exclusively expressed in FSHD1 samples (miR-330, miR-331-5p, miR-34a, miR-380-3p, miR-516b, miR-582-5p, miR-517* and miR-625) which could represent new biomarkers for this disease. Their putative targets are mainly involved in muscle development and morphogenesis. Interestingly, these FSHD1 specific miRNAs do not target the genes previously described to be involved in FSHD.

Conclusions: This work provides new candidate mechanisms potentially involved in the onset of FSHD pathology. Whether these FSHD specific miRNAs cause deregulations during fetal development, or protect against the appearance of the FSHD phenotype until the second decade of life still needs to be investigated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116853PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333765PMC
November 2015

miR-98 delays skeletal muscle differentiation by down-regulating E2F5.

Biochem J 2015 Feb;466(1):85-93

*CNRS FRE 3377, CEA Saclay, F-91191 Gif-sur-Yvette, France.

A genome-wide screen had previously shown that knocking down miR-98 and let-7g, two miRNAs of the let-7 family, leads to a dramatic increase in terminal myogenic differentiation. In the present paper, we report that a transcriptomic analysis of human myoblasts, where miR-98 was knocked down, revealed that approximately 240 genes were sensitive to miR-98 depletion. Among these potential targets of miR-98, we identified the transcriptional repressor E2F5 and showed that it is a direct target of miR-98. Knocking down simultaneously E2F5 and miR-98 almost fully restored normal differentiation, indicating that E2F5 is involved in the regulation of skeletal muscle differentiation. We subsequently show that E2F5 can bind to the promoters of two inhibitors of terminal muscle differentiation, ID1 (inhibitor of DNA binding 1) and HMOX1 (heme oxygenase 1), which decreases their expression in skeletal myoblasts. We conclude that miR-98 regulates muscle differentiation by altering the expression of the transcription factor E2F5 and, in turn, of multiple E2F5 targets.
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http://dx.doi.org/10.1042/BJ20141175DOI Listing
February 2015

Regulation by miR181 family of the dependence receptor CDON tumor suppressive activity in neuroblastoma.

J Natl Cancer Inst 2014 Nov 13;106(11). Epub 2014 Oct 13.

Apoptosis, Cancer and Development Laboratory-Equipe labellisée 'La Ligue,' LabEx DEVweCAN, Centre de Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Université de Lyon, Centre Léon Bérard, Lyon, France (BG, CDB, OM, SLG, JF, BD, FL, MC, AMN, PM); CNRS UMR 8126, University Paris-Sud 11, Institut Gustave Roussy, Villejuif, France (C-HG, JB); Stem Cell and Brain Research Institute, INSERM U846, Bron, France (FB); INSERM, U830, Génétique et Biologie des Cancers, Institut Curie, Paris, France (IJL, OD); Department Epigenetics and Cancer FRE 3377, Centre National de la Recherche Scientifique, Commissariat à l'Energie Atomique Saclay, Gif-sur-Yvette, France (AHB); Université Paris-Sud, Gif-sur-Yvette, France (AH-B); Present address: INSERM UMR 1078, Etablissement Français du Sang, Centre Hospitalier Régional Universitaire de Brest, SFR ScInBioS, Université de Bretagne Occidentale, Faculté de Médecine, Brest, France (C-HG).

Background: The Sonic Hedgehog (SHH) signaling pathway plays an important role in neural crest cell fate during embryonic development and has been implicated in the progression of multiple cancers that include neuroblastoma, a neural crest cell-derived disease. While most of the SHH signaling is mediated by the well-described canonical pathway leading to the activation of Smoothened and Gli, it has recently been shown that cell-adhesion molecule-related/downregulated by oncogenes (CDON) serves as a receptor for SHH and contributes to SHH-induced signaling. CDON has also been recently described as a dependence receptor, triggering apoptosis in the absence of SHH. This CDON proapoptotic activity has been suggested to constrain tumor progression.

Methods: CDON expression was analyzed by quantitative-reverse transcription-polymerase chain reaction in a panel of 226 neuroblastoma patients and associated with stages, overall survival, and expression of miR181 family members using Kaplan Meier and Pearson correlation methods. Cell death assays were performed in neuroblastoma cell lines and tumor growth was investigated in the chick chorioallantoic model. All statistical tests were two-sided.

Results: CDON expression was inversely associated with neuroblastoma aggressiveness (P < .001). Moreover, re-expression of CDON in neuroblastoma cell lines was associated with apoptosis in vitro and tumor growth inhibition in vivo. We show that CDON expression is regulated by the miR181 miRNA family, whose expression is directly associated with neuroblastoma aggressiveness (survival: high miR181-b 73.2% vs low miR181-b 54.6%; P = .03).

Conclusions: Together, these data support the view that CDON acts as a tumor suppressor in neuroblastomas, and that CDON is tightly regulated by miRNAs.
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http://dx.doi.org/10.1093/jnci/dju318DOI Listing
November 2014

ALDH1-positive cancer stem cells predict engraftment of primary breast tumors and are governed by a common stem cell program.

Cancer Res 2013 Dec 18;73(24):7290-300. Epub 2013 Oct 18.

Authors' Affiliations: INSERM, U1068, CRCM, Molecular Oncology, Institut Paoli-Calmettes, Biopathology, Aix-Marseille Univ, UM 105, F-13284, Départements d'Oncologie Médicale, Chirugie oncologique, and Radiologie, Institut Paoli-Calmettes, Marseille; and Plateforme ARN interference PArI, CEA SACLAY, Gif-sur-Yvette, France.

Cancer stem-like cells (CSC) have been widely studied, but their clinical relevance has yet to be established in breast cancer. Here, we report the establishment of primary breast tumor-derived xenografts (PDX) that encompass the main diversity of human breast cancer and retain the major clinicopathologic features of primary tumors. Successful engraftment was correlated with the presence of ALDH1-positive CSCs, which predicted prognosis in patients. The xenografts we developed showed a hierarchical cell organization of breast cancer with the ALDH1-positive CSCs constituting the tumorigenic cell population. Analysis of gene expression from functionally validated CSCs yielded a breast CSC signature and identified a core transcriptional program of 19 genes shared with murine embryonic, hematopoietic, and neural stem cells. This generalized stem cell program allowed the identification of potential CSC regulators, which were related mainly to metabolic processes. Using an siRNA genetic screen designed to target the 19 genes, we validated the functional role of this stem cell program in the regulation of breast CSC biology. Our work offers a proof of the functional importance of CSCs in breast cancer, and it establishes the reliability of PDXs for use in developing personalized CSC therapies for patients with breast cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-4704DOI Listing
December 2013

Genome-wide exploration of miRNA function in mammalian muscle cell differentiation.

PLoS One 2013 21;8(8):e71927. Epub 2013 Aug 21.

Department Epigenetics and Cancer FRE 3377, Centre National de la Recherche Scientifique, Commissariat à l'Energie Atomique Saclay, Gif-sur-Yvette, France ; Université Paris-Sud, Gif-sur-Yvette, France.

MiRNAs impact on the control of cell fate by regulating gene expression at the post-transcriptional level. Here, using mammalian muscle differentiation as a model and a phenotypic loss-of-function screen, we explored the function of miRNAs at the genome-wide level. We found that the depletion of a high number of miRNAs (63) impacted on differentiation of human muscle precursors, underscoring the importance of this post-transcriptional mechanism of gene regulation. Interestingly, a comparison with miRNA expression profiles revealed that most of the hit miRNAs did not show any significant variations of expression during differentiation. These constitutively expressed miRNAs might be required for basic and/or essential cell function, or else might be regulated at the post-transcriptional level. MiRNA inhibition yielded a variety of phenotypes, reflecting the widespread miRNA involvement in differentiation. Using a functional screen (the STarS--Suppressor Target Screen--approach, i. e. concomitant knockdown of miRNAs and of candidate target proteins), we discovered miRNA protein targets that are previously uncharacterized controllers of muscle-cell terminal differentiation. Our results provide a strategy for functional annotation of the human miRnome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071927PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749189PMC
April 2014

10-million-years AGO: argonaute on chromatin in yeast and human, a conserved mode of action?

Transcription 2013 May-Jun;4(3):89-91. Epub 2013 Apr 12.

Université Paris Sud; Laboratoire Epigenetique et Cancer; Formation de Recherche en Evolution 3377; Gif-Sur-Yvette, France; Centre National de la Recherche Scientifique (CNRS); Gif-Sur-Yvette, France; Commissariat à l'Energie Atomique (CEA); Saclay; Gif-sur-Yvette, France.

Whereas in yeast the function and mode of action of nuclear RNAi are well documented, mammalian nuclear RNAi is a matter of debates. Several papers support a role for nuclear Argonaute in alternative splicing. However, the molecular mechanism remains elusive. Here, we discuss the human nuclear RNAi mechanism in light of what is known of the yeast process.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042589PMC
http://dx.doi.org/10.4161/trns.24582DOI Listing
March 2015

Argonaute proteins couple chromatin silencing to alternative splicing.

Nat Struct Mol Biol 2012 Oct 9;19(10):998-1004. Epub 2012 Sep 9.

Université Paris Sud, Laboratoire Epigenetique et Cancer, Formation de Recherche en Evolution, Gif-Sur-Yvette, France.

Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1γ, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification.
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http://dx.doi.org/10.1038/nsmb.2373DOI Listing
October 2012

Prognostic impact of vitamin B6 metabolism in lung cancer.

Cell Rep 2012 Aug 26;2(2):257-69. Epub 2012 Jul 26.

INSERM, U, Villejuif, France.

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.
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http://dx.doi.org/10.1016/j.celrep.2012.06.017DOI Listing
August 2012

Kinetic signatures of microRNA modes of action.

RNA 2012 Sep 31;18(9):1635-55. Epub 2012 Jul 31.

CNRS FRE 3377, CEA Saclay, France.

MicroRNAs (miRNAs) are key regulators of all important biological processes, including development, differentiation, and cancer. Although remarkable progress has been made in deciphering the mechanisms used by miRNAs to regulate translation, many contradictory findings have been published that stimulate active debate in this field. Here we contribute to this discussion in three ways. First, based on a comprehensive analysis of the existing literature, we hypothesize a model in which all proposed mechanisms of microRNA action coexist, and where the apparent mechanism that is detected in a given experiment is determined by the relative values of the intrinsic characteristics of the target mRNAs and associated biological processes. Among several coexisting miRNA mechanisms, the one that will effectively be measurable is that which acts on or changes the sensitive parameters of the translation process. Second, we have created a mathematical model that combines nine known mechanisms of miRNA action and estimated the model parameters from the literature. Third, based on the mathematical modeling, we have developed a computational tool for discriminating among different possible individual mechanisms of miRNA action based on translation kinetics data that can be experimentally measured (kinetic signatures). To confirm the discriminatory power of these kinetic signatures and to test our hypothesis, we have performed several computational experiments with the model in which we simulated the coexistence of several miRNA action mechanisms in the context of variable parameter values of the translation.
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http://dx.doi.org/10.1261/rna.032284.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425779PMC
September 2012

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats.

Mol Vis 2011 18;17:2228-40. Epub 2011 Aug 18.

Department of Biochemistry and Immunology, School of Medicine University of São Paulo, Ribeirão Preto, Brazil.

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats.

Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis.

Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b.

Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164688PMC
January 2012

A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease.

Nat Genet 2011 Mar 30;43(3):242-5. Epub 2011 Jan 30.

INSERM ERI-21, EA4319, Faculty of Medicine, Nice, France.

Susceptibility to Crohn's disease, a complex inflammatory disease, is influenced by common variants at many loci. The common exonic synonymous SNP (c.313C>T) in IRGM, found in strong linkage disequilibrium with a deletion polymorphism, has been classified as non-causative because of the absence of an alteration in the IRGM protein sequence or splice sites. Here we show that a family of microRNAs (miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohn's disease and downregulates the IRGM protective variant (c.313C) but not the risk-associated allele (c.313T). Subsequent loss of tight regulation of IRGM expression compromises control of intracellular replication of Crohn's disease-associated adherent invasive Escherichia coli by autophagy. These results suggest that the association of IRGM with Crohn's disease arises from a miRNA-based alteration in IRGM regulation that affects the efficacy of autophagy, thereby implicating a synonymous polymorphism as a likely causal variant.
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http://dx.doi.org/10.1038/ng.762DOI Listing
March 2011

[Small non-coding RNAs: new targets and new therapeutic possibilities].

Bull Acad Natl Med 2010 Feb;194(2):319-24; discussion 324-5

CNRS FRE 2944, Institut Andre Lwoff, Villejuif, F-94801 and Universit6 Paris-Sud, Villejuif F-94801, France.

The discovery of regulatory small non-coding RNAs represents a revolution in our understanding of gene regulation. These small non-coding RNAs are powerful tools for exploring cellular pathways and for artificially controlling gene expression. Natural small RNAs also represent potential therapeutic targets.
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February 2010

Preferential association of irreversibly silenced E2F-target genes with pericentromeric heterochromatin in differentiated muscle cells.

Epigenetics 2010 Nov-Dec;5(8):704-9. Epub 2010 Nov 1.

Institut André Lwoff, Centre National de la Recherche Scientifique (CNRS), FRE2944, Université Paris-Sud, Villejuif, France.

The heterochromatin-associated H3K9 tri-methylase Suv39h1 is involved in the permanent silencing of E2F target genes in differentiating but not in quiescent cells. Here, we tested the hypothesis that permanent silencing of E2F target genes is associated with their subnuclear positioning close to the pericentromeric heterochromatin compartment, enriched in Suv39h1. Using fluorescence in situ hybridization, we analyzed the subnuclear localization of three E2F target genes relative to the pericentromeric heterochromatin, in cycling fibroblasts or differentiating myoblasts. We observed that all three E2F-target genes have a tendency to relocate closer to the pericentromeric heterochromatin, only when cells differentiate and undergo an irreversible cell cycle withdrawal. These data suggest that repression of E2F target genes in cycling or in differentiating cells is achieved through distinct mechanisms. In differentiating cells, permanent silencing is driven by a Suv39h1-dependent H3K9 tri-methylation and positioning close to the heterochromatin compartment, whereas repression in cycling cells seems independent from subnuclear positioning and requires distinct H3K9 methylation levels.
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http://dx.doi.org/10.4161/epi.5.8.13025DOI Listing
March 2011

[Metamorphoses].

Bull Cancer 2010 Mar;97(3):287

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March 2010

Dynamical modeling of microRNA action on the protein translation process.

BMC Syst Biol 2010 Feb 24;4:13. Epub 2010 Feb 24.

Institut Curie, Bioinformatics and Computational Systems Biology of Cancer, Paris, France.

Background: Protein translation is a multistep process which can be represented as a cascade of biochemical reactions (initiation, ribosome assembly, elongation, etc.), the rate of which can be regulated by small non-coding microRNAs through multiple mechanisms. It remains unclear what mechanisms of microRNA action are the most dominant: moreover, many experimental reports deliver controversial messages on what is the concrete mechanism actually observed in the experiment. Nissan and Parker have recently demonstrated that it might be impossible to distinguish alternative biological hypotheses using the steady state data on the rate of protein synthesis. For their analysis they used two simple kinetic models of protein translation.

Results: In contrary to the study by Nissan and Parker, we show that dynamical data allow discriminating some of the mechanisms of microRNA action. We demonstrate this using the same models as developed by Nissan and Parker for the sake of comparison but the methods developed (asymptotology of biochemical networks) can be used for other models. We formulate a hypothesis that the effect of microRNA action is measurable and observable only if it affects the dominant system (generalization of the limiting step notion for complex networks) of the protein translation machinery. The dominant system can vary in different experimental conditions that can partially explain the existing controversy of some of the experimental data.

Conclusions: Our analysis of the transient protein translation dynamics shows that it gives enough information to verify or reject a hypothesis about a particular molecular mechanism of microRNA action on protein translation. For multiscale systems only that action of microRNA is distinguishable which affects the parameters of dominant system (critical parameters), or changes the dominant system itself. Dominant systems generalize and further develop the old and very popular idea of limiting step. Algorithms for identifying dominant systems in multiscale kinetic models are straightforward but not trivial and depend only on the ordering of the model parameters but not on their concrete values. Asymptotic approach to kinetic models allows putting in order diverse experimental observations in complex situations when many alternative hypotheses co-exist.
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http://dx.doi.org/10.1186/1752-0509-4-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2847993PMC
February 2010

miR-181a and miR-630 regulate cisplatin-induced cancer cell death.

Cancer Res 2010 Mar 9;70(5):1793-803. Epub 2010 Feb 9.

Institut National de la Sante et de la Recherche Medicale, U848, Villejuif, France.

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non-small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27(Kip1) as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-3112DOI Listing
March 2010

A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex.

Mol Cell 2010 Jan;37(1):46-56

Institut André Lwoff, Centre National de la Recherche Scientifique, FRE2944, Université Paris-Sud, 94800 Villejuif, France.

Lysine 9 of histone 3 (H3K9) can be mono-, di-, or trimethylated, inducing distinct effects on gene expression and chromatin compaction. H3K9 methylation can be mediated by several histone methyltransferases (HKMTs) that possess mono-, di-, or trimethylation activities. Here we provide evidence that a subset of each of the main H3K9 HKMTs, G9a/KMT1C, GLP/KMT1D, SETDB1/KMT1E, and Suv39h1/KMT1A, coexist in the same megacomplex. Moreover, in Suv39h or G9a null cells, the remaining HKMTs are destabilized at the protein level, indicating that the integrity of these HKMTs is interdependent. The four HKMTs are recruited to major satellite repeats, a known Suv39h1 genomic target, but also to multiple G9a target genes. Moreover, we report a functional cooperation between the four H3K9 HKMTs in the regulation of known G9a target genes. Altogether, our data identify a H3K9 methylation multimeric complex.
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http://dx.doi.org/10.1016/j.molcel.2009.12.017DOI Listing
January 2010

Tandem affinity purification of miRNA target mRNAs (TAP-Tar).

Nucleic Acids Res 2010 Mar 2;38(4):e20. Epub 2009 Dec 2.

CNRS FRE 2944, Institut André Lwoff, Villejuif F-94801 and Université Paris-Sud, Villejuif F-94801, France.

MicroRNAs (miRNAs) bind to Argonaute proteins, and together they form the RISC complex and regulate target mRNA translation and/or stability. Identification of mRNA targets is key to deciphering the physiological functions and mode of action of miRNAs. In mammals, miRNAs are generally poorly homologous to their target sequence, and target identification cannot be based solely on bioinformatics. Here, we describe a biochemical approach, based on tandem affinity purification, in which mRNA/miRNA complexes are sequentially pulled down, first via the Argonaute moiety and then via the miRNA. Our 'TAP-Tar' procedure allows the specific pull down of mRNA targets of miRNA. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.
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http://dx.doi.org/10.1093/nar/gkp1100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2831319PMC
March 2010

Physical and functional interaction between heterochromatin protein 1alpha and the RNA-binding protein heterogeneous nuclear ribonucleoprotein U.

J Biol Chem 2009 Oct 17;284(41):27974-27979. Epub 2009 Jul 17.

CNRS FRE 2944, Institut André Lwoff; Université Paris-Sud, Villejuif F-94801, France. Electronic address:

By combining biochemical purification and mass spectrometry, we identified proteins associated with human heterochromatin protein 1alpha (HP1alpha) both in the nucleoplasm and in chromatin. Some of these are RNA-binding proteins, and among them is the protein heterogeneous nuclear ribonucleoprotein U (hnRNP U)/SAF-A, which is linked to chromatin organization and transcriptional regulation. Here, we demonstrate that hnRNP U is a bona fide HP1alpha-interacting molecule. More importantly, hnRNP U depletion reduces HP1alpha-dependent gene silencing and disturbs HP1alpha subcellular localization. Thus, our data demonstrate that hnRNP U is involved in HP1alpha function, shedding new light on the mode of action of HP1alpha and on the function of hnRNP U.
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http://dx.doi.org/10.1074/jbc.M109.037929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788849PMC
October 2009

Differential cooperation between heterochromatin protein HP1 isoforms and MyoD in myoblasts.

J Biol Chem 2008 Aug 2;283(35):23692-700. Epub 2008 Jul 2.

Institut André Lwoff, CNRS, FRE 2944, 7 rue Guy Moquet, Villejuif, France.

Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1alpha, HP1beta, and HP1gamma play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1alpha and HP1beta isoforms, but not HP1gamma, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1alpha and HP1beta, but not HP1gamma, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner.
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http://dx.doi.org/10.1074/jbc.M802647200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259757PMC
August 2008

[Micro-RNAs and muscle differentiation].

J Soc Biol 2007 5;201(4):367-76. Epub 2008 Mar 5.

CNRS FRE 2944 Epigénétique et Cancer, Institut André Lwoff, 7 rue Guy Môquet, BP8, 94801 Villejuif Cedex, France.

Deciphering the mechanisms underlying skeletal muscle differentiation in mammals is an important challenge. Cell differentiation involves complex pathways regulated at both transcriptional and post-transcriptional levels. Recent observations have revealed the importance of small (20-25 base pairs) non-coding RNAs (microRNAs or miRNAs) that are expressed in both lower organisms and in mammals. miRNAs modulate gene expression by affecting mRNA translation or stability. In lower organisms, miRNAs are essential for cell differentiation during development; some miRNAs are involved in maintenance of the differentiated state. We have shown that miR-181, a microRNA that is strongly upregulated during differentiation, participates in establishing the muscle phenotype. Moreover, our results suggest that miR-181 downregulates the homeobox protein Hox-A11 (a repressor of the differentiation process), thus establishing a functional link between miR-181 and the complex process of mammalian skeletal muscle differentiation. Therefore, miRNAs can be involved in the establishment of a differentiated phenotype - even when they are not expressed in the corresponding fully differentiated tissue.
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http://dx.doi.org/10.1051/jbio:2007902DOI Listing
December 2015