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Genetics studies the inheritance of genetic information encoded by the base pair sequence and its variants. Sequence variants can have severe consequences as seen in genetically inherited diseases (e. g. osteogenesis Imperfecta, hypophosphatasia). On the other hand, epigenetics deals with inherited and dynamically reversible modifications of chromatin without changing the base pair sequence, resulting in a change in phenotype without a change in genotype. These modifications primarily exert their effects by influencing gene expression. Initially, the definition of epigenetics exclusively comprised inherited changes that persist across several generations without changes in the DNA sequence. This definition has been extended to include also dynamic and partially reversible changes that occur more short-term. These gene modulatory effects introduce new levels of complexity and are crucial determinants of cell fate and organismal development. With its length of approximately two meters, human DNA has to be compacted to fit into the nuclei and fulfill its functions. DNA is wrapped around histone octamers into so-called nucleosomes. DNA, histones, and other DNA-associated proteins together form what is called chromatin. DNA packaging is achieved by variable degrees of chromatin condensation depending on cell type and context. Epigenetic transcriptional regulation modifies the affinity and accessibility of cis-regulatory elements (CREs) for transcription factors and the basic transcriptional machinery and governs interaction between CREs. CREs include promoters, enhancers, silencers, and insulators and are potent modulators of gene expression impacting core cell biological processes such as proliferation and differentiation. Chromatin looping and remodeling by differential covalent modifications of DNA (e. g., methylation or hydroxylation) and histone tails (e. g., acetylation or methylation) elicit fundamental changes in CRE accessibility, thus impacting gene expression. Chromatin looping depends on a specialized machinery including cohesins. Chromatin modifications are mediated by specific enzymes like DNA methylases (DNMTs), histone-modifying enzymes, like histone methyl- and acetyltransferases (KMTs, HATs/KATs), and histone demethylases and deacetylases (KDMs, HDACs). It becomes increasingly evident that epigenetic (dys)regulation plays a decisive role in physiology and pathophysiology, impacting many age-related diseases like cancer and degenerative pathologies (e. g., osteoporosis, Alzheimer’s, or Parkinson’s) in a significant fashion. Recently, small-molecule inhibitors of chromatin-modifying enzymes (e. g., vorinostat) have been identified and successfully introduced in therapy. Significant progress in high-throughput sequencing technologies and big data analysis has broadened our understanding of noncoding (nc) RNAs and DNA sequence regions in (post-)transcriptional regulation and disease development. Among ncRNAs that play vital roles in gene expression are micro- (miRs) and long noncoding RNAs (lncRNAs; e. g., XIST or HOTAIR). By interacting with the coding genome, these RNAs modulate important genetic programs. Interfering RNAs can, for example, enhance the post-transcriptional degradation of transcripts, altering their translation, or assist in the recruitment of chromatin-modifying enzymes to regulate transcription. They can also be packaged into extracellular vesicles as cargo and thus deliver critical information to the microenvironment or even systemically to distant tissues. Therefore, ncRNAs represent a novel playground for therapeutical investigations and supplement epigenetic mechanisms of gene regulation while being subject to epigenetic regulation themselves. Last but not least, dysregulated ncRNAs can also propagate disease. Until recently, the detection of epigenetic phenomena necessitated invasive diagnostic interventions. However, with the arrival of so-called “liquid biopsies” an analysis of circulating cell-free DNA fragments (cfDNA) and RNAs as well as vesicle-packed RNAs through minimal invasively drawn blood samples can be obtained. Such “fragmentomics” and RNAomics approaches on peripheral blood will ultimately serve as diagnostic tools for personalized clinical interventions.

International audience; Senescent cells affect many physiological and pathophysiological processes. While select genetic and epigenetic elements for senescence induction have been identified, the dynamics, epigenetic mechanisms and regulatory networks defining senescence competence, induction and maintenance remain poorly understood, precluding the deliberate therapeutic targeting of senescence for health benefits. Here, we examined the possibility that the epigenetic state of enhancers determines senescent cell fate. We explored this by generating time-resolved transcriptomes and epigenome profiles during oncogenic RAS-induced senescence and validating central findings in different cell biology and disease models of senescence. Through integrative analysis and functional validation, we reveal links between enhancer chromatin, transcription factor recruitment and senescence competence. We demonstrate that activator protein 1 (AP-1) 'pioneers' the senescence enhancer landscape and defines the organizational principles of the transcription factor network that drives the transcriptional programme of senescent cells. Together, our findings enabled us to manipulate the senescence phenotype with potential therapeutic implications.

Bone marrow-derived mesenchymal stromal cells (hMSCs) are capable of differentiating into the osteogenic lineage, and for osteogenic differentiation, mechanical loading is a relevant stimulus. Mechanotransduction leads to the formation of second messengers such as cAMP, cGMP, or Ca2+ influx resulting in the activation of transcription factors mediating gene regulation. The second messengers cAMP and cGMP are degraded by phosphodiesterase isoenzymes (PDE), but the role of these enzymes during osteogenic differentiation or mechanotransduction remains unclear. Here, we focused on the isoenzyme phosphodiesterase 10A (PDE10A) and its role during osteogenic commitment and mechanotransduction. We observed a time-dependent decrease of PDE10A expression in hMSC undergoing differentiation towards the osteogenic lineage. PDE10A inhibition by papaverine diminished osteogenic differentiation. While applying mechanical strain via cyclic stretching of hMSCs led to an upregulation of PDE10A gene expression, inhibition of PDE10A using the drug papaverine repressed expression of mechanoresponsive genes. We conclude that PDE10A is a modulator of osteogenic differentiation as well as mechanotransduction in hMSCs. Our data further suggests that the relative increase of cAMP, rather than the absolute cAMP level, is a key driver of the observed effects.

SUMMARYSenescent cells play important physiological- and pathophysiological roles in tumor suppression, tissue regeneration, and aging. While select genetic and epigenetic elements crucial for senescence induction were identified, the dynamics, underlying epigenetic mechanisms, and regulatory networks defining senescence competence, induction and maintenance remain poorly understood, precluding a deliberate therapeutic manipulation of these dynamic processes. Here, we show, using dynamic analyses of transcriptome and epigenome profiles, that the epigenetic state of enhancers predetermines their sequential activation during senescence. We demonstrate that activator protein 1 (AP-1) ‘imprints’ the senescence enhancer landscape effectively regulating transcriptional activities pertinent to the timely execution of the senescence program. We define and validate a hierarchical transcription factor (TF) network model and demonstrate its effectiveness for the design of senescence reprogramming experiments. Together, our findings define the dynamic nature and organizational principles of gene-regulatory elements driving the senescence program and reveal promising inroads for therapeutic manipulation of senescent cells.

International audience; Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers. We also present a resource tool to facilitate the identification of genes linked with senescence, SeneQuest (available at http://Senequest.net). Lastly, we propose an algorithm to accurately assess and quantify senescence, both in cultured cells and in vivo.

Cellular senescence acts as a potent barrier for tumour initiation and progression. Previous studies showed that the PML tumour suppressor promotes senescence, although the precise mechanisms remain to be elucidated. Combining gene expression profiling with chromatin-binding analyses and promoter reporter studies, we identify TBX2, a T-box transcription factor frequently overexpressed in cancer, as a novel and direct PML-repressible E2F-target gene in senescence but not quiescence. Recruitment of PML to the TBX2 promoter is dependent on a functional p130/E2F4 repressor complex ultimately implementing a transcriptionally inactive chromatin environment at the TBX2 promoter. TBX2 repression actively contributes to senescence induction as cells depleted for TBX2 trigger PML pro-senescence function(s) and enter senescence. Reciprocally, elevated TBX2 levels antagonize PML pro-senescence function through direct protein–protein interaction. Collectively, our findings indicate that PML and TBX2 act in an autoregulatory loop to control the effective execution of the senescence program.

On considere a l’heure actuelle que la senescence cellulaire, tout comme l’apoptose, represente un veritable mecanisme de suppression des tumeurs chez les mammiferes. La senescence cellulaire a ete identifiee a l’origine comme phenomene consecutif au raccourcissement telomerique et limitant la duree de vie des cellules humaines en culture primaire. Depuis, de nouvelles donnees ont etabli qu’a cote de cette senescence replicative (RS) existait egalement la senescence prematuree induite par le stress (SIPS) dont le phenotype est impossible a distinguer de celui de la RS. La SIPS peut etre induite aussi bien dans des cellules normales que dans des cellules tumorales en l’absence de nombreux cycles de division cellulaire, et ce en reponse a divers stress et/ou signaux inappropries. En depit de l’activite oncosuppressive potentielle du processus de senescence, des resultats recents indiquent que les cellules senescentes pourraient, dans certaines circonstances, favoriser l’emergence de cellules tumorales ou promouvoir la croissance de cellules preneoplasiques. La senescence agirait alors en veritable « cheval de Troie » biologique. Nous tenterons dans cet article de faire le point des connaissances actuelles sur les causes et les consequences de la senescence cellulaire in vitro et in vivo .

Cellular senescence is a stress response to a variety of extrinsic and intrinsic insults that cause genomic or epigenomic perturbations. It is now widely recognized as a potent tumor suppressor mechanism as well as a biological process impacting aging and organismal development. Like other cell fate decisions, senescence is executed and maintained by an intricate network of transcription factors (TFs), chromatin modifiers, and noncoding RNAs (ncRNAs). Altogether, these factors cooperate to implement the gene expression program that initiates and sustains the senescent phenotype. In the context of senescence, microRNAs (miRs) and long ncRNAs have been found to play regulatory roles at both the transcriptional and post‐transcriptional levels. In this review, we discuss recent developments in the field and point toward future research directions to gain a better understanding of ncRNAs in senescence. © 2015 IUBMB Life, 67(4):255–267, 2015

Cellular senescence is a stable cell cycle arrest that limits the proliferation of pre-cancerous cells. Here we demonstrate that scaffold-attachment-factor A (SAFA) and the long noncoding RNA PANDA differentially interact with polycomb repressive complexes (PRC1 and PRC2) and the transcription factor NF-YA to either promote or suppress senescence. In proliferating cells, SAFA and PANDA recruit PRC complexes to repress the transcription of senescence-promoting genes. Conversely, the loss of SAFA–PANDA–PRC interactions allows expression of the senescence programme. Accordingly, we find that depleting either SAFA or PANDA in proliferating cells induces senescence. However, in senescent cells where PANDA sequesters transcription factor NF-YA and limits the expression of NF-YA-E2F-coregulated proliferation-promoting genes, PANDA depletion leads to an exit from senescence. Together, our results demonstrate that PANDA confines cells to their existing proliferative state and that modulating its level of expression can cause entry or exit from senescence., The gene-regulatory circuits that establish and maintain senescence remain incompletely understood. Here, the authors show that the long noncoding RNA PANDA and scaffold-attachment-factor A (SAFA) regulate entry and exit from senescence through context-specific interactions with PRC 1/2 and the transcription factor NF-YA.

International audience; We report here the cloning and characterization of a novel KRAB zinc finger gene, ZFQR, which has eight tandemly repeated zinc fingers, a complete KRAB box at the N-terminal region, and a unique C-terminal sequence. It is expressed in a variety of human tissues, and mRNA levels are upregulated in nondividing senescent and quiescent human fibroblasts. Overexpression of the protein in quiescent cells stimulated with serum growth factors results in inhibition of entry into the cell cycle. The latter activity is lost when the N-terminal KRAB domain is deleted. The KRAB domain is also required for the transcriptional repression activity of ZFQR and in maintaining association of the protein with the nuclear matrix. The gene has been mapped to human chromosome 19q13.4. The association of ZFQR with the nuclear matrix, transcriptional repression activity, increased expression in senescent and quiescent cells, and the ability to inhibit quiescent cells stimulated with growth factors from entering the cell cycle suggests a role for ZFQR in the maintenance of the nondividing state of normal human cells.

Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.

The c- ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type β transforming growth factor (TGF-β) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-β. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-β-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-β-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-β-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-β-mediated growth inhibition in a prostate-derived epithelial cell line.

International audience; Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2–iminothiolane as cross-linkers. Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, andL36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified. Further, the binding sites of puromycin and spiramycin were established at die peptide level in several proteins that were found to constitute me antibiotic-binding sites. Peptide stretches of puromycin binding were identified from proteins S7, S14, S18, L18, and L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35. Comparison of the RNA–peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides. Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E. coli ribosomes that were active. The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes. These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis. Incorporation studies with a mutant of HmaL2 widi a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome. Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.Key words: antibiotic-binding site, RNA–peptide-binding sites, protein–RNA interaction in ribosomes, functional role of protein L2.; Les sites de contact entre les protéines et l'ARNr dans les sous-unités ribosomiques 30 S et 50S de Escherichia coli et Bacillus stearothermophilus ont été étudiés par réticulation à l'aide du 2-iminothiolane et des ultraviolets. Treize protéines ribosomiques (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 et L36) de ces bactéries ont été liées de façon covalente aux ARNr, puis les peptides et les acides aminés intervenant dans ces liaisons ont été identifiés. De plus, les segments peptidiques de plusieurs protéines des sites de liaison de la puromycine et de la spiramycine ont été identifiés. Les segments peptidiques liant la puromycine se trouvent dans les protéines S7, S14, S18, L18 et L29; ceux liant la spiramycine se trouvent dans les protéines S12, S14, L17, L18, L27 et L35. La comparaison des peptides en contact avec l'ARNr, des peptides liant les antibiotiques et des peptides modifiés chez des mutants résistants à ces antibiotiques démontre clairement que des séquences peptidiques intervenant dans ces liaisons sont identiques, ce qui démontre l'importance fonctionnelle de ces peptides. Des expériences de complémentation dans lesquelles la protéine L2 de Halobacterium marismortui a été incorporée dans des ribosomes de E. coli qui sont actifs apportent une autre preuve que des protéines ribosomiques participent à la traduction. La protéine L2 a été incorporée dans les sous-unités 50S, les ribosomes 70S, les disomes et les polysomes. Ces résultats indiquent clairement que la protéine L2 joue un rôle dans la synthèse des protéines. Les ribosomes dans lesquels une protéine L2 mutante de H. marismortui, ayant une glycine à la place de l'histidine 229, a été incorporée sont complètement inactifs. Donc, la protéine L2 et l'histidine 229 est cruciale pour le système de traduction.Mots clés : site de liaison d'un antibiotique, site de liaison peptide–ARNr, interaction protéine–ARNr, ribosomes, fonction de la protéine L2.

> La proteine PML (promyelocytic leukemia protein) a ete identifiee il y a 20 ans dans le produit de la translocation t(15,17) causant la leucemie aigue promyelocytaire [1, 2]. La proteine de fusion PML-RAR (retinoic acid receptor ) issue de cette translocation bloque la differenciation des cellules progenitrices hematopoietiques. PML fait partie, avec p53 et Rb (retinoblastoma protein) notamment, de facteurs suppresseurs de tumeur induisant la senescence cellulaire. Celle-ci est caracterisee par un arret permanent du cycle cellulaire en reponse a divers signaux tels que l’erosion des telomeres ou l’activation d’oncogenes. En empechant la proliferation illimitee des cellules, la senescence constitue une puissante barriere antitumorale (➜) [3]. Depuis la decouverte du role de PML dans la senescence cellulaire en 2000, la comprehension des mecanismes impliques dans sa fonction de suppresseur de tumeur constitue un enjeu majeur.

International audience; Cellular senescence is a tumour-suppressor mechanism that is triggered by cancer-initiating or promoting events in mammalian cells. The molecular underpinnings for this stable arrest involve transcriptional repression of proliferation-promoting genes regulated by the retinoblastoma (RB1)/E2F repressor complex. Here, we demonstrate that AGO2, RB1 and microRNAs (miRNAs), as exemplified here by let-7, physically and functionally interact to repress RB1/E2F-target genes in senescence, a process that we call senescence-associated transcriptional gene silencing (SA-TGS). Herein, AGO2 acts as the effector protein for let-7-directed implementation of silent-state chromatin modifications at target promoters, and inhibition of the let-7/AGO2 effector complex perturbs the timely execution of senescence. Thus, we identify cellular senescence as the an endogenous signal of miRNA/AGO2-mediated TGS in human cells. Our results suggest that miRNA/AGO2-mediated SA-TGS may contribute to tumour suppression by stably repressing proliferation-promoting genes in premalignant cancer cells.

International audience; Heat shock and other environmental stresses rapidly induce transcriptional responses subject to regulation by a variety of post-translational modifications. Among these, poly(ADP-ribosyl)ation and sumoylation have received growing attention. Here we show that the SUMO E3 ligase PIASy interacts with the poly(ADP-ribose) polymerase PARP-1, and that PIASy mediates heat shock-induced poly-sumoylation of PARP-1. Furthermore, PIASy, and hence sumoylation, appears indispensable for full activation of the inducible HSP70.1 gene. Chromatin immunoprecipitation experiments show that PIASy, SUMO and the SUMO-conjugating enzyme Ubc9 are rapidly recruited to the HSP70.1 promoter upon heat shock, and that they are subsequently released with kinetics similar to PARP-1. Finally, we provide evidence that the SUMO-targeted ubiquitin ligase RNF4 mediates heat-shock-inducible ubiquitination of PARP-1, regulates the stability of PARP-1, and, like PIASy, is a positive regulator of HSP70.1 gene activity. These results, thus, point to a novel mechanism for regulating PARP-1 transcription function, and suggest crosstalk between sumoylation and RNF4-mediated ubiquitination in regulating gene expression in response to heat shock.

International audience; Cellular senescence is an irreversible cell cycle arrest that curbs unrestrained, aberrant proliferation of mammalian cells and is now recognized as a prominent tumor suppressive mechanism. We have shown recently that the E3 SUMO ligase PIASy actively contributes to execution of the senescence program, thus, providing the first evidence for a direct role of SUMO modification in this process. Here, we discuss some of the implications for this novel connection and future directions to study in more detail the importance of the SUMO pathway in senescence and tumorigenesis.

International audience; p53 functions to prevent malignant progression, in part by inhibiting proliferation or inducing the death of potential tumour cells. One of the most important regulators of p53 is MDM2, a RING domain E3 ligase that ubiquitinates p53, leading to both proteasomal degradation and relocation of p53 from the nucleus to the cytoplasm. Previous studies have suggested that although polyubiquitination is required for degradation, monoubiquitination of p53 is sufficient for nuclear export. Using a p53-ubiquitin fusion protein we show that ubiquitination contributes to two steps before export: exposure of a carboxy-terminal nuclear export sequence (NES), and dissociation of MDM2. Monoubiquitination can directly promote further modifications of p53 with ubiquitin-like proteins and MDM2 promotes the interaction of the SUMO E3 ligase PIASy with p53, enhancing both sumoylation and nuclear export. Our results suggest that modifications such as sumoylation can regulate the strength of the p53-MDM2 interaction and participate in driving the export of p53.

International audience; SUMO modification (sumoylation) plays important roles in nucleo-cytoplasmic transport, maintenance of sub-nuclear architecture, the regulation of gene expression and in DNA replication, repair and recombination. Here we review recent evidence for SUMO's role in protecting genomic integrity at both the chromosomal and the DNA level. Furthermore, the involvement of sumoylation and of specific SUMO targets in cancer is discussed.

International audience; The function of the subnuclear structure the promyelocytic leukaemia (PML) body is unclear largely because of the functional heterogeneity of its constituents. Here, we provide the evidence for a direct link between PML, higher-order chromatin organization and gene regulation. We show that PML physically and functionally interacts with the matrix attachment region (MAR)-binding protein, special AT-rich sequence binding protein 1 (SATB1) to organize the major histocompatibility complex (MHC) class I locus into distinct higher-order chromatin-loop structures. Interferon gamma (IFNgamma) treatment and silencing of either SATB1 or PML dynamically alter chromatin architecture, thus affecting the expression profile of a subset of MHC class I genes. Our studies identify PML and SATB1 as a regulatory complex that governs transcription by orchestrating dynamic chromatin-loop architecture.

International audience; In this study, we investigated the subcellular and molecular mechanisms underlying promyelocytic leukemia (PML)-induced premature senescence. We demonstrate that intact PML nuclear bodies are not required for the induction of senescence. We have determined further that of seven known PML isoforms, only PML IV is capable of causing premature senescence, providing the first evidence for functional differences among these isoforms. Of interest is the fact that in contrast to PML(+/+) fibroblasts, PML(-/-) cells are resistant to PML IV-induced senescence. This suggests that although PML IV is necessary for this process to occur, it is not sufficient and requires other components for activity. Finally, we provide evidence that PML IV-induced senescence involves stabilization and activation of p53 through phosphorylation at Ser46 and acetylation at Lys382, and that it occurs independently of telomerase and differs from that elicited by oncogenic Ras. Taken together, our data assign a specific pro-senescent activity to an individual PML isoform that involves p53 activation and is independent from PML nuclear bodies.

International audience; Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix–bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix–based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.

International audience; The basic helix-loop-helix/leucine zipper (bHLH/ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF. The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168-325) encoded the ubiquitin conjugating enzyme hUBC9. The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay. Although hUBC9 is known to interact preferentially with SENTRIN/SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN. Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation. MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin-proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermore, we identified lysine 201 as a potential ubiquitination site. A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo. Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation.

International audience; In an attempt to gain information about the peptidyl transferase center at the peptide level we cross-linked the spiramycin derivative dihydrospiramycin to its functional binding site in the 70 S ribosome of Escherichia coli. In this manner ribosomal proteins S12, S14, L17, L18, L27 and L35 were found specifically affinity-labeled. Proteolytic fragmentation of these proteins, separation by C18 reversed-phase high performance liquid chromatography of the peptide mixtures, and subsequent sequence analysis of labeled peptides revealed peptide regions at positions Ala1-Lys9 and Tyr116-Lys119 of S12, Leu47-Asp53 of protein S14, Ser6-Lys35 of protein L17, Ala57-Lys63 of protein L18, Ala5-Lys18 and Val66-Lys71 of protein L27, and Thr5-Lys11 of protein L35. This approach is a valuable tool to characterize the binding site of spiramycin as well as the peptidyl transferase center at the molecular level.

International audience; We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.

International audience; Photoinduced cross-linking of Escherichia coli 70 S ribosomes with [3H]puromycin has led to the labeling of ribosomal proteins S7, S14, S18, L18, and L29. Proteolytic fragmentation of these proteins and separation of the peptide mixtures by C18 reversed-phase high performance liquid chromatography resulted in six puromycin-labeled peptides which were applied to sequence analysis. The following peptides were found labeled: Pro1-Lys10 of S7, Ala28-Lys46 and Ala7-Lys11 of S14, Asp24-Lys29 of S18, Tyr64-Lys68 of L18, and Thr55-Lys60 of L29. For the first time the molecular environment of an antibiotic in the procaryotic ribosome is presented at the peptide level.

International audience; Immunoprecipitated p100Gag-Mil protein kinase from MH2-transformed quail embryo fibroblasts is associated with an 89 kDa protein. The molar ratio between p89 and Gag-Mil in the immunocomplex is 0.72, indicating that the majority of Gag-Mil is complexed with p89. During mitosis part of Gag-Mil is shifted to a form with reduced electrophoretic mobility, p102Gag-Mil. Appearance of p102Gag-Mil leads to a reduced association with p89 indicating that p102 is not associated with p89. Microsequencing of p89 isolated from immunoprecipitates of Gag-Mil identified the protein as the quail homologue of chicken hsp90. Our results show that p100Gag-Mil is associated with hsp90 with a high stoichiometry and that upshifted p102Gag-Mil is released from the complex with hsp90.

International audience; Hepatocellular carcinoma (HCC) is a heterogeneous disease triggered by various risk factors and frequently characterized by chromosome instability. This instability is considered to be caused primarily by Hepatitis B virus (HBV), although aflatoxin B1 (AFB1), a potent fungal mutagen is also suspected to influence chromosomal repair. We studied 90 HCCs from Italy, the country with the highest incidence of hepatocellular carcinoma in Europe, 81 samples from France and 52 specimens from Shanghai, in a region where intake of AFB1 via the diet is known to be high. All 223 tumours were characterized for 15 different genomic targets, including allelic loss at 13 chromosome arms and mutations of beta-catenin and p53 genes. Despite disparity in risk-factor distribution, Italian and French cases did not significantly differ for 14 of the 15 targets tested. beta-Catenin and p53 displayed moderate and similar mutation rates (18-29% of cases) in European series. By contrast, tumours from Shanghai were significantly different, with a lower mutation rate for beta-catenin (4% vs. 26%, p