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SummaryAlthough oncogene-induced senescence (OIS) is a potent tumor-suppressor mechanism, recent studies revealed that cells can escape from OIS with features of transformed cells. However, the mechanisms that promote OIS escape remain unclear, and evidence of post-senescent cells in human cancers is missing. Here, we unravel the regulatory mechanisms underlying OIS escape using dynamic multidimensional profiling. We demonstrate a critical role for AP1 and POU2F2 transcription factors for escape from OIS and identify ‘senescence-associated chromatin scars (SACS)’ as an epigenetic memory of OIS, detectable during colorectal cancer progression. POU2F2 levels are elevated already in precancerous lesions and as cells escape from OIS, and its expression and binding activity to cis-regulatory elements are associated with decreased patient survival. Our results support a model in which POU2F2 exploits a precoded enhancer landscape to promote senescence escape and reveal POU2F2 gene signatures and SACS as valuable biomarkers with diagnostic and prognostic potential.

Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase (AHCY) inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global Histone H3 Lysine 36 trimethylation (H3K36me3), an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Remarkably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood (UCB) cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence.

Fragile bone is the root cause of osteoporosis. For inherited or acquired reasons, the fragile bone does not provide sufficient fracture resistance to withstand the physical strains of a normal lifestyle. Accordingly, clinical characteristics consist of fragility fractures that occur during daily life activities or low energy trauma. Hip fractures and vertebral fractures are so called "major osteoporotic fractures”, that also cause the highest burden of disease. Although the clinical osteoporosis manifestations are relatively uniform, there is a vast spectrum of underlying molecular causes. Impaired bone formation, accelerated bone loss, and impaired lifetime adaptive regeneration according to physical impact characterize the cruder facets of osteoporosis. The signaling cascades that govern bone formation and metabolism may be altered by genetically or epigenetically inherited defects or acquired epigenetic changes due to tissue aging and/or underlying diseases. While molecular genetics and mechanisms and specific osteoporosis treatments have made impressive progress over the last three decades, there is still an urgent need to better understand the role of epigenetics in this disease.Epigenetic mechanisms such as covalent modifications of DNA, histones, or essential core factors like the osteogenic transcription factors (e. g., RUNX2) and inhibitory modulators of osteogenic WNT-signaling (e. g., Dickkopf-1 (DKK-1), sclerostin (SOST)) are all intricately implicated in developmental bone formation and adaptive regeneration and remodeling processes throughout adult life. These mechanisms are accompanied by chromatin architecture and gene expression changes of small (e. g., microRNAs (miRs)) and long, noncoding RNAs (lncRNAs). The timely execution of these mechanisms either facilitates or inhibits bone formation and remodeling. Together, epigenetic mechanisms controlling bone homeostasis widen the spectrum of potential dysregulations that can cause osteoporosis and open new avenues for therapeutic interventions.Apart from the core mechanisms of bone formation and regeneration, recent research revealed that tissue-resident cells of the immune system such as tissue-specific macrophages, myeloid precursors, and lymphocytes have surprisingly fundamental influence on tissue regeneration, including bone. Those tissue resident cells are also subject to epigenetic changes and may substantially contribute to the development of disease. Epigenetic constellations can be inherited, but the dynamic epigenetic mechanisms involved in physiological processes of tissue regeneration may also be affected by pathologies such as cellular aging and senescence. Recently, several studies aimed at identifying DNA methylation signatures in peripheral blood leukocytes from osteoporosis patients that reveal novel disease mechanisms and potential targets for diagnosis and treatment. Overall, these studies rendered, however, yet inconclusive results.By contrast, studies using bone marrow-derived skeletal progenitors identified transcriptome changes in osteoporosis patients, which could have epigenetic reasons in the absence of genetic causes. Respective changes may be related to the local milieu in bone and bone marrow as a kind of segmental attitude of a specific tissue acquired through tissue aging and/or supported by underlying aging-associated diseases such as arteriosclerosis or aging of cells of the immune system.In summary, there is cumulating evidence linking epigenetic factors to the pathogenesis of aging-associated osteoporosis. However, we are currently still limited in our knowledge with respect to the causal traits that are common, inherited, or acquired in a lifetime in the respective tissues and cells involved in bone formation and regeneration. During the following years, the field will most certainly learn more about molecular processes and factors that can be targeted therapeutically and/or used as biomarkers for risk assessment.

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; Cellular senescence is a cell fate that prominently impacts physiological and pathophysiological processes. Diverse cellular stresses induce it, and dramatic gene expression changes accompany it. However, determining the interactions comprising the gene regulatory network (GRN) governing senescence remains a challenge. Recent advances in signal processing techniques provide opportunities to reconstruct GRNs. Here, we describe a GRN controlling senescence integrating time-series transcriptome and transcription factor depletion datasets. We infer a set of differential equations modeling the CS transcriptome using the “Sparse Identification of Nonlinear Dynamics” (SINDy) algorithm, discriminate genes with potential hidden regulators, validating the inferred GRN for time points not included in the training data. Our work is a proof of concept of a data-based method for GRN reconstruction, consolidating an iterative, powerful mathematical platform for more comprehensive senescence models that can be used to test hypotheses in silico and has the potential for future discoveries of clinical impact.

Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase (AHCY) inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global Histone H3 Lysine 36 trimethylation (H3K36me3), an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Remarkably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood (UCB) cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence. We thank O.C. Bing (BRC, A*STAR) for the histopathology scoring of liver sections. For the purpose of open access, the author has applied a Creative Commons Attribution license. Core support from MRC (MC_U120085810), a Development Gap Fund grant from LifeArc and Cancer Research UK (C15075/A28647) funded this research in J. Gil’s laboratory. P.M.-C. acknowledges funding from RTI2018-096068-B-I00, ERC-2016-AdG-741966, La Caixa HR17-00040, UPGRADE-H2020-825825, MWRF, Fundació La Marató-TV3, AFM, MDA and DPP-E. This work was supported by grants from the Deutsche Krebshilfe (to J.J.), the Dutch Cancer Society (to G.d.H.) and the Tekke Huizinga Fund (S.B. and G.d.H.). L.R. was supported by the Pasteur - Paris University International PhD Program and by the Fondation pour la Recherche Médicale. O.B was supported by Fondation ARC pour la Recherche sur le Cancer, INSERM-AGEMED and ANR S-ENCODE - 19-CE13-0017-01. O.B. is a Centre National de la Recherche Scientifique Research Director DR2. T.W. was funded by National Medical Research Council, Singapore through NMRC/OFLCG/003b/2018 and A*STAR through the Central Research Fund for Applied/Translational Research.

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.

Oncogene-induced senescence (OIS) is a potent barrier for the transformation of pre-cancerous cells. The molecular pathways involved in the execution of OIS are still incompletely understood, but they include chronic DNA damage signaling and post-translational modifications of key factors. Here, we show that OIS-associated transcriptional downregulation of deubiquitinating enzyme USP1 triggers and maintains a DNA damage checkpoint response with atypical DNA lesions that is dependent on functional FANCD2-FI-ATR-CHK1-p53-CDKN1A signaling. We find that a reduced USP1 level causes aberrant aggregation of its target FANCD2 concomitant with replication stress, accumulation, and colocalization of γ-H2Ax and p53-binding protein 1 (53BP1) in large and unusual sparse DNA damage foci and an increased number of polyploid cells and cells arrested in G2/M, as well as a sensitization of senescence-bypassing cells to DNA interstrand crosslinking-mediated cell death. Our study identifies USP1 as a key senescence regulator controlling genomic integrity and a promising target for anti-cancer therapy.

Author(s): Bischof, Oliver; Galande, Sanjeev; Farzaneh, Farzin; Kohwi-Shigematsu, Terumi; Campisi, Judith | Abstract: This article has been withdrawn by the authors except Dr. Kohwi- Shigematsu, who could not be reached. Fig. 3B has a duplication of the top band in lanes 1 and 3. Fig. 5D has a duplication between the top bands and a horizontal flip of the bottom bands. Fig. 5E likewise has a duplication of the top bands excluding the probe lanes and a horizontal flip of the bottom bands. Fig. 6A has smaller microscopy images pasted on the background of larger microscopy images.

Volume 25, no. 3, p. 1013–1024, 2005, [https://doi.org/10.1128/MCB.25.3.1013-1024.2005][1]. We hereby retract this article. We were recently made aware of the presence of irregularities associated with the Western blots shown in several figures. We have further investigated the matter and found

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 and apoptosis have evolved to restrain unwarranted proliferation of potentially tumorigenic cells. Here we show that overexpression of the E3 SUMO ligase PIASy in normal human fibroblasts recruits the p53 and Rb tumor suppressor pathways to provoke a senescence arrest. By contrast, in Rb-deficient fibroblasts, expression of PIASy leads to p53-dependent apoptosis. Induction of senescence requires PIASy E3 activity and is specific for this member of the PIAS ligase family. PIASy stimulates sumoylation and transcriptional activity of p53 and increases Rb-dependent corepression through recruitment to E2F-responsive promoters. Viral oncoprotein E6 suppresses both PIASy-induced senescence and sumoylation of PIASy substrates. Finally, we show that fibroblasts lacking PIASy exhibit a highly reduced propensity to undergo senescence in response to a prosenescence stimulus. Altogether, these data provide the first evidence for a direct role of an E3 SUMO ligase, and by implication of the SUMO pathway, in cellular senescence and apoptosis.

INTRODUCTION. 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 that is presumed to function in mammalian DNA replication, recombination or repair. Because of its importance in maintaining genomic stability, we asked whether BLM was among the proteins targeted for selective degradation during the execution phase of apoptosis as has been shown recently for PARP and ATM (1, 2), among others. We show here that BLM is rapidly cleaved in cells undergoing apoptosis. RESULTS. BLM was cleaved to 47 and 110 kDa major fragments, with kinetics similar to the apoptotic cleavage of poly-ADP ribose polymerase (only ∆BLM of 47 kDa is shown in the Fig.). BLM cleavage was prevented by a caspase 3 inhibitor, and did not occur in caspase 3deficient cells. Moreover, recombinant BLM was cleaved to 47 and 110 kDa fragments by caspase 3, but not caspase 6, in vitro. The caspase 3 recognition sequence 412TEVD415 was verified by mutating aspartate-415 to glycine, and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Apoptotic cleavage did not abolish the BLM helicase activity, but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM is an early selected target during the execution of apoptosis.

Comment in :- Neighbourhood deaths cause a switch in cancer subtype. [Nature. 2018]- Neighbourly deaths dictate fate. [Nat Rev Cancer. 2018]- Bad neighborhoods: apoptotic and necroptotic microenvironments determine liver cancer subtypes. [Hepatobiliary Surg Nutr. 2019]- Viewpoint: necroptosis influences the type of liver cancer via changes of hepatic microenvironment. [Hepatobiliary Surg Nutr. 2019]; International audience; Primary liver cancer represents a major health problem. It comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which differ markedly with regards to their morphology, metastatic potential and responses to therapy. However, the regulatory molecules and tissue context that commit transformed hepatic cells towards HCC or ICC are largely unknown. Here we show that the hepatic microenvironment epigenetically shapes lineage commitment in mosaic mouse models of liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment determines ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, a function that is conserved in humans. Together, our results provide insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC.

International audience; Transcriptional deregulation caused by epigenetic or genetic alterations is a major cause of leukemic transformation. The Spi1/PU.1 transcription factor is a key regulator of many steps of hematopoiesis, and limits self-renewal of hematopoietic stem cells. The deregulation of its expression or activity contributes to leukemia, in which Spi1 can be either an oncogene or a tumor suppressor. Herein we explored whether cellular senescence, an anti-tumoral pathway that restrains cell proliferation, is a mechanism by which Spi1 limits hematopoietic cell expansion, and thus prevents the development of leukemia. We show that Spi1 overexpression triggers cellular senescence both in primary fibroblasts and hematopoietic cells. Erythroid and myeloid lineages are both prone to Spi1-induced senescence. In hematopoietic cells, Spi1-induced senescence requires its DNA-binding activity and a functional p38MAPK14 pathway but is independent of a DNA-damage response. In contrast, in fibroblasts, Spi1-induced senescence is triggered by a DNA-damage response. Importantly, using our well-established Spi1 transgenic leukemia mouse model, we demonstrate that Spi1 overexpression also induces senescence in erythroid progenitors of the bone marrow in vivo before the onset of the pre-leukemic phase of erythroleukemia. Remarkably, the senescence response is lost during the progression of the disease and erythroid blasts do not display a higher expression of Dec1 and CDKN1A, two of the induced senescence markers in young animals. These results bring indirect evidence that leukemia develops from cells which have bypassed Spi1-induced senescence. Overall, our results reveal senescence as a Spi1-induced anti-proliferative mechanism that may be a safeguard against the development of acute myeloid leukemia.

The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases., Senescence of mammalian cells is characterized by proliferative arrest and expression of an inflammatory phenotype. Here the authors show the H2A variant H2A.J, found only in mammals, accumulates following persistent DNA damage or natural aging.

In this Article, the pCaMIN construct consisted of ‘mouse MYC and mouse NrasG12V’ instead of ‘mouse Myc and human NRASG12V; and the pCAMIA construct consisted of ‘mouse Myc and human AKT1’ instead of ‘mouse Myc and Akt1’ this has been corrected online.

International audience; Oncogene-induced senescence (OIS) is a critical tumor-suppressing mechanism that restrains cancer progression at premalignant stages, in part by causing telomere dysfunction. Currently it is unknown whether this proliferative arrest presents a stable and therefore irreversible barrier to cancer progression. Here we demonstrate that cells frequently escape OIS induced by oncogenic H-Ras and B-Raf, after a prolonged period in the senescence arrested state. Cells that had escaped senescence displayed high oncogene expression levels, retained functional DNA damage responses, and acquired chromatin changes that promoted c-Myc–dependent expression of the human telomerase reverse transcriptase gene ( hTERT ). Telomerase was able to resolve existing telomeric DNA damage response foci and suppressed formation of new ones that were generated as a consequence of DNA replication stress and oncogenic signals. Inhibition of MAP kinase signaling, suppressing c-Myc expression, or inhibiting telomerase activity, caused telomere dysfunction and proliferative defects in cells that had escaped senescence, whereas ectopic expression of hTERT facilitated OIS escape. In human early neoplastic skin and breast tissue, hTERT expression was detected in cells that displayed features of senescence, suggesting that reactivation of telomerase expression in senescent cells is an early event during cancer progression in humans. Together, our data demonstrate that cells arrested in OIS retain the potential to escape senescence by mechanisms that involve derepression of hTERT expression.

Cellular senescence is a stable cell cycle arrest that inhibits the outgrowth of pre-cancerous cells but is also implicated in wound healing, embryonic development, aging and age-related pathologies. Our knowledge on gene regulatory circuits that establish and maintain the senescence phenotype is highly fragmentary. Here, we provide several lines of evidence supporting a critical and novel function of scaffolding-attachment-factor A SAF-A and long, noncoding RNA PANDA in the establishment and maintenance of the senescence phenotype. First, we demonstrate that SAF-A and PANDA are differentially expressed in presenescent compared to senescent cells. Second, we show that both SAF-A and PANDA actively contribute to senescence induction and maintenance. Finally, we establish that SAF-A and PANDA physically and functionally interact to directly repress senescence- and proliferation-promoting genes by regulating access of polycomb repressive complexes PRC1 and PRC2 as well as transcription factor NF-YA to their cognate target genes. Together, our data identify DNA-RNA-binding protein SAF-A and long, noncoding RNA PANDA as key actors in senescence cell fate decision and unravel the importance of cell fate dependent target gene changes of transcription factors and noncoding RNAs.

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

Oncogene-induced senescence (OIS) is a potent barrier for the transformation of pre-cancerous cells. The molecular pathways involved in the execution of OIS are still incompletely understood, but they include chronic DNA damage signaling and post-translational modifications of key factors. Here, we show that OIS-associated transcriptional downregulation of deubiquitinating enzyme USP1 triggers and maintains a DNA damage checkpoint response with atypical DNA lesions that is dependent on functional FANCD2-FI-ATR-CHK1-p53-CDKN1A signaling. We find that a reduced USP1 level causes aberrant aggregation of its target FANCD2 concomitant with replication stress, accumulation, and colocalization of γ-H2Ax and p53-binding protein 1 (53BP1) in large and unusual sparse DNA damage foci and an increased number of polyploid cells and cells arrested in G2/M, as well as a sensitization of senescence-bypassing cells to DNA interstrand crosslinking-mediated cell death. Our study identifies USP1 as a key senescence regulator controlling genomic integrity and a promising target for anti-cancer therapy.

Cellular senescence and apoptosis have evolved to restrain unwarranted proliferation of potentially tumorigenic cells. Here we show that overexpression of the E3 SUMO ligase PIASy in normal human fibroblasts recruits the p53 and Rb tumor suppressor pathways to provoke a senescence arrest. By contrast, in Rb-deficient fibroblasts, expression of PIASy leads to p53-dependent apoptosis. Induction of senescence requires PIASy E3 activity and is specific for this member of the PIAS ligase family. PIASy stimulates sumoylation and transcriptional activity of p53 and increases Rb-dependent corepression through recruitment to E2F-responsive promoters. Viral oncoprotein E6 suppresses both PIASy-induced senescence and sumoylation of PIASy substrates. Finally, we show that fibroblasts lacking PIASy exhibit a highly reduced propensity to undergo senescence in response to a prosenescence stimulus. Altogether, these data provide the first evidence for a direct role of an E3 SUMO ligase, and by implication of the SUMO pathway, in cellular senescence and apoptosis.

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; Cellular senescence is a fundamental cell fate, playing important physiological and pathophysiological roles. This SnapShot focuses on major signaling pathways and transcriptional control mechanisms that consolidate the senescence phenotype.

International audience; Cellular senescence is a fundamental cell fate, important both in physiological and pathophysiological processes. This SnapShot focuses on the role of cellular senescence in health, disease, and aging.

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 that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6, in vitro. The caspase 3 recognition sequence (412)TEVD(415) was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.

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.