Role of Histone Variant H2A.J in Fine-Tuning Chromatin Organization for the Establishment of Ionizing Radiation-Induced Senescence

Purpose: Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNASegments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-AssociatedSecretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. Experimental Design: Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. Results: Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to proinflammatory tissue reactions. Conclusions: The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiationinduced injury

Reduced RNA turnover as a driver of cellular senescence

Accumulation of senescent cells is an important contributor to chronic inflammation upon aging. The inflammatory phenotype of senescent cells was previously shown to be driven by cytoplasmic DNA. Here, we propose that cytoplasmic double-stranded RNA has a similar effect. We find that several cell types driven into senescence by different routes share an accumulation of long promoter RNAs and 3' gene extensions rich in retrotransposon sequences. Accordingly, these cells display increased expression of genes involved in response to double stranded RNA of viral origin downstream of the interferon pathway. The RNA accumulation is associated with evidence of reduced RNA turnover, including in some cases, reduced expression of RNA exosome subunits. Reciprocally, depletion of RNA exosome subunit EXOSC3 accelerated expression of multiple senescence markers. A senescence-like RNA accumulation was also observed in cells exposed to oxidative stress, an important trigger of cellular senescence. Altogether, we propose that in a subset of senescent cells, repeat-containing transcripts stabilized by oxidative stress or reduced RNA exosome activity participate in driving and maintaining the permanent inflammatory state characterizing cellular senescence

Histone Variant H2A.J Is Enriched in Luminal Epithelial Gland Cells

H2A.J is a poorly studied mammalian-specific variant of histone H2A. We used immuno histochemistry to study its localization in various human and mouse tissues. H2A.J showed cell-type specific expression with a striking enrichment in luminal epithelial cells of multiple glands including those of breast, prostate, pancreas, thyroid, stomach, and salivary glands. H2A.J was also highly ex pressed in many carcinoma cell lines and in particular, those derived from luminal breast and prostate cancer. H2A.J thus appears to be a novel marker for luminal epithelial cancers. Knocking-out the H2AFJ gene in T47D luminal breast cancer cells reduced the expression of several estrogen-responsive genes which may explain its putative tumorigenic role in luminal-B breast cancer

Analyse fonctionnelle du variant d’histone H2A.J impliqué dans le développement d’un phénotype pro-inflammatoire en sénescence induite par des dommages à l’ADN

Cellular senescence is a stress response characterized by a stable proliferative arrest accompanied by, among others, the secretion of pro-inflammatory factors constituting what is called the SASP and an important chromatin remodelling.This PhD hosting laboratory is interested in the identification of novel regulators and biomarkers of cellular senescence. Recent data showed a specific accumulation of the histone variant H2A.J in DNA damage induced senescence and its implication in the expression of pro-inflammatory genes.This PhD project is aiming to understand the mechanisms by which H2A.J accumulation promotes the expression of pro-inflammatory genes and to identify novel physiological or organ/tissue/cell functions for H2A.J.We showed that H2A.J accumulation contributes to weakening the association of linker histone H1 to chromatin. Decreased H1 in senescence was correlated with increased expression of some repeated DNA sequences and activation of the interferon signalling pathway. Moreover, this H1 loss in senescence and its consequences could be partially recovered by H2A.J depletion by RNA interference. A mutational analysis also showed the functional importance of H2A.J sequence specificities: a moderation of its activity by the Val-11 and a potentiation by the phosphorylation of the Ser-123.Additionally, we developed a H2A.J-KO mouse model and preliminary results indicate a potential implication of H2A.J in the predisposition to obesity and the associated pathologies (diabete, chronic inflammation, etc.) and in erythropoïesis.La sénescence cellulaire est une réponse au stress des cellules caractérisée par un arrêt prolifératif stable accompagné, en outre, de la sécrétion de facteurs pro-inflammatoires constituant ce que l’on appelle le SASP ainsi que d’un remodelage important de la chromatine.Le laboratoire d’accueil de ce doctorat s’intéresse à l’identification de nouveaux régulateurs et biomarqueurs de la sénescence cellulaire. Des résultats récents ont permis de mettre en évidence l’accumulation spécifique d’un variant d’histone, H2A.J, en sénescence induite par des dommages à l’ADN et de montrer son implication dans l’expression de gènes pro-inflammatoires.Le but de ce projet de doctorat est de comprendre les mécanismes par lesquels l’accumulation de H2A.J favorise l’expression de gènes pro-inflammatoires ainsi que d’identifier de nouvelles fonctions physiologiques ou organe/tissu/cellule spécifique de H2A.J.Nous avons montré que l’accumulation de H2A.J contribue à affaiblir l’association de l’histone de liaison H1 avec la chromatin. La perte de H1 en sénescence a été corrélée avec une augmentation de l’expression de séquences ADN répétées et une activation de la voie interféron. De plus, cette perte de H1 en sénescence et ses conséquences ont pu être partiellement compensées par la déplétion de H2A.J par interférence ARN. Une analyse mutationnelle nous a également permis de montrer l’importance fonctionnelle des spécificités de séquence de H2A.J : une modération de son activité par la Val-11 et une potentiation par la phosphorylation de la Ser-123.Par ailleurs, un modèle de souris H2A.J-KO a été développé et des résultats préliminaires indiquent une potentielle implication de H2A.J dans la prédisposition à l’obésité et les pathologies associées (diabète, inflammation chronique, etc.) ainsi que dans l’érythropoïèse

Analyse fonctionnelle du variant d’histone H2A.J impliqué dans le développement d’un phénotype pro-inflammatoire en sénescence induite par des dommages à l’ADN

Cellular senescence is a stress response characterized by a stable proliferative arrest accompanied by, among others, the secretion of pro-inflammatory factors constituting what is called the SASP and an important chromatin remodelling.This PhD hosting laboratory is interested in the identification of novel regulators and biomarkers of cellular senescence. Recent data showed a specific accumulation of the histone variant H2A.J in DNA damage induced senescence and its implication in the expression of pro-inflammatory genes.This PhD project is aiming to understand the mechanisms by which H2A.J accumulation promotes the expression of pro-inflammatory genes and to identify novel physiological or organ/tissue/cell functions for H2A.J.We showed that H2A.J accumulation contributes to weakening the association of linker histone H1 to chromatin. Decreased H1 in senescence was correlated with increased expression of some repeated DNA sequences and activation of the interferon signalling pathway. Moreover, this H1 loss in senescence and its consequences could be partially recovered by H2A.J depletion by RNA interference. A mutational analysis also showed the functional importance of H2A.J sequence specificities: a moderation of its activity by the Val-11 and a potentiation by the phosphorylation of the Ser-123.Additionally, we developed a H2A.J-KO mouse model and preliminary results indicate a potential implication of H2A.J in the predisposition to obesity and the associated pathologies (diabete, chronic inflammation, etc.) and in erythropoïesis.La sénescence cellulaire est une réponse au stress des cellules caractérisée par un arrêt prolifératif stable accompagné, en outre, de la sécrétion de facteurs pro-inflammatoires constituant ce que l’on appelle le SASP ainsi que d’un remodelage important de la chromatine.Le laboratoire d’accueil de ce doctorat s’intéresse à l’identification de nouveaux régulateurs et biomarqueurs de la sénescence cellulaire. Des résultats récents ont permis de mettre en évidence l’accumulation spécifique d’un variant d’histone, H2A.J, en sénescence induite par des dommages à l’ADN et de montrer son implication dans l’expression de gènes pro-inflammatoires.Le but de ce projet de doctorat est de comprendre les mécanismes par lesquels l’accumulation de H2A.J favorise l’expression de gènes pro-inflammatoires ainsi que d’identifier de nouvelles fonctions physiologiques ou organe/tissu/cellule spécifique de H2A.J.Nous avons montré que l’accumulation de H2A.J contribue à affaiblir l’association de l’histone de liaison H1 avec la chromatin. La perte de H1 en sénescence a été corrélée avec une augmentation de l’expression de séquences ADN répétées et une activation de la voie interféron. De plus, cette perte de H1 en sénescence et ses conséquences ont pu être partiellement compensées par la déplétion de H2A.J par interférence ARN. Une analyse mutationnelle nous a également permis de montrer l’importance fonctionnelle des spécificités de séquence de H2A.J : une modération de son activité par la Val-11 et une potentiation par la phosphorylation de la Ser-123.Par ailleurs, un modèle de souris H2A.J-KO a été développé et des résultats préliminaires indiquent une potentielle implication de H2A.J dans la prédisposition à l’obésité et les pathologies associées (diabète, inflammation chronique, etc.) ainsi que dans l’érythropoïèse

Synergistic Anti-Tumor Effect of Simvastatin Combined to Chemotherapy in Osteosarcoma

International audienceContext: Osteosarcoma is the most common primary solid malignancy of the bone, mainly affecting pediatric patients. The main clinical issues are chemoresistance and metastatic spread, leading to a survival rate stagnating around 60% for four decades. Purpose: Here, we investigated the effect of simvastatin as adjuvant therapy on chemotherapy. Methods: Cell viability was assessed by the MTT test, and a combination index was evaluated by an isobologram approach. Cell motility was assessed by wound-healing assay. Cell-derived xenograft models were established in mice. FFPE tumor samples were assessed by immunohistochemistry. Results: In vitro experiments indicate that simvastatin synergized the conventional chemotherapy drugs’ inhibitory effect on cell viability. Functional assays reveal that simvastatin supplementation favored the anticancer mechanism of action of the tested chemotherapy drugs, such as DNA damage through intercalation or direct alkylation and disorganization of microtubules. Additionally, we show that even though simvastatin alone did not modify tumor behavior, it potentiated the inhibitory effect of doxorubicin on primary tumor growth (+50%, p < 0.05) and metastatic spread (+50%, p < 0.05). Our results provide evidence that simvastatin exerted an anti-tumor effect combined with chemotherapy in the preclinical murine model and represents valuable alternative adjuvant therapy that needs further investigation in clinical trials

MT2A is an early predictive biomarker of response to chemotherapy and a potential therapeutic target in osteosarcoma

International audienceOsteosarcoma is the most prevalent primary bone malignancy in children and young adults. Resistance to chemotherapy remains a key challenge for effective treatment of patients with osteosarcoma. The aim of the present study was to investigate the preventive role of metallothionein-2A (MT2A) in response to cytotoxic effects of chemotherapy. A panel of human and murine osteosarcoma cell lines, modified for MT2A were evaluated for cell viability, and motility (wound healing assay). Cell-derived xenograft models were established in mice. FFPE tumour samples were assessed by IHC. In vitro experiments indicated a positive correlation between half-maximal inhibitory concentration (IC50) for drugs in clinical practice, and MT2A mRNA level. This reinforced our previously reported correlation between MT2A mRNA level in tumour samples at diagnosis and overall survival in patients with osteosarcoma. In addition, MT2A/MT2 silencing using shRNA strategy led to a marked reduction of IC50 values and to enhanced cytotoxic effect of chemotherapy on primary tumour. Our results show that MT2A level could be used as a predictive biomarker of resistance to chemotherapy, and provide with preclinical rational for MT2A targeting as a therapeutic strategy for enhancing anti-tumour treatment of innate chemo-resistant osteosarcoma cells

Reduced RNA turnover as a driver of cellular senescence

International audienceAccumulation of senescent cells is an important contributor to chronic inflammation upon aging. The inflammatory phenotype of senescent cells was previously shown to be driven by cytoplasmic DNA. Here, we propose that cytoplasmic double-stranded RNA has a similar effect. We find that several cell types driven into senescence by different routes share an accumulation of long promoter RNAs and 3′ gene extensions rich in retrotransposon sequences. Accordingly, these cells display increased expression of genes involved in response to double stranded RNA of viral origin downstream of the interferon pathway. The RNA accumulation is associated with evidence of reduced RNA turnover, including in some cases, reduced expression of RNA exosome subunits. Reciprocally, depletion of RNA exosome subunit EXOSC3 accelerated expression of multiple senescence markers. A senescence-like RNA accumulation was also observed in cells exposed to oxidative stress, an important trigger of cellular senescence. Altogether, we propose that in a subset of senescent cells, repeat-containing transcripts stabilized by oxidative stress or reduced RNA exosome activity participate in driving and maintaining the permanent inflammatory state characterizing cellular senescence