Kinetics of DNA methylation inheritance by the Dnmt1-including complexes during the cell cycle

<p>Abstract</p> <p>Background</p> <p>The clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during the cellular division is a crucial biological process controlled by the DNA methyltransferase Dnmt1, mainly. To investigate possible dynamic mechanisms of DNA methylation inheritance during the cell cycle, we used a Proximity Ligation <it>In Situ </it>Assay (P-LISA) to analyze the kinetic of formation and DNA recruitment of Dnmt1-including complexes.</p> <p>Results</p> <p>P-LISA, sequential chromatin immunoprecipitation and quantitative methylation specific PCR revealed that the Dnmt1/PCNA/UHRF1-including complexes are mainly formed and recruited on DNA during the S-phase of cell cycle, while the formation and the DNA recruitment of several Dnmt1/transcription factors-including complexes are not S-phase dependent but are G0/G1 and/or G2/M phases dependent.</p> <p>Conclusion</p> <p>Our data confirm that DNA methylation inheritance occurs in S-phase, and demonstrate that DNA methylation inheritance can also occur in G0/G1 and G2/M phases of the cell cycle.</p

Impact of pH on Bax α conformation, oligomerisation and mitochondrial integration

AbstractThe change in the conformation of Bax at the onset of apoptosis is a determinant for the execution of this cell death programme. However, very few models can account for this modification and the factors involved in this process remain elusive. We have analysed the modifications in the conformation induced by a variation in pH using a cell-free assay. We show that a moderate basic or acidic pH can induce apoptotic-like changes in the conformation of Bax, such as the exposure of the N-terminal or the BH3 domain. These changes in the conformation are associated with the binding of Bax to mitochondria and an enhanced Bax homo- and oligomerisation. Our results suggest that variations in the pH, in a range consistent with that often observed during apoptosis, are sufficient to trigger Bax translocation to mitochondria and the subsequent release of apoptogenic factors from this organelle

Opposite role of Bax and BCL-2 in the anti-tumoral responses of the immune system

BACKGROUND: The relative role of anti apoptotic (i.e. Bcl-2) or pro-apoptotic (e.g. Bax) proteins in tumor progression is still not completely understood. METHODS: The rat glioma cell line A15A5 was stably transfected with human Bcl-2 and Bax transgenes and the viability of theses cell lines was analyzed in vitro and in vivo. RESULTS: In vitro, the transfected cell lines (huBax A15A5 and huBcl-2 A15A5) exhibited different sensitivities toward apoptotic stimuli. huBax A15A5 cells were more sensitive and huBcl-2 A15A5 cells more resistant to apoptosis than mock-transfected A15A5 cells (pCMV A15A5). However, in vivo, in syngenic rat BDIX, these cell lines behaved differently, as no tumor growth was observed with huBax A15A5 cells while huBcl-2 A15A5 cells formed large tumors. The immune system appeared to be involved in the rejection of huBax A15A5 cells since i) huBax A15A5 cells were tumorogenic in nude mice, ii) an accumulation of CD8+ T-lymphocytes was observed at the site of injection of huBax A15A5 cells and iii) BDIX rats, which had received huBax A15A5 cells developed an immune protection against pCMV A15A5 and huBcl-2 A15A5 cells. CONCLUSIONS: We show that the expression of Bax and Bcl-2 controls the sensitivity of the cancer cells toward the immune system. This sensitization is most likely to be due to an increase in immune induced cell death and/or the amplification of an anti tumour immune respons

Disruption of Dnmt1/PCNA/UHRF1 Interactions Promotes Tumorigenesis from Human and Mice Glial Cells

Global DNA hypomethylation is a hallmark of cancer cells, but its molecular mechanisms have not been elucidated. Here, we show that the disruption of Dnmt1/PCNA/UHRF1 interactions promotes a global DNA hypomethylation in human gliomas. We then demonstrate that the Dnmt1 phosphorylations by Akt and/or PKC abrogate the interactions of Dnmt1 with PCNA and UHRF1 in cellular and acelluar studies including mass spectrometric analyses and the use of primary cultured patient-derived glioma. By using methylated DNA immunoprecipitation, methylation and CGH arrays, we show that global DNA hypomethylation is associated with genes hypomethylation, hypomethylation of DNA repeat element and chromosomal instability. Our results reveal that the disruption of Dnmt1/PCNA/UHRF1 interactions acts as an oncogenic event and that one of its signatures (i.e. the low level of mMTase activity) is a molecular biomarker associated with a poor prognosis in GBM patients. We identify the genetic and epigenetic alterations which collectively promote the acquisition of tumor/glioma traits by human astrocytes and glial progenitor cells as that promoting high proliferation and apoptosis evasion

Specific inhibition of DNMT1/CFP1 reduces cancer phenotypes and enhances chemotherapy effectiveness

International audienceAim: DNA methylation is a fundamental biologic process of genomes and is a candidate for pharmacological manipulation that might have important therapeutic advantages. Thus, DNA methyltransferases (DNMTs) appear to be ideal targets for drug intervention. Materials & methods: To develop a new generation of DNMT inhibitor, we analyzed the ability of peptides to selectively inhibit certain DNMT1-incuding complexes. Results: Our study demonstrates that the disruption of DNMT1/CFP1-including complexes increases the efficiency of chemotherapeutic treatment on established tumors in mice. Conclusion: Our data opens a promising and innovative alternative to the development of DNMT inhibitor

Diuron exposure and Akt overexpression promote glioma formation through DNA hypomethylation

International audienceBackground: Diuron is an environmental component listed as a likely human carcinogen. Several other studies report that diuron can be oncogenic for bladder, urothelial, skin, and mammary cells. No study mentions the putative effect of diuron on the glioma occurrence. Objectives: We here wanted to investigate the effects of diuron exposure on the glioma occurrence while wishing to incriminate a putative implication of DNA methylation modulation in this process. Methods: In in vivo model of glioma, diuron exposure was firstly compared or combined with oncogenic overexpressions already known to promote gliomagenesis. ELISA quantifying the 5-methylcytosine level on DNA was performed to examine the global DNA methylation level. Quantitative real-time polymerase chain reaction and proximity ligation in situ assay were performed to identify the molecular causes of the diuron-induced changes of DNA methylation. The signatures diuron-induced changes of DNA methylation were analyzed in a cohort of 23 GBM patients. Results: Diuron exposure is not sufficient to promote glioma, such as the oncogenic overexpression of Akt or Ras. However, the combination of diuron exposure and Akt overexpression promotes glioma. We observed that the diuron/Akt-induced glioma is characterized by three phenotypic signatures characterizing cancer cells: a global DNA hypomethylation, a loss of sensitivity to cell death induction, and a gain of signals of immune escape. Our data associated these phenotypes with three aberrant DNA methylation signatures: the LLT1, PD-L1, and Bcl-w hypomethylations. Strikingly, we observed that these three concomitant hypomethylations were only observed in GBM patients having a potential exposure to diuron via their professional activity. Conclusions: As single player, diuron is not an oncogenic of glioma, but it can participate to the glioma formation in association with other events (also devoid of oncogenic property as single player) such as Akt overexpression

The phosphorylation of Metaxin 1 controls Bak activation during TNFα induced cell death

International audienceThe proapoptotic protein Bak is implicated in the execution phase of apoptosis, a cell death program. Bak is essentially mitochondrial and during early steps of apoptosis undergoes conformational changes that lead to its full membrane integration in mitochondria and the subsequent liberation of pro-apoptotic mitochondrial proteins. Little is known about the partners and mechanisms implicated in the activation of Bak. We have recently shown that Bak is incorporated into a Voltage dependent anionic channel of type 2 (VDAC2)/Metaxin 1(Mtx1)/Metaxin 2 (Mtx2) multi-protein complex in both resting and dying cells. Here, we show that, after the induction of apoptosis, Bak switches from its association with Mtx2 and VDAC2 to a closer association with Mtx1. This change of partners is under the control of a tyrosine phosphorylation of Mtx1 by c-Abl

Dnmt1/Transcription Factor Interactions: An Alternative Mechanism of DNA Methylation Inheritance

DNA methylation inheritance is the process of copying, via the DNA methyltransferase 1 (Dnmt1), the pre-existing methylation patterns onto the new DNA strand during DNA replication. Experiments of chromatin immunoprecipitation, measurement of maintenance methyltransferase activity, proximity ligation in situ assays (P-LISA, Duolink/Olink), and transcription factor arrays demonstrate that Dnmt1 interacts with transcription factors to promote site-specific DNA methylation inheritance, while the Dnmt1-PCNA-UHRF1 complex promotes the DNA methylation inheritance without site preference. We also show that the Dnmt1-PCNA-UHRF1 and Dnmt1/transcription factor complexes methylate DNA by acting as a single player or in cooperation. Thus, our data establish that the copying of the pre-existing methylation pattern is governed by the orchestration of the untargeted and the targeted mechanisms of DNA methylation inheritance, which are themselves dictated by the partners of Dnmt1

Méthylation/déméthylation de l’ADN et expression du génome

National audienceMethylation/demethylation phenomenons in DNA are master pieces of the maintenance and regulation of the genome. These chemical modifications which do not alter the natural genomic sequence, are mediated by many players. Cytosines within CpG dinucleotides are generally the target of these changes. DNA methylation (by adding a CH3 group in position 5 of the pyrimidine ring) involves DNA methyltransferases (DNMTs) that do not have all the same role. This methylation is not an aleatory mechanism and will involve several parameters. Intrinsic properties of DNMTs, interaction partners of these (RNAs, transcription factor ...) and the direct environment of the DNA (chromatin state, ...) will therefore influence the methylation system. But in parallel to this, there are also demethylation processes. They are usually either passive or active. They will involve new actors such as Ten-Eleven-Translocation proteins (TETs), resulting in new chemical modification of cytosine (e.g., hydroxylation of the methyl group, formylation, carboxylation ...). Many different proteins interact together and affect the demethylation mechanism of DNA. Combined together these mechanisms will have an impact on both non-coding regions of DNA and the coding regions. The consequence is a fine regulation of genomic stability but also of the expression or not of genes. In this review, we will reference and discuss the various aspects of methylation/demethylation of DNALes phénomènes de méthylation/déméthylation de l’ADN sont des pièces maîtresses du maintien et de la régulation du génome. Ces modifications chimiques, qui n’altèrent en rien la séquence génomique propre, vont être médiées par de nombreux acteurs. Les cytosines au sein de dinucléotides CpG sont en règle générale la cible de ces modifications. La méthylation de l’ADN (par ajout d’un groupement CH3 en position 5 du cycle pyrimidique) fait intervenir des DNA méthyltransférases (DNMTs) qui n’auront pas toutes le même rôle. Cette méthylation n’est pas aléatoire et va faire intervenir plusieurs paramètres. Les propriétés intrinsèques des DNMTs, les partenaires d’interactions de ces dernières (ARNs, facteur de transcription,…) et l’environnement direct de l’ADN (état chromatinien,…) vont donc influencer les mécanismes de méthylation. Mais en parallèle de cela, il existe aussi des processus de déméthylation. Ils sont en général soit passifs, soit actifs. Ils feront intervenir de nouveaux acteurs comme les protéines ten-eleven-translocation (TETs), entraînant de nouvelles modifications chimiques de la cytosine (par exemple, l’hydroxylation du groupement méthyle, formylation, carboxylation,…). Là aussi, de nombreux partenaires de natures diverses vont interagir ensemble et influer sur la déméthylation de l’ADN. Tout cela va avoir un impact aussi bien sur des régions non codantes de l’ADN, que sur des régions codantes. La conséquence est une régulation fine de la stabilité du génome mais aussi de l’expression ou non des gènes qui le compose. Dans cette revue, nous allons référencer et discuter ces différents aspects de la méthylation/déméthylation de l’ADN

Proximity ligation in situ assay for monitoring the global DNA methylation in cells.

International audienceABSTRACT: BACKGROUND: DNA methylation has a central role in the epigenetic control of mammalian gene expression, and is required for X inactivation, genomics imprinting and silencing of retrotransposons and repetitive sequences. Thus, several technologies have been developed to measure the degree of DNA methylation. RESULTS: We here present the development of the detection of protein-protein interactions via the adaptation of the proximity ligation in situ technology to evaluate the DNA methylation status in cells since the quantification of Dnmt1/PCNA interaction in cells reflects the degree of DNA methylation. CONCLUSION: This method being directly realizable on cells, it appears that it could suggest a wide range of applications in basic research and drug development. More particularly, this method is specially adapted for the investigations realized from a weak quantity of biologic materiel such as stem cells or primary cultured tumor cells for examples