Search

International audience; Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.

International audience; Promoter methylation of the MGMT gene, encoding the enzyme O6-methylguanine-ubiquitous methyltransferase, is a theranostic good prognosis marker of glioblastomas treated with alkylating chemotherapy (temozolomide, Temodal(®)). Among the methylation analysis techniques, pyrosequencing is a reproducible and sensitive quantitative method. As part of the accreditation of the hospital platform of molecular genetics of cancer, Besançon, our objective was to verify the performance of the pyrosequencing commercial kit therascreen(®) MGMT Pyro(®) (Qiagen) in terms of repeatability, reproducibility, limit of blank (LOB), limit of detection (LOD), linearity and contamination by the guide SH GTA 04 delivered by the Cofrac. The repeatability tests show an average methylation of 3.22% [standard deviation (SD) = 0.41, coefficient of variation (CV) = 12.75%] for the unmethylated control and 70.16% (SD = 2.20, CV = 3.14%) for the methylated control. Reproducibility demontrates an average methylation of 1.39% (SD = 0.25, CV = 18.25%) for the unmethylated control and of 94.03% (SD = 2.56, CV = 2.73%) for the methylated control. The percentages of LOB and LOD are respectively 3.43% and 6.22% methylation. The regression coefficient of 0,983 confirms the linearity of the assay from 0% to 100% methylation. No contamination has been observed. Over 40% of glioblastomas studied in 2013 in our laboratory have shown a methylated MGMT gene. Our results confirms that the theraScreen(®) MGMT Pyro(®) kit (Qiagen) is performant in compliance with the quality requirements of the NF EN ISO 15189 for the routine analysis of methylation status of MGMT in glioblastomas.

Multiple myeloma is a plasma cell cancer with poor survival, characterized by the clonal expansion of multiple myeloma cells (MMC), primarily in the bone marrow. Novel compounds are currently tested in this disease, but partial or minor patients' responses are observed for most compounds used as a single agent. The design of predictors for drug efficacy could be most useful to better understand basic mechanisms targeted by these drugs and design clinical trials. In the current study, we report the building of a DNA methylation score (DM score) predicting the efficacy of decitabine, an inhibitor of DNA methyltransferase (DNMT), targeting methylation-regulated gene expression. DM score was built by identifying 47 genes regulated by decitabine in human myeloma cell lines and the expression of which in primary MMCs of previously untreated patients is predictive for overall survival. A high DM score predicts patients' poor survival, and, of major interest, high sensitivity of primary MMCs or human myeloma cell lines to decitabine in vitro. Thus, DM score could be useful to design novel treatments with DMNT inhibitor in multiple myeloma and has highlighted 47 genes, the gene products of which could be important for multiple myeloma disease development. Mol Cancer Ther; 11(12); 2685–92. ©2012 AACR.

International audience; Epigenetics signifies stable and heritable changes in gene expression without changes in the genetic code. There is a wealth of emerging evidence for such processes in promoting autoimmunity. The first clue is that inhibition of DNA methyl transferases (DNMTs) induces systemic lupus erythematosus (SLE) in animals. Similar immune-mediated disorders have been generated by injecting normal T cells incubated with DNMT inhibitors into healthy mice. Further, monozygotic twins display differences in DNA methylation that parallel discordances in SLE. Moreover, defects in DNA methylation characterize lymphocytes from SLE, synoviocytes from rheumatoid arthritis, and neural cells from multiple sclerosis patients. It has also been shown that DNA hypomethylation of T and B cells correlates with reduced DNMT efficacy and histone acetylation in SLE. Once a gene promoter has been demethylated, the gene recovers its capacity to be transcribed, e.g., genes for cytokines, activation receptors on cells, and endogenous retroviruses. This outcome has been associated with a blockage of the Erk pathway and/or a growth arrest at the G0/G1 interface of the cell cycle. Of importance is the fact that these changes can be reversed. For example, blockade of the interleukin-6 autocrine loop in SLE B cells restores DNA methylation status, thus opening new perspectives for therapy.

International audience; Major histocompatibility complex (MHC) class I-specific inhibitory natural killer receptors (iNKRs) are expressed by subsets of T cells but the mechanisms inducing their expression are poorly understood, particularly for killer-cell immunoglobulin-like receptors (KIRs). The iNKRs are virtually absent from the surface of cord blood T cells but we found that KIR expression could be induced upon interleukin-2 stimulation in vitro. In addition, KIR expression was enhanced after treatment with 5-aza-2'-deoxycytidine, suggesting a role for DNA methylation. In vivo induction of KIR expression on cord blood T cells was also observed during a human congenital infection with Trypanosoma cruzi which triggers activation of fetal CD8(+) T cells. These KIR(+) T cells had an effector and effector/memory phenotype suggesting that KIR expression was consecutive to the antigenic stimulation; however, KIR was not preferentially found on parasite-specific CD8(+) T cells secreting interferon-gamma upon in vitro restimulation with live T. cruzi. These findings show that KIR expression is likely regulated by epigenetic mechanisms that occur during the maturation process of cord blood T cells. Our data provide a molecular basis for the appearance of KIRs on T cells with age and they have implications for T-cell homeostasis and the regulation of T-cell-mediated immune responses.

Prokaryote DNA methyltransferases (MTases) of the Dam family (including those of bacteriophages T2 and T4) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. Dam DNA MTases, as all site-specific enzymes interacting with polymeric DNA, require a mechanism of action that ensures a rapid search for specific targets for catalytic action, during both the initial and subsequent rounds of methylation. The results of pre-steady-state (reaction burst) and steady-state methylation analyses of individual targets permitted us to monitor the action of T4Dam, which has three degrees of freedom: sliding, reorientation and adaptation to the canonical GATC sequence. The salient results are as follows: (i) 40mer substrate duplexes containing two canonical GATC sites showed differential methylation of the potential targets, i.e., T4Dam exhibited a preference for one site/target, which may present the better ‘kinetic trap’ for the enzyme. (ii) Prior hemimethylation of the two sites made both targets equally capable of being methylated during the pre-steady-state reaction. (iii) Although capable of moving in either direction along double-stranded DNA, there are some restrictions on T4Dam reorientation/adaptation on 40mer duplexes.