When our genome is targeted by pathogenic bacteria.

Eukaryotic cells repair thousands of lesions arising in the genome at each cell cycle. The most hazardous damage is likely DNA double-strand breaks (DSB) that cleave the double helix backbone. DSBs occur naturally during T cell receptor and immunoglobulin gene recombination in lymphocytes. DSBs can also arise as a consequence of exogenous stresses (e.g., ionizing irradiation, chemotherapeutic drugs, viruses) or oxidative processes. An increasing number of studies have reported that infection with pathogenic bacteria also alters the host genome, producing DSB and other modifications on DNA. This review focuses on recent data on bacteria-induced DNA damage and the known strategies used by these pathogens to maintain a physiological niche in the host. Even after DNA repair in infected cells, "scars" often remain on chromosomes and might generate genomic instability at the next cell division. Chronic inflammation in tissue, combined with infection and DNA damage, can give rise to genomic instability and eventually cancer. A functional link between the DNA damage response and the innate immune response has been recently established. Pathogenic bacteria also highjack the host cell cycle, often acting on the stability of the master regulator p53, or dampen the DNA damage response to support bacterial replication in an appropriate reservoir. Except in a few cases, the molecular mechanisms responsible for DNA lesions are poorly understood, although ROS release during infection is a serious candidate for generating DNA breaks. Thus, chronic or repetitive infections with genotoxic bacteria represent a common source of DNA lesions that compromise host genome integrity.

A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation.

A general radioprotective effect by fibroblast growth factor (FGF) has been extensively described since the early 1990s; however, the molecular mechanisms involved remain largely unknown. Radiation-induced DNA double-strand breaks (DSBs) lead to a complex set of responses in eukaryotic cells. One of the earliest consequences is phosphorylation of histone H2AX to form nuclear foci of the phosphorylated form of H2AX (γH2AX) in the chromatin adjacent to sites of DSBs and to initiate the recruitment of DNA-repair molecules. Upon a DSB event, a rapid signaling network is activated to coordinate DNA repair with the induction of cell-cycle checkpoints. To date, three kinases (ATM, ATR, and DNA-PK) have been shown to phosphorylate histone H2AX in response to irradiation. Here, we report a kinome-targeted small interfering RNA (siRNA) screen to characterize human kinases involved in H2AX phosphorylation. By analyzing γH2AX foci at a single-nucleus level, we identified 46 kinases involved either directly or indirectly in H2AX phosphorylation in response to irradiation in human keratinocytes. Furthermore, we demonstrate that in response to irradiation, the FGFR4 signaling cascade promotes JNK1 activation and direct H2AX phosphorylation leading, in turn, to more efficient DNA repair. This can explain, at least partially, the radioprotective effect of FGF.

[Bacterial infections as seen from the eukaryotic genome: DNA double strand breaks, inflammation and cancer]. / Les infections bactériennes vues du génome eucaryote - cassures double-brin, inflammation et cancer.

An increasing number of studies report that infection by pathogenic bacteria alters the host genome, producing highly hazardous DNA double strand breaks for the eukaryotic cell. Even when DNA repair occurs, it often leaves "scars" on chromosomes that might generate genomic instability at the next cell division. Chronic intestinal inflammation promotes the expansion of genotoxic bacteria in the intestinal microbiote which in turn triggers tumor formation and colon carcinomas. Bacteria act at the level of the host DNA repair machinery. They also highjack the host cell cycle to allow themselves time for replication in an appropriate reservoir. However, except in the case of bacteria carrying the CDT nuclease, the molecular mechanisms responsible for DNA lesions are not well understood, even if reactive oxygen species released during infection make good candidates.

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells.

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

Identification of three tomato flower and fruit MADS-box proteins with a putative histone deacetylase binding domain.

MADS-box transcription factors play crucial roles in organ and cell differentiation in organisms ranging from yeast to humans. Most of the work on plant MADS-box proteins focused on their roles in floral development whereas less information is available on their function in fruit maturation. We cloned three distinct tomato cDNAs using a RT-PCR approach, encoding LeMADS1, LeMADS5 and LeMADS6 factors and whose mRNAs mostly accumulate in tomato flowers and fruits. Phylogeny analysis indicates that LeMADS1, 5 and 6 belong to the MEF2-like family. When transiently expressed in tobacco leaves or in human cells, LeMADS1, 5 and 6 are targeted to the cell nucleus. As the endogenous target genes of these putative transcription factors are unknown, the transcriptional activity of these proteins was characterized in a heterologous system and we showed that, when fused to a Gal4-DNA-binding domain, they repress the transcription of heterologous reporter genes. Since histone deacetylases control MEF2 transcriptional activity and since a putative histone deacetylase binding site was present in LeMADS1, 5 and 6, we tested the potential interaction between these factors and HDAC5 deacetylase. Surprisingly, in this heterologous system, LeMADS1, 5 and 6 interacted with HDAC5 N-terminal region. Our data suggest that, like mammalian MEF2A, plant MADS-box transcriptional activity might be regulated by enzymes controlling chromatin acetylation.

Dual roles for MEF2A and MEF2D during human macrophage terminal differentiation and c-Jun expression.

Recent reports have evidenced a role for MEF2C (myocyte enhancer factor 2C) in myelopoiesis, although the precise functions of this transcription factor are still unclear. We show in the present study that MEF2A and MEF2D, two other MEF2 family members, are expressed in human primary monocytes and in higher amounts in monocyte-derived macrophages. High levels of MEF2A-MEF2D heterodimers are found in macrophage-differentiated HL60 cells. Chromatin immunoprecipitations demonstrate that MEF2A is present on the c-Jun promoter, both in undifferentiated and in macrophage-differentiated cells. Moreover, c-Jun expression is derepressed in undifferentiated cells in the presence of HDAC (histone deacetylase) inhibitor, indicating the importance of chromatin acetylation in this process. We show that MEF2A/D dimers strongly interact with HDAC1, and to a lesser extent with HDAC7 in macrophages, whereas low levels of MEF2A/D-HDAC1 complexes are found in undifferentiated cells or in monocytes. Since trichostatin A does not disrupt MEF2A/D-HDAC1 complexes, we analysed the potential interaction of MEF2A with p300 histone acetyltransferase, whose expression is up-regulated in macrophages. Interestingly, endogenous p300 only associates with MEF2A in differentiated macrophages, indicating that MEF2A/D could activate c-Jun expression in macrophages through a MEF2A/D-p300 activator complex. The targets of MEF2A/D-HDAC1-HDAC7 multimers remain to be identified. Nevertheless, these data highlight for the first time the possible dual roles of MEF2A and MEF2D in human macrophages, as activators or as repressors of gene transcription.

HOX11L2/TLX3 is transcriptionally activated through T-cell regulatory elements downstream of BCL11B as a result of the t(5;14)(q35;q32).

The t(5;14)(q35;q32) chromosomal translocation is specifically observed in up to 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL). It affects the BCL11B/CTIP2 locus on chromosome 14 and the RANBP17-TLX3/HOX11L2 region on chromosome 5. It leads to ectopic activation of TLX3/HOX11L2. To investigate the reasons of the association between t(5;14) and T-ALL, we isolated the translocation breakpoints in 8 t(5;14) patients. Sequence analyses did not involve recombinase activity in the genesis of the translocation. We used DNAse1 hypersensitive experiments to locate transcriptional regulatory elements downstream of BCL11B. By transient transfection experiments, 2 of the 6 regions demonstrated cis-activation properties in T cells and were also effective on the TLX3 promoter. Our data indicate that the basis of the specific association between t(5;14) and T-ALL lies on the juxtaposition of TLX3 to long-range cis-activating regions active during T-cell differentiation.

Monitoring the interaction between DNA and a transcription factor (MEF2A) using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is an analytical method that allows distinguishing different populations of fluorescent probes in solution and provides data on their concentrations and their diffusion coefficients. FCS was used to characterize the interaction of the transcription factor (MEF2A) with its DNA target sequence. The myocyte enhancer factor 2 (MEF2) belongs to the MADS-box family and activates transcription of numerous muscle genes during myogenesis. Measurements were made using TAMRA-labelled oligonucleotide duplexes derived from a wild type (WT) or a mutated MEF2 target gene. Binding of the protein to the WT DNA resulted in significant changes of the diffusion. Specificity of the interaction was confirmed using the mutated DNA. Bound to free probe ratios were determined at different MEF2A concentrations and the apparent equilibrium dissociation constant K(D) for the full-length MEF2A was estimated.

Tip60 acetyltransferase activity is controlled by phosphorylation.

Here we show that the phosphorylation of histone acetyltransferase Tip60, a target of human immunodeficiency virus, type 1-encoded transactivator Tat, plays a crucial role in the control of its catalytic activity. Baculovirus-based expression and purification of Tip60 combined with mass spectrometry allowed the identification of serines 86 and 90 as two major sites of phosphorylation in vivo. The phosphorylation of Tip60 was found to modulate its histone acetyltransferase activity. One of the identified phosphorylated serines, Ser-90, was within a consensus cyclin B/Cdc2 site. Ser-90 was specifically phosphorylated in vitro by the cyclin B/Cdc2 complex. Accordingly, the phosphorylation of Tip60 was enhanced after drug-induced arrest of cells in G(2)/M. This G(2)/M-dependent phosphorylation of Tip60 was abolished by treating cells with a specific inhibitor of the cyclin-dependent kinase, roscovitin. All together, these results strongly suggest a G(2)/M-dependent control of Tip60 activity.

Point mutations in BCL6 DNA-binding domain reveal distinct roles for the six zinc fingers.

The B-cell lymphoma 6 (BCL6) gene encodes a transcriptional repressor containing six C-terminal Krüppel-like zinc fingers. The zinc finger (ZF) cluster is necessary and sufficient for interaction with both DNA and several proteins and for nuclear targeting. However, the functional specificity of the six ZFs in these cellular roles is unknown. To characterize this domain, we mutated individually each ZF of BCL6. Our results reveal that mutation of the two N-terminal ZFs does not impair cognate DNA-binding, cellular localization of the protein nor the transcriptional repression capacity of BCL6. By contrast, mutation of any of the remaining ZFs abolishes the binding of BCL6 to DNA in vitro and the transrepressive function of the protein in vivo. Finally, none of the six mutations affect the interaction between BCL6 and class II histone deacetylases. Thus our experiments demonstrate that BCL6 uses each of the four C-terminus ZFs for binding to a target sequence while the two amino terminal fingers are likely engaged in other unknown function(s).

Tip60 is targeted to proteasome-mediated degradation by Mdm2 and accumulates after UV irradiation.

Acetylation is a prominent post-translational modification of nucleosomal histone N-terminal tails, which regulates chromatin accessibility. Accordingly, histone acetyltransferases (HATs) play major roles in processes such as transcription. Here, we show that the HAT Tip60, which is involved in DNA repair and apoptosis following gamma irradiation, is subjected to proteasome-dependent proteolysis. Furthermore, we provide evidence that Mdm2, the ubiquitin ligase of the p53 tumour suppressor, interacts physically with Tip60 and induces its ubiquitylation and proteasome-dependent degradation. Moreover, a ubiquitin ligase-defective mutant of Mdm2 had no effect on Tip60 stability. Our results indicate that Mdm2 targets both p53 and Tip60, suggesting that these two proteins could be co-regulated with respect to protein stability. Consistent with this hypothesis, Tip60 levels increased significantly upon UV irradiation of Jurkat cells. Collectively, our results suggest that degradation of Tip60 could be part of the mechanism leading to cell transformation by Mdm2.

Class II histone deacetylases are directly recruited by BCL6 transcriptional repressor.

BCL6 is a member of the POZ/zinc finger (POK) family involved in survival and/or differentiation of a number of cell types and in B cell lymphoma upon chromosomal alteration. Transcriptional repression by BCL6 is thought to be achieved in part by recruiting a repressor complex containing two class I histone deacetylases (HDACs). In this study we investigated whether BCL6 could also target members of class II HDACs. Our results indicate that three related class II deacetylases, HDAC4, HDAC5, and HDAC7 can associate with BCL6 in vivo and in vitro. Using electron microscopy, we found that endogenous BCL6 and class II HDACs partially co-localize in the nucleus. Overexpression experiments showed that BCL6 and HDAC4, -5, or -7 are intermingled onto common nuclear substructures and form stable complexes. A highly conserved domain in the N-terminal region of HDAC5 and HDAC7 as well as the zinc finger region of BCL6 were found necessary for the complex formation in vivo and in vitro. Moreover, our data point to the zinc finger region of BCL6 as a multifunctional domain which, beside its known capacity to bind DNA, is involved in the nuclear targeting of the protein and in the recruitment of the class II HDACs, and hence constitutes an autonomous repressor domain. Since PLZF, a BCL6 relative, could also interact with HDAC4, -5, and 7, we suggest that class II HDACs are largely involved in the control of the POK transcription factors activity.