Functional analysis of a conserved DNA methyltransferase in caulobacter crescentus

CcrM is a DNA methyltransferase that methylates the adenine in GANTC motifs in the chromo-some of the bacterial model Caulobacter crescentus. The loss of the CcrM homolog is lethal in C. crescentus and in several other species of Alphaproteobacteria. In this research, we used different experimental and bioinformatic approaches to determine why CcrM is so critical to the physiology of C. crescentus. We first showed that CcrM is a resident orphan DNA methyltransferase in non-Rickettsiales Alphaproteobacteria and that its gene is strictly conserved in this clade (with only one ex¬ception among the genomes sequenced so far). In C. crescentus, cells depleted in CcrM in rich medium quickly lose viability and present an elongated phenotype characteristic of an im¬pairment in cell division. Using minimal medium instead of rich medium as selective and main¬tenance substrate, we could generate a AccrM mutant that presents a viability comparable to the wild type strain and only mild morphological defects. On the basis of a transcriptomic ap¬proach, we determined that several genes essential for cell division were downregulated in the AccrM strain in minimal medium. We offered decisive arguments to support that the efficient transcription of two of these genes, ftsZ and mipZ, coding respectively for the Z-ring forming GTPase FtsZ and an inhibitor of FtsZ polymerization needed for the correct positioning of the Z- ring at mid-cell, requires the methylation of an adenine in a conserved GANTC motif located in their core promoter region. We propose a model, according to which the genome of C. crescentus encodes a transcriptional activator that requires a methylated adenine in a GANTC context to bind to DNA and suggest that this transcriptional regulator might be the global cell-cycle regulator GcrA. In addition, combining a classic genetic approach and in vitro evolution experiments, we showed that the mortality and cell division defects of the AccrM strain in rich medium are mainly due to limiting intracellular levels of the FtsZ protein. We also studied the dynamics of GANTC methylation in C. crescentus using the SMRT technol¬ogy developed by Pacific Biosciences. Our findings support the commonly accepted model, accord¬ing to which the methylation state of GANTC motifs varies during the cell cycle of C. crescentus: before the initiation of DNA replication, the GANTC motifs are fully-methylated (methylated on both strands); when the DNA gets replicated, the GANTC motifs become hemi-methylated (methyl¬ated on one strand only) and this occurs at different times during replication for different loci along the chromosome depending on their position relative to the origin of replication; the GANTC mo¬tifs are only remethylated after DNA replication has finished as a consequence of the massive and short-lived expression of CcrM in predivisional cells. About 30 GANTC motifs in the C. crescentus chromosome were found to be undermethylated in most of the bacterial population; these might be protected from CcrM activity by DNA binding proteins and some of them could be involved in methylation-based bistable transcriptional switches. - CcrM est une ADN méthyltransférase qui méthyle les adénines dans le contexte GANTC dans le génome de la bactérie modèle Caulobacter crescentus. La perte de l'homologue de CcrM chez C. crescentus et chez plusieurs autres espèces d'Alphaproteobactéries est létale. Dans le courant de cette recherche, nous tentons de déterminer pourquoi la protéine CcrM est cruciale pour la survie de C. crescentus. Nous démontrons d'abord que CcrM est une adénine méthyltransférase orpheline résidente, dont le gène fait partie du génome minimal partagé par les Alphaprotéobactéries non-Rickettsiales (à une exception près). Lorsqu'une souche de C. crescentus est privée de CcrM, sa viabilité décroît rapi¬dement et ses cellules présentent une morphologie allongée qui suggère que la division cellulaire est inhibée. Nous sommes parvenus à créer une souche AccrM en utilisant un milieu minimum, au lieu du milieu riche classiquement employé, comme milieu de sélection et de maintenance pour la souche. Lorsque nous avons étudié le transcriptome de cette souche de C. crescentus privée de CcrM, nous avons pu constater que plusieurs gènes essentiels pour le bon déroulement de la division cellulaire bactérienne étaient réprimés. En particulier, l'expression adéquate des gènes ftsZ et mipZ - qui codent, respectivement, pour FtsZ, la protéine qui constitue, au milieu de la cellule, un anneau protéique qui initie le processus de division et pour MipZ, un inhibiteur de la polymérisation de FtsZ qui est indispensable pour le bon positionnement de l'anneau FtsZ - est dépendante de la présence d'une adénine méthylée dans un motif GANTC conservé situé dans leur région promotrice. Nous présentons un modèle selon lequel le génome de C. crescentus code pour un facteur de transcription qui exige la présence d'une adénine méthylée dans un contexte GANTC pour s'attacher à l'ADN et nous suggérons qu'il pourrait s'agir du régulateur global du cycle cellulaire GcrA. En outre, nous montrons, en combinant la génétique classique et une approche basée sur l'évolution expérimentale, que la mortalité et l'inhibition de la division cellulaire caractéristiques de la souche àccrMeη milieu riche sont dues à des niveaux excessivement bas de protéine FtsZ. Nous avons aussi étudié la dynamique de la méthylation du chromosome de C. crescentus sur la base de la technologie SMRT développée par Pacific Biosciences. Nous confirmons le modèle communément accepté, qui affirme que l'état de méthylation des motifs GANTC change durant le cycle cellulaire de C. crescentus: les motifs GANTC sont complètement méthylés (méthylés sur les deux brins) avant de début de la réplication de l'ADN; ils deviennent hémi-méthylés (méthylés sur un brin seulement) une fois répliqués, ce qui arrive à différents moments durant la réplication pour différents sites le long du chromosome en fonction de leur position par rapport à l'origine de répli-cation; finalement, les motifs GANTC sont reméthylés après la fin de la réplication du chromosome lorsque la protéine CcrM est massivement, mais très transitoirement, produite. Par ailleurs, nous identifions dans le chromosome de C. crescentus environ 30 motifs GANTC qui restent en perma-nence non-méthylés dans une grande partie de la population bactérienne; ces motifs sont probable-ment protégés de l'action de CcrM par des protéines qui s'attachent à l'ADN et certains d'entre eux pourraient être impliqués dans des mécanismes de régulation générant une transcription bistable.

NCoR1 is a conserved physiological modulator of muscle mass and oxidative function.

Transcriptional coregulators control the activity of many transcription factors and are thought to have wide-ranging effects on gene expression patterns. We show here that muscle-specific loss of nuclear receptor corepressor 1 (NCoR1) in mice leads to enhanced exercise endurance due to an increase of both muscle mass and of mitochondrial number and activity. The activation of selected transcription factors that control muscle function, such as MEF2, PPARβ/δ, and ERRs, underpins these phenotypic alterations. NCoR1 levels are decreased in conditions that require fat oxidation, resetting transcriptional programs to boost oxidative metabolism. Knockdown of gei-8, the sole C. elegans NCoR homolog, also robustly increased muscle mitochondria and respiration, suggesting conservation of NCoR1 function. Collectively, our data suggest that NCoR1 plays an adaptive role in muscle physiology and that interference with NCoR1 action could be used to improve muscle function.

Conserved chromosomal clustering of genes governed by chromatin regulators in Drosophila

BACKGROUND: The trithorax group (trxG) and Polycomb group (PcG) proteins are responsible for the maintenance of stable transcriptional patterns of many developmental regulators. They bind to specific regions of DNA and direct the post-translational modifications of histones, playing a role in the dynamics of chromatin structure. RESULTS: We have performed genome-wide expression studies of trx and ash2 mutants in Drosophila melanogaster. Using computational analysis of our microarray data, we have identified 25 clusters of genes potentially regulated by TRX. Most of these clusters consist of genes that encode structural proteins involved in cuticle formation. This organization appears to be a distinctive feature of the regulatory networks of TRX and other chromatin regulators, since we have observed the same arrangement in clusters after experiments performed with ASH2, as well as in experiments performed by others with NURF, dMyc, and ASH1. We have also found many of these clusters to be significantly conserved in D. simulans, D. yakuba, D. pseudoobscura and partially in Anopheles gambiae. CONCLUSION: The analysis of genes governed by chromatin regulators has led to the identification of clusters of functionally related genes conserved in other insect species, suggesting this chromosomal organization is biologically important. Moreover, our results indicate that TRX and other chromatin regulators may act globally on chromatin domains that contain transcriptionally co-regulated genes.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

Combining MHC tetramer and intracellular cytokine staining for CD8(+) T cells to reveal antigenic epitopes naturally presented on tumor cells.

As more tumor antigens are discovered and as computer-guided T cell epitope prediction programs become more sophisticated, many potential T cell epitopes are synthesized and demonstrated to be antigenic in vitro. However, it is estimated that about 50% of such tumor antigen-specific T cells have not been demonstrated to recognize the naturally presented epitopes due to either technical difficulties, such as T cell cloning which is still challenging for many laboratories; or the predicted T cell epitopes are not generated or not generated in sufficient amounts by the antigen processing machinery. However, to potentially identify clinically relevant vaccine candidate epitopes, it is essential to demonstrate natural antigen presentation. Here we combine the advantages of MHC tetramer and intracellular cytokine staining to sensitively detect natural antigen presentation by tumor cells for epitopes of interest. The novel method does not require T cell cloning or long-term T cell culture. Because the antigen-specific T cells are positively identified, this method is much less influenced by IFNgamma producing cells with unknown specificities and should be widely applicable.

Assaying the regulatory potential of mammalian conserved non-coding sequences in human cells.

BACKGROUND: Conserved non-coding sequences in the human genome are approximately tenfold more abundant than known genes, and have been hypothesized to mark the locations of cis-regulatory elements. However, the global contribution of conserved non-coding sequences to the transcriptional regulation of human genes is currently unknown. Deeply conserved elements shared between humans and teleost fish predominantly flank genes active during morphogenesis and are enriched for positive transcriptional regulatory elements. However, such deeply conserved elements account for <1% of the conserved non-coding sequences in the human genome, which are predominantly mammalian. RESULTS: We explored the regulatory potential of a large sample of these 'common' conserved non-coding sequences using a variety of classic assays, including chromatin remodeling, and enhancer/repressor and promoter activity. When tested across diverse human model cell types, we find that the fraction of experimentally active conserved non-coding sequences within any given cell type is low (approximately 5%), and that this proportion increases only modestly when considered collectively across cell types. CONCLUSIONS: The results suggest that classic assays of cis-regulatory potential are unlikely to expose the functional potential of the substantial majority of mammalian conserved non-coding sequences in the human genome.

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.

Chicken BAFF--a highly conserved cytokine that mediates B cell survival.

Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the chicken homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS). By searching a chicken EST database we identified two overlapping cDNA clones that code for the entire open reading frame of chicken BAFF (chBAFF), which contains a predicted transmembrane domain and a putative furin protease cleavage site like its mammalian counterparts. The amino acid identity between soluble chicken and human BAFF is 76%, considerably higher than for most other known cytokines. The chBAFF gene is most strongly expressed in the bursa of Fabricius. Soluble recombinant chBAFF produced by human 293T cells interacted with the mammalian cell-surface receptors TACI, BCMA and BAFF-R. It bound to chicken B cells, but not to other lymphocytes, and it promoted the survival of splenic chicken B cells in culture. Furthermore, bacterially expressed chBAFF induced the selective expansion of B cells in the spleen and cecal tonsils when administered to young chicks. Our results suggest that like its mammalian counterpart, chBAFF plays an important role in survival and/or proliferation of chicken B cells.

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.

Boosting of Replication Competent NYVAC Primed HIV-1-Specific T-Cell Responses by HIV Synthetic Long Peptides (SLP)

Background: To enhance the induction of insert specific immune responses, a new generation of replication competent poxvirus vectors was designed and evaluated against non-replicating poxvirus vectors in a HIV vaccine study in non human primates.Methods: Rhesus macaques were immunized with either the non-replicating variant NYVAC-GagPolNef HIV-1 clade C or the replicating NYVAC-GagPolNef-C-KC, boosted with HIVGag- PolEnv-SLP and immune responses were monitored.Results: Gag-specific T-cell responses were only detected in animals immunized with the replicating NYVAC-GagPolNef-C-KC variant. Further enhancement and broadening of the immune response was studied by boosting the animals with novel T-cell immunogens HIVconsv synthetic long peptides (SLP), which direct vaccine-induced responses to the most conserved regions of HIV and contain both CD4 T-helper and CD8 CTL epitopes. The adjuvanted (Montanide ISA-720) SLP divided into subpools and delivered into anatomically separate sites enhanced the Gag-specific T-cell responses in 4 out of 6 animals, to more than 1000 SFC/106 PBMC in some animals. Furthermore, the SLP immunization broadened the immune response in 4 out of 6 animals to multiple Pol epitopes. Even Env-specific responses, to which the animals had not been primed, were induced by SLP in 2 out of 6 animals.Conclusion: This new immunization strategy of priming with replicating competent poxvirus NYVAC-HIVGagPolNef and boosting with HIVGagPolEnv-SLP, induced strong and broad Tcell responses and provides a promising new HIV vaccine approach. This study was performed within the Poxvirus T-cell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Unique and conserved functions of B cell-activating factor of the TNF family (BAFF) in the chicken.

The chicken represents the best-characterized animal model for B cell development in the so-called gut-associated lymphoid tissue (GALT) and the molecular processes leading to B cell receptor diversification in this species are well investigated. However, the mechanisms regulating B cell development and homeostasis in GALT species are largely unknown. Here we investigate the role played by the avian homologue of B cell-activating factor of the tumor necrosis factor family (BAFF). Flow cytometric analysis showed that the receptor for chicken B cell-activating factor of the tumor necrosis factor family (chBAFF) is expressed by mature and immature B cells. Unlike murine and human BAFF, chBAFF is primarily produced by B cells both in peripheral lymphoid organs and in the bursa of Fabricius, the chicken's unique primary lymphoid organ. In vitro and in vivo studies revealed that chBAFF is required for mature B cell survival. In addition, in vivo neutralization with a decoy receptor led to a reduction of the size and number of B cell follicles in the bursa, demonstrating that, in contrast to humans and mice, in chickens BAFF is also required for the development of immature B cells. Collectively, we show that chBAFF has phylogenetically conserved functions in mature B cell homeostasis but displays unique and thus far unknown properties in the regulation of B cell development in birds.

Comparison of human chromosome 21 conserved nongenic sequences (CNGs) with the mouse and dog genomes shows that their selective constraint is independent of their genic environment.

The analysis of conservation between the human and mouse genomes resulted in the identification of a large number of conserved nongenic sequences (CNGs). The functional significance of this nongenic conservation remains unknown, however. The availability of the sequence of a third mammalian genome, the dog, allows for a large-scale analysis of evolutionary attributes of CNGs in mammals. We have aligned 1638 previously identified CNGs and 976 conserved exons (CODs) from human chromosome 21 (Hsa21) with their orthologous sequences in mouse and dog. Attributes of selective constraint, such as sequence conservation, clustering, and direction of substitutions were compared between CNGs and CODs, showing a clear distinction between the two classes. We subsequently performed a chromosome-wide analysis of CNGs by correlating selective constraint metrics with their position on the chromosome and relative to their distance from genes. We found that CNGs appear to be randomly arranged in intergenic regions, with no bias to be closer or farther from genes. Moreover, conservation and clustering of substitutions of CNGs appear to be completely independent of their distance from genes. These results suggest that the majority of CNGs are not typical of previously described regulatory elements in terms of their location. We propose models for a global role of CNGs in genome function and regulation, through long-distance cis or trans chromosomal interactions.

Hierarchal utilization of different T-cell receptor Vbeta gene segments in the CD8(+)-T-cell response to an immunodominant Moloney leukemia virus-encoded epitope in vivo.

The CD8(+)-T-cell response to Moloney murine leukemia virus (M-MuLV)-associated antigens in C57BL/6 mice is directed against an immunodominant gag-encoded epitope (CCLCLTVFL) presented in the context of H-2D(b) and is restricted primarily to cytotoxic T lymphocytes (CTL) expressing the Valpha3.2 and Vbeta5.2 gene segments. We decided to examine the M-MuLV response in congenic C57BL/6 Vbeta(a) mice which are unable to express the dominant Valpha3.2(+) Vbeta5.2(+) T-cell receptor (TCR) due to a large deletion at the TCR locus that includes the Vbeta5.2 gene segment. Interestingly, M-MuLV-immune C57BL/6 Vbeta(a) mice were still able to reject M-MuLV-infected tumor cells and direct ex vivo analysis of peripheral blood lymphocytes from these immune mice revealed a dramatic increase in CD8(+) cells utilizing the same Valpha3.2 gene segment in association with two different Vbeta segments (Vbeta3 and Vbeta17). Surprisingly, all these CTL recognized the same immunodominant M-MuLV gag epitope. Analysis of the TCR repertoire of individual M-MuLV-immune (C57BL/6 x C57BL/6 Vbeta(a))F(1) mice revealed a clear hierarchy in Vbeta utilization, with a preferential usage of the Vbeta17 gene segment, whereas Vbeta3 and especially Vbeta5.2 were used to much lesser extents. Sequencing of TCRalpha- and -beta-chain junctional regions of CTL clones specific for the M-MuLV gag epitope revealed a diverse repertoire of TCRbeta chains in Vbeta(a) mice and a highly restricted TCRbeta-chain repertoire in Vbeta(b) mice, whereas TCRalpha-chain sequences were highly conserved in both cases. Collectively, our data indicate that the H-2D(b)-restricted M-MuLV gag epitope can be recognized in a hierarchal fashion by different Vbeta domains and that the degree of beta-chain diversity varies according to Vbeta utilization.

Native homing endonucleases can target conserved genes in humans and in animal models.

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays.

NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.