Implication of DNA methylation, CTCF and BORIS factors in the transcriptional regulation of the human telomerase catalytic subunit hTERT

RESUME La télomérase confère une durée de vie illimitée et est réactivée dans la plupart des cellules tumorales. Sa sous-unité catalytique hTERT est définie comme le facteur limitant pour son activation. De l'identification de facteurs liant la région régulatrice d'hTERT, au rôle de la méthylation de l'ADN et de la modification des histones, de nombreux modèles de régulation ont été suggérés. Cependant, aucun de ces modèles n'a pu expliquer l'inactivation de la télomérase dans la plupart des cellules somatiques et sa réactivation dans la majorité des cellules tumorales. De plus, les observations contradictoires entre le faible niveau d'expression d'ARN messager d'hTERT dans les cellules télomérase-positives et la très forte activité transcriptionnelle du promoteur d'hTERT en transfection restent incomprises. Dans cette étude, nous avons montré que la région proximale du gène hTERT (exon 1 et 2) était impliquée dans la répression de l'activité de son promoteur. Nous avons identifié le facteur CTCF comme étant un inhibiteur du promoteur d'hTERT, en se liant au niveau de son premier exon. La méthylation de l'exon 1 du gène hTERT, couramment observée dans les tumeurs mais pas dans les cellules normales, empêcherait la liaison de CTCF. L'étude du profil de méthylation du promoteur d'hTERT indique qu'une partie du promoteur reste déméthylée et qu'elle semble suffisante pour permettre une faible activité transcriptionnelle du gène hTERT. Ainsi, la méthylation particulière des régions régulatrices d'hTERT inhibe la liaison de CTCF tout en permettant une faible transcription du gène. Cependant, dans certaines cellules tumorales, le promoteur et la région proximale du gène hTERT ne sont pas méthylés. Dans les lignées cellulaires tumorales de tesitcules et d'ovaires, l'inhibition de CTCF est contrée par son paralogue BORIS, qui se lie aussi au niveau de l'exon 1 d'hTERT, mais permet ainsi l'activation du promoteur. L'étude de l'expression du gène BORIS montre qu'il est exclusivement exprimé dans les tissus normaux de testicules et d'ovaires jeunes, ainsi qu'à différents niveaux dans la plupart des tumeurs. Sa transcription est sous le contrôle de deux promoteurs. Le promoteur proximal est régulé par méthylation et un transcrit alternatif majoritaire, délété de l'exon 6, est trouvé lorsque ce promoteur est actif. Tous ces résultats conduisent à un modèle de régulation du gène hTERT qui tient compte du profil épigénétique du gène et qui permet d'expliquer le faible taux de transcription observé in vivo. De plus, l'expression de BORIS dans les cancers et son implication dans l'activation du gène hTERT pourrait permettre de comprendre les phénomènes de dérégulation épigénétique et d'immortalisation qui ont lieu durant la tumorigenèse. SUMMARY Telomerase confers an unlimited lifespan, and is reactivated in most tumor cells. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors that bind to the hTERT 5' regulatory region, and the role of CpG methylation and histone acetylation, an abundance of regulatory models have been suggested. None of these models can explain the silence of telomerase in most somatic cells and its reactivation in tumor cells. Moreover, the contradictory observations of the low level of hTERT mRNA in telomerase-positive cells and the high transcriptional activity of the hTERT promoter in transfection experiments remain unresolved. In this study, we demonstrated that the proximal exonic region of the hTERT gene (exon 1 and 2) is involved in the inhibition of its promoter. We identified the protein CTCF as the inhibitor of the hTERT promoter, through its binding to the first exon. The methylation of the first exon region, which is often observed in cancer cells but not in noimal cells, represses CTCF binding. Study of hTERT promoter methylation shows a partial demethylation sufficient to activate the transcription of the hTERT gene. Therefore, we demonstrated that the particular methylation profile of the hTERT regulatory sequences inhibits the binding of CTCF, while it allows a low transcription of the gene. Nevertheless, in some tumor cells, the promoter and the proximal exonic region of hTERT are unmethylated. In testicular and ovarian cancer cell lines, CTCF inhibition is counteracted by its BORIS paralogue that also binds the hTERT first exon but allows the promoter activation. The study of BORIS gene regulation showed that this factor is exclusively expressed in normal tissue of testis and ovary of young woman, as well as in almost all tumors with different levels. Two promoters were found to induce its transcription. The proximal promoter was regulated by methylation. Moreover, a major alternative transcript, deleted of the exon 6, is detected when this promoter is active. All these results lead to a model for hTERT regulation that takes into account the epigenetic profile of the gene and provides an explanation for the low transcriptional level observed in vivo. BORIS expression in cancers and its implication in hTERT activation might also permit the understanding of epigenetic deregulation and immortalization phenomena that occur during tumorigenesis.

T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension

This article introduces a new interface for T-Coffee, a consistency-based multiple sequence alignment program. This interface provides an easy and intuitive access to the most popular functionality of the package. These include the default T-Coffee mode for protein and nucleic acid sequences, the M-Coffee mode that allows combining the output of any other aligners, and template-based modes of T-Coffee that deliver high accuracy alignments while using structural or homology derived templates. These three available template modes are Expresso for the alignment of protein with a known 3D-Structure, R-Coffee to align RNA sequences with conserved secondary structures and PSI-Coffee to accurately align distantly related sequences using homology extension. The new server benefits from recent improvements of the T-Coffee algorithm and can align up to 150 sequences as long as 10 000 residues and is available from both http://www.tcoffee.org and its main mirror http://tcoffee.crg.cat.

Dyrk1A is dynamically expressed on subsets of motor neurons and in the neuromuscular junction: possible role in Down syndrome

Individuals with Down syndrome (DS) present important motor deficits that derive from altered motor development of infants and young children. DYRK1A, a candidate gene for DS abnormalities has been implicated in motor function due to its expression in motor nuclei in the adult brain, and its overexpression in DS mouse models leads to hyperactivity and altered motor learning. However, its precise role in the adult motor system, or its possible involvement in postnatal locomotor development has not yet been clarified. During the postnatal period we observed time-specific expression of Dyrk1A in discrete subsets of brainstem nuclei and spinal cord motor neurons. Interestingly, we describe for the first time the presence of Dyrk1A in the presynaptic terminal of the neuromuscular junctions and its axonal transport from the facial nucleus, suggesting a function for Dyrk1A in these structures. Relevant to DS, Dyrk1A overexpression in transgenic mice (TgDyrk1A) produces motor developmental alterations possibly contributing to DS motor phenotypes and modifies the numbers of motor cholinergic neurons, suggesting that the kinase may have a role in the development of the brainstem and spinal cord motor system.

Neural detection of complex sound sequences in the absence of consciousness.

The neural response to a violation of sequences of identical sounds is a typical example of the brain's sensitivity to auditory regularities. Previous literature interprets this effect as a pre-attentive and unconscious processing of sensory stimuli. By contrast, a violation to auditory global regularities, i.e. based on repeating groups of sounds, is typically detectable when subjects can consciously perceive them. Here, we challenge the notion that global detection implies consciousness by testing the neural response to global violations in a group of 24 patients with post-anoxic coma (three females, age range 45-87 years), treated with mild therapeutic hypothermia and sedation. By applying a decoding analysis to electroencephalographic responses to standard versus deviant sound sequences, we found above-chance decoding performance in 10 of 24 patients (Wilcoxon signed-rank test, P < 0.001), despite five of them being mildly hypothermic, sedated and unarousable. Furthermore, consistently with previous findings based on the mismatch negativity the progression of this decoding performance was informative of patients' chances of awakening (78% predictive of awakening). Our results show for the first time that detection of global regularities at neural level exists despite a deeply unconscious state.

Comparison of Immunogenicity in Rhesus Macaques of Transmitted-Founder, HIV-1 Group M Consensus, and Trivalent Mosaic Envelope Vaccines Formulated as a DNA Prime, NYVAC, and Envelope Protein Boost.

An effective human immunodeficiency virus type 1 (HIV-1) vaccine must induce protective antibody responses, as well as CD4(+) and CD8(+) T cell responses, that can be effective despite extraordinary diversity of HIV-1. The consensus and mosaic immunogens are complete but artificial proteins, computationally designed to elicit immune responses with improved cross-reactive breadth, to attempt to overcome the challenge of global HIV diversity. In this study, we have compared the immunogenicity of a transmitted-founder (T/F) B clade Env (B.1059), a global group M consensus Env (Con-S), and a global trivalent mosaic Env protein in rhesus macaques. These antigens were delivered using a DNA prime-recombinant NYVAC (rNYVAC) vector and Env protein boost vaccination strategy. While Con-S Env was a single sequence, mosaic immunogens were a set of three Envs optimized to include the most common forms of potential T cell epitopes. Both Con-S and mosaic sequences retained common amino acids encompassed by both antibody and T cell epitopes and were central to globally circulating strains. Mosaics and Con-S Envs expressed as full-length proteins bound well to a number of neutralizing antibodies with discontinuous epitopes. Also, both consensus and mosaic immunogens induced significantly higher gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) responses than B.1059 immunogen. Immunization with these proteins, particularly Con-S, also induced significantly higher neutralizing antibodies to viruses than B.1059 Env, primarily to tier 1 viruses. Both Con-S and mosaics stimulated more potent CD8-T cell responses against heterologous Envs than did B.1059. Both antibody and cellular data from this study strengthen the concept of using in silico-designed centralized immunogens for global HIV-1 vaccine development strategies. IMPORTANCE: There is an increasing appreciation for the importance of vaccine-induced anti-Env antibody responses for preventing HIV-1 acquisition. This nonhuman primate study demonstrates that in silico-designed global HIV-1 immunogens, designed for a human clinical trial, are capable of eliciting not only T lymphocyte responses but also potent anti-Env antibody responses.

NLRP3 Inflammasome Is Expressed and Functional in Mouse Brain Microglia but Not in Astrocytes.

Neuroinflammation is the local reaction of the brain to infection, trauma, toxic molecules or protein aggregates. The brain resident macrophages, microglia, are able to trigger an appropriate response involving secretion of cytokines and chemokines, resulting in the activation of astrocytes and recruitment of peripheral immune cells. IL-1β plays an important role in this response; yet its production and mode of action in the brain are not fully understood and its precise implication in neurodegenerative diseases needs further characterization. Our results indicate that the capacity to form a functional NLRP3 inflammasome and secretion of IL-1β is limited to the microglial compartment in the mouse brain. We were not able to observe IL-1β secretion from astrocytes, nor do they express all NLRP3 inflammasome components. Microglia were able to produce IL-1β in response to different classical inflammasome activators, such as ATP, Nigericin or Alum. Similarly, microglia secreted IL-18 and IL-1α, two other inflammasome-linked pro-inflammatory factors. Cell stimulation with α-synuclein, a neurodegenerative disease-related peptide, did not result in the release of active IL-1β by microglia, despite a weak pro-inflammatory effect. Amyloid-β peptides were able to activate the NLRP3 inflammasome in microglia and IL-1β secretion occurred in a P2X7 receptor-independent manner. Thus microglia-dependent inflammasome activation can play an important role in the brain and especially in neuroinflammatory conditions.

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease.

The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.

Coal deposition in carbonate rich shallow Lacustrine systems: The Calaf and Mequinenza sequences (Oligocene, eastern Ebro Basin, NE Spain)

Two main coal-bearing sequences developed during the Oligocene in the Tertiary Ebro Basin, the Calaf (early Oligocene) and Mequinenza (late Oligocene) coal basins. Coal deposition took place in shallow marsh-swamp-lake complexes which sometimes became closed and evolved under warm climatic conditions with fluctuating humidity. These shallow lacustrine systems are closely interrelated with the terminal parts of the distributive fluvial systems which spread from the tectonically active Ebro basin margins. Laterally extensive lignite-bearing sequences, including rather thin, lenticular autochthonous and/or hypautochthonous coal seams with high ash and sulphur contents, characterized coal deposition in the shallow lacustrine systems. Coal seam geometry, which makes them nearly subeconomic, resulted from the tectonic instability during basin margin evolution and the sometimes closed, arid conditions under which the lacustrine systems evolved. High ash and sulphur contents resulted from the inadequate isolation of peat forming environments from clastic influx and from the very low acidity and sometimes high sulphate contents of the lacustrine waters. Coal exploration in shallow lacustrine sequences similar to those described here must take into account that the spread of coal-forming environments and maxima of coal deposition are usually coincident with lake expansions and retraction or shifting of the terminal fluvial zones interrelated with the lacustrine areas.

Molecular phylogenetics and temporal diversification in the genus Aeromonas based on the sequences of five housekeeping genes

Several approaches have been developed to estimate both the relative and absolute rates of speciation and extinction within clades based on molecular phylogenetic reconstructions of evolutionary relationships, according to an underlying model of diversification. However, the macroevolutionary models established for eukaryotes have scarcely been used with prokaryotes. We have investigated the rate and pattern of cladogenesis in the genus Aeromonas (γ-Proteobacteria, Proteobacteria, Bacteria) using the sequences of five housekeeping genes and an uncorrelated relaxed-clock approach. To our knowledge, until now this analysis has never been applied to all the species described in a bacterial genus and thus opens up the possibility of establishing models of speciation from sequence data commonly used in phylogenetic studies of prokaryotes. Our results suggest that the genus Aeromonas began to diverge between 248 and 266 million years ago, exhibiting a constant divergence rate through the Phanerozoic, which could be described as a pure birth process.

Quantitative TCR:pMHC Dissociation Rate Assessment by NTAmers Reveals Antimelanoma T Cell Repertoires Enriched for High Functional Competence.

Experimental models demonstrated that therapeutic induction of CD8 T cell responses may offer protection against tumors or infectious diseases providing that T cells have sufficiently high TCR/CD8:pMHC avidity for efficient Ag recognition and consequently strong immune functions. However, comprehensive characterization of TCR/CD8:pMHC avidity in clinically relevant situations has remained elusive. In this study, using the novel NTA-His tag-containing multimer technology, we quantified the TCR:pMHC dissociation rates (koff) of tumor-specific vaccine-induced CD8 T cell clones (n = 139) derived from seven melanoma patients vaccinated with IFA, CpG, and the native/EAA or analog/ELA Melan-A(MART-1)(26-35) peptide, binding with low or high affinity to MHC, respectively. We observed substantial correlations between koff and Ca(2+) mobilization (p = 0.016) and target cell recognition (p < 0.0001), with the latter independently of the T cell differentiation state. Our strategy was successful in demonstrating that the type of peptide impacted on TCR/CD8:pMHC avidity, as tumor-reactive T cell clones derived from patients vaccinated with the low-affinity (native) peptide expressed slower koff rates than those derived from patients vaccinated with the high-affinity (analog) peptide (p < 0.0001). Furthermore, we observed that the low-affinity peptide promoted the selective differentiation of tumor-specific T cells bearing TCRs with high TCR/CD8:pMHC avidity (p < 0.0001). Altogether, TCR:pMHC interaction kinetics correlated strongly with T cell functions. Our study demonstrates the feasibility and usefulness of TCR/CD8:pMHC avidity assessment by NTA-His tag-containing multimers of naturally occurring polyclonal T cell responses, which represents a strong asset for the development of immunotherapy.

Anti-MPER antibodies with heterogeneous neutralization capacity are detectable in most untreated HIV-1 infected individuals

Background The MPER region of the HIV-1 envelope glycoprotein gp41 is targeted by broadly neutralizing antibodies. However, the localization of this epitope in a hydrophobic environment seems to hamper the elicitation of these antibodies in HIV infected individuals. We have quantified and characterized anti-MPER antibodies by ELISA and by flow cytometry using a collection of mini gp41-derived proteins expressed on the surface of 293T cells. Longitudinal plasma samples from 35 HIV-1 infected individuals were assayed for MPER recognition and MPER-dependent neutralizing capacity using HIV-2 viruses engrafted with HIV-1 MPER sequences. Results Miniproteins devoid of the cysteine loop of gp41 exposed the MPER on 293T cell membrane. Anti-MPER antibodies were identified in most individuals and were stable when analyzed in longitudinal samples. The magnitude of the responses was strongly correlated with the global response to the HIV-1 envelope glycoprotein, suggesting no specific limitation for anti-MPER antibodies. Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope. However, antibody titers failed to correlate with 2F5-blocking activity and, more importantly, with the specific neutralization of HIV-2 chimeric viruses bearing the HIV-1 MPER sequence; suggesting a strong functional heterogeneity in anti-MPER humoral responses. Conclusions Anti-MPER antibodies can be detected in the vast majority of HIV-1 infected individuals and are generated in the context of the global anti-Env response. However, the neutralizing capacity is heterogeneous suggesting that eliciting neutralizing anti-MPER antibodies by immunization might require refinement of immunogens to skip nonneutralizing responses.

A highly expressed miR-101 isomiR is a functional silencing small RNA

Background MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression usually producing translational repression and gene silencing. High-throughput sequencing technologies have revealed heterogeneity at length and sequence level for the majority of mature miRNAs (IsomiRs). Most isomiRs can be explained by variability in either Dicer1 or Drosha cleavage during miRNA biogenesis at 5" or 3" of the miRNA (trimming variants). Although isomiRs have been described in different tissues and organisms, their functional validation as modulators of gene expression remains elusive. Here we have characterized the expression and function of a highly abundant miR-101 5"-trimming variant (5"-isomiR-101). Results The analysis of small RNA sequencing data in several human tissues and cell lines indicates that 5"-isomiR-101 is ubiquitously detected and a highly abundant, especially in the brain. 5"- isomiR-101 was found in Ago-2 immunocomplexes and complementary approaches showed that 5"-isomiR-101 interacted with different members of the silencing (RISC) complex. In addition, 5"-isomiR-101 decreased the expression of five validated miR-101 targets, suggesting that it is a functional variant. Both the binding to RISC members and the degree of silencing were less efficient for 5"-isomiR-101 compared with miR-101. For some targets, both miR-101 and 5"-isomiR-101 significantly decreased protein expression with no changes in the respective mRNA levels. Although a high number of overlapping predicted targets suggest similar targeted biological pathways, a correlation analysis of the expression profiles of miR-101 variants and predicted mRNA targets in human brains at different ages, suggest specific functions for miR-101- and 5"-isomiR-101. Conclusions These results suggest that isomiRs are functional variants and further indicate that for a given miRNA, the different isomiRs may contribute to the overall effect as quantitative and qualitative fine-tuners of gene expression.

Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas

BACKGROUND: The bacterial flagellum is the most important organelle of motility in bacteria and plays a key role in many bacterial lifestyles, including virulence. The flagellum also provides a paradigm of how hierarchical gene regulation, intricate protein-protein interactions and controlled protein secretion can result in the assembly of a complex multi-protein structure tightly orchestrated in time and space. As if to stress its importance, plants and animals produce receptors specifically dedicated to the recognition of flagella. Aside from motility, the flagellum also moonlights as an adhesion and has been adapted by humans as a tool for peptide display. Flagellar sequence variation constitutes a marker with widespread potential uses for studies of population genetics and phylogeny of bacterial species. RESULTS: We sequenced the complete flagellin gene (flaA) in 18 different species and subspecies of Aeromonas. Sequences ranged in size from 870 (A. allosaccharophila) to 921 nucleotides (A. popoffii). The multiple alignment displayed 924 sites, 66 of which presented alignment gaps. The phylogenetic tree revealed the existence of two groups of species exhibiting different FlaA flagellins (FlaA1 and FlaA2). Maximum likelihood models of codon substitution were used to analyze flaA sequences. Likelihood ratio tests suggested a low variation in selective pressure among lineages, with an omega ratio of less than 1 indicating the presence of purifying selection in almost all cases. Only one site under potential diversifying selection was identified (isoleucine in position 179). However, 17 amino acid positions were inferred as sites that are likely to be under positive selection using the branch-site model. Ancestral reconstruction revealed that these 17 amino acids were among the amino acid changes detected in the ancestral sequence. CONCLUSION: The models applied to our set of sequences allowed us to determine the possible evolutionary pathway followed by the flaA gene in Aeromonas, suggesting that this gene have probably been evolving independently in the two groups of Aeromonas species since the divergence of a distant common ancestor after one or several episodes of positive selection. REVIEWERS: This article was reviewed by Alexey Kondrashov, John Logsdon and Olivier Tenaillon (nominated by Laurence D Hurst).

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

Cytosine-and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34 -bases long cytosine-rich sequence and its complementary guanin e-rich strand corresponding to the first intron of the n-mycgene were studied. Both sequences , not yet studied, contain a 12 - base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively ...