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1.
Sci Rep ; 11(1): 17571, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475432

RESUMO

Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/-p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/-p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/-p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/-p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/-p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/-p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.

2.
Methods Mol Biol ; 2318: 187-208, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019291

RESUMO

MYC is a transcription factor playing multiple functions both in physiological and pathological settings. Biochemical characterizations, combined with the analyses of MYC chromatin binding, have shown that its pleiotropic activity depends on the chromatin context and its protein-protein interactions with different cofactors. In order to determine the contribution of MYC in a certain biological condition, it would be relevant to analyze the concomitant binding of MYC and its associated proteins, in relationship to the chromatin environment. To this end, we here provide a simple method to parallel map the genome-wide binding of MYC-associated proteins, together with the chromatin profiling of multiple histone modifications. We detail the procedure to perform high-throughput ChIP-seq (HT-ChIP-seq) with a variety of biological samples. In addition, we describe simple bioinformatic steps to determine the distribution of MYC binding with respect to the chromatin context and the association of its cofactors. The described approach will permit the reproducible characterization of MYC activity in different biological contexts.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Epigenômica/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Biologia Computacional/métodos , DNA/genética , Epigênese Genética/genética , Genes myc/genética , Genes myc/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Código das Histonas/genética , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Análise de Sequência de DNA/métodos , Fatores de Transcrição/metabolismo
3.
Gut ; 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436496

RESUMO

OBJECTIVE: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy. DESIGN: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance. RESULTS: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy. CONCLUSIONS: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

4.
Nat Genet ; 52(12): 1397-1411, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33169020

RESUMO

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.


Assuntos
Anormalidades Múltiplas/genética , Núcleo Celular/fisiologia , Cromatina/metabolismo , Face/anormalidades , Haploinsuficiência/genética , Doenças Hematológicas/genética , Histona-Lisina N-Metiltransferase/genética , Doenças Vestibulares/genética , Células 3T3 , Animais , Linhagem Celular , Linhagem da Célula/genética , Condrócitos/citologia , Condrogênese/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Camundongos , Osteócitos/citologia , Osteogênese/genética , Proteínas do Grupo Polycomb/genética , Estresse Mecânico
5.
Sci Adv ; 6(39)2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32978159

RESUMO

Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.

6.
Cell Rep ; 29(12): 4036-4052.e10, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851932

RESUMO

The transition of neural progenitors to differentiated postmitotic neurons is mainly considered irreversible in physiological conditions. In the present work, we show that Shh pathway activation through SmoM2 expression promotes postmitotic neurons dedifferentiation, re-entering in the cell cycle and originating medulloblastoma in vivo. Notably, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here, we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Indeed, postmitotic neurons re-entered the cell cycle and proliferated as a result of chromatin remodeling of neurons by BRPF1. Our model of brain cancer explains the onset of a subset of human medulloblastoma in adult individuals where granule neuron progenitors are no longer present.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Mutação , Neurônios/patologia , Receptor Smoothened/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Animais , Apoptose , Proliferação de Células , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Feminino , Proteínas Hedgehog/genética , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Nus , Neurônios/metabolismo , Receptor Smoothened/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Neuron ; 104(2): 271-289.e13, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31515109

RESUMO

Mutations in one SETD5 allele are genetic causes of intellectual disability and autistic spectrum disorders. However, the mechanisms by which SETD5 regulates brain development and function remain largely elusive. Herein, we found that Setd5 haploinsufficiency impairs the proliferative dynamics of neural progenitors and synaptic wiring of neurons, ultimately resulting in behavioral deficits in mice. Mechanistically, Setd5 inactivation in neural stem cells, zebrafish, and mice equally affects genome-wide levels of H3K36me3 on active gene bodies. Notably, we demonstrated that SETD5 directly deposits H3K36me3, which is essential to allow on-time RNA elongation dynamics. Hence, Setd5 gene loss leads to abnormal transcription, with impaired RNA maturation causing detrimental effects on gene integrity and splicing. These findings identify SETD5 as a fundamental epigenetic enzyme controlling the transcriptional landscape in neural progenitors and their derivatives and illuminate the molecular events that connect epigenetic defects with neuronal dysfunctions at the basis of related human diseases.


Assuntos
Encéfalo/embriologia , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Código das Histonas/genética , Metiltransferases/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Cognição , Epigênese Genética , Histona Metiltransferases/genética , Metiltransferases/fisiologia , Camundongos , Mutação , Células-Tronco Neurais/metabolismo , Splicing de RNA/genética , RNA-Seq , Comportamento Social , Elongação da Transcrição Genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
8.
Cell Death Differ ; 26(9): 1813-1831, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30538287

RESUMO

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinogênese/genética , Proteínas de Ciclo Celular/genética , Glioblastoma/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Autorrenovação Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais
9.
Nat Commun ; 9(1): 3921, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237396

RESUMO

The original version of this Article contained an error in the spelling of the author Miriam Gaggianesi, which was incorrectly given as Miriam Giaggianesi. Furthermore, the affiliation details for Gabriella Gaudioso, Valentina Vaira, and Silvano Bosari incorrectly omitted 'Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, 20122, Italy'. Finally, the affiliation details for Alice Turdo, Miriam Gaggianesi, Aurora Chinnici and Elisa Lipari were incorrectly given as 'Dipartimento di Biotecnologie Mediche e Medicina Legale Sezione di Biochimica Medica, Facoltà di Medicina e Chirurgia, Policlinico "P.Giaccone", Università di Palermo, Palermo, 90127, Italy'. The correct affiliation is 'Department of Surgical, Oncological and Stomatological Sciences, University of Palermo, Palermo, 90127, Italy'. These errors have now been corrected in both the PDF and HTML versions of the Article.

10.
Stem Cells Int ; 2018: 4598195, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29853913

RESUMO

Accumulating evidences indicate that many tumors rely on subpopulations of cancer stem cells (CSCs) with the ability to propagate malignant clones indefinitely and to produce an overt cancer. Of importance, CSCs seem to be more resistant to the conventional cytotoxic treatments, driving tumor growth and contributing to relapse. CSCs can originate from normal committed cells which undergo tumor-reprogramming processes and reacquire a stem cell-like phenotype. Increasing evidences also show how tumor homeostasis and progression strongly rely on the capacity of nontumorigenic cancer cells to dedifferentiate to CSCs. Both tumor microenvironment and epigenetic reprogramming drive such dynamic mechanisms, favoring cancer cell plasticity and tumor heterogeneity. Here, we report new developments which led to an advancement in the CSC field, elucidating the concepts of cancer cell of origin and CSC plasticity in solid tumor initiation and maintenance. We further discuss the main signaling pathways which, under the influence of extrinsic environmental factors, play a critical role in the formation and maintenance of CSCs. Moreover, we propose a review of the main epigenetic mechanisms whose deregulation can favor the onset of CSC features both in tumor initiation and tumor maintenance. Finally, we provide an update of the main strategies that could be applied to target CSCs and cancer cell plasticity.

11.
Cell Mol Life Sci ; 75(14): 2537-2555, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29691590

RESUMO

Cancer heterogeneity arises during tumor progression as a consequence of genetic insults, environmental cues, and reversible changes in the epigenetic state, favoring tumor cell plasticity. The role of enhancer reprogramming is emerging as a relevant field in cancer biology as it supports adaptation of cancer cells to those environmental changes encountered during tumor progression and metastasis seeding. In this review, we describe the cancer-related alterations that drive oncogenic enhancer activity, leading to dysregulated transcriptional programs. We discuss the molecular mechanisms of both cis- and trans-factors in overriding the regulatory circuits that maintain cell-type specificity and imposing an alternative, de-regulated enhancer activity in cancer cells. We further comment on the increasing evidence which implicates stress response and aging-signaling pathways in the enhancer landscape reprogramming during tumorigenesis. Finally, we focus on the potential therapeutic implications of these enhancer-mediated subverted transcriptional programs, putting particular emphasis on the lack of information regarding tumor progression and the metastatic outgrowth, which still remain the major cause of mortality related to cancer.


Assuntos
Transformação Celular Neoplásica/genética , Reprogramação Celular/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Animais , Plasticidade Celular/genética , Progressão da Doença , Humanos , Neoplasias/patologia , Transdução de Sinais/genética
12.
Nat Commun ; 9(1): 1024, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523784

RESUMO

Breast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes are difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Overexpression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor-initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers.


Assuntos
Neoplasias da Mama/metabolismo , Epigênese Genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Carcinogênese , Linhagem Celular Tumoral , Reprogramação Celular , Elementos Facilitadores Genéticos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/citologia
13.
Front Cell Dev Biol ; 5: 7, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28217689

RESUMO

Pluripotent stem cells (PSCs) are defined by their self-renewal potential, which permits their unlimited propagation, and their pluripotency, being able to generate cell of the three embryonic lineages. These properties render PSCs a valuable tool for both basic and medical research. To induce and stabilize the pluripotent state, complex circuitries involving signaling pathways, transcription regulators and epigenetic mechanisms converge on a core transcriptional regulatory network of PSCs, thus determining their cell identity. Among the transcription factors, MYC represents a central hub, which modulates and integrates multiple mechanisms involved both in the maintenance of pluripotency and in cell reprogramming. Indeed, it instructs the PSC-specific cell cycle, metabolism and epigenetic landscape, contributes to limit exit from pluripotency and modulates signaling cascades affecting the PSC identity. Moreover, MYC extends its regulation on pluripotency by controlling PSC-specific non-coding RNAs. In this report, we review the MYC-controlled networks, which support the pluripotent state and discuss how their perturbation could affect cell identity. We further discuss recent finding demonstrating a central role of MYC in triggering epigenetic memory in PSCs, which depends on the establishment of a WNT-centered self-reinforcing circuit. Finally, we comment on the therapeutic implications of the role of MYC in affecting PSCs. Indeed, PSCs are used for both disease and cancer modeling and to derive cells for regenerative medicine. For these reasons, unraveling the MYC-mediated mechanism in those cells is fundamental to exploit their full potential and to identify therapeutic targets.

14.
Nat Commun ; 7: 11903, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27301576

RESUMO

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/ß-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/ß-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity.


Assuntos
Redes Reguladoras de Genes/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Autorrenovação Celular/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Proteínas do Grupo Polycomb/metabolismo , Transcrição Genética/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
15.
PLoS Pathog ; 12(4): e1005557, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-27105075

RESUMO

Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.


Assuntos
Proteínas de Bactérias/química , Meningite Meningocócica/imunologia , Proteínas Repressoras/química , Fatores de Virulência/química , Adesinas Bacterianas/biossíntese , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Western Blotting , Varredura Diferencial de Calorimetria , Cromatografia Líquida de Alta Pressão , Regulação Bacteriana da Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Neisseria meningitidis Sorogrupo B/química , Neisseria meningitidis Sorogrupo B/imunologia , Conformação Proteica , Proteínas Repressoras/imunologia , Proteínas Repressoras/metabolismo , Ressonância de Plasmônio de Superfície , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo , Difração de Raios X
16.
Stem Cells Int ; 2016: 8652748, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26798364

RESUMO

Stem cells balance their self-renewal and differentiation potential by integrating environmental signals with the transcriptional regulatory network. The maintenance of cell identity and/or cell lineage commitment relies on the interplay of multiple factors including signaling pathways, transcription factors, and the epigenetic machinery. These regulatory modules are strongly interconnected and they influence the pattern of gene expression of stem cells, thus guiding their cellular fate. Embryonic stem cells (ESCs) represent an invaluable tool to study this interplay, being able to indefinitely self-renew and to differentiate towards all three embryonic germ layers in response to developmental cues. In this review, we highlight those mechanisms of signaling to chromatin, which regulate chromatin modifying enzymes, histone modifications, and nucleosome occupancy. In addition, we report the molecular mechanisms through which signaling pathways affect both the epigenetic and the transcriptional state of ESCs, thereby influencing their cell identity. We propose that the dynamic nature of oscillating signaling and the different regulatory network topologies through which those signals are encoded determine specific gene expression programs, leading to the fluctuation of ESCs among multiple pluripotent states or to the establishment of the necessary conditions to exit pluripotency.

17.
Front Mol Biosci ; 3: 82, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28066774

RESUMO

PIPE-chipSAD is a pipeline for bacterial transcriptome studies based on high-density microarray experiments. The main algorithm chipSAD, integrates the analysis of the hybridization signal with the genomic position of probes and identifies portions of the genome transcribing for mRNAs. The pipeline includes a procedure, align-chipSAD, to build a multiple alignment of transcripts originating in the same locus in multiple experiments and provides a method to compare mRNA expression across different conditions. Finally, the pipeline includes anno-chipSAD a method to annotate the detected transcripts in comparison to the genome annotation. Overall, our pipeline allows transcriptional profile analysis of both coding and non-coding portions of the chromosome in a single framework. Importantly, due to its versatile characteristics, it will be of wide applicability to analyse, not only microarray signals, but also data from other high throughput technologies such as RNA-sequencing. The current PIPE-chipSAD implementation is written in Python programming language and is freely available at https://github.com/silviamicroarray/chipSAD.

18.
PLoS One ; 10(5): e0126325, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25951061

RESUMO

Most bacterial small RNAs (sRNAs) are post-transcriptional regulators involved in adaptive responses, controlling gene expression by modulating translation or stability of their target mRNAs often in concert with the RNA chaperone Hfq. Neisseria meningitides, the leading cause of bacterial meningitis, is able to adapt to different host niches during human infection. However, only a few sRNAs and their functions have been fully described to date. Recently, transcriptional expression profiling of N. meningitides in human blood ex vivo revealed 91 differentially expressed putative sRNAs. Here we expanded this analysis by performing a global transcriptome study after exposure of N. meningitides to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation). and we identified putative sRNAs that were differentially expressed in vitro. A set of 98 putative sRNAs was obtained by analyzing transcriptome data and 8 new sRNAs were validated, both by Northern blot and by primer extension techniques. Deletion of selected sRNAs caused attenuation of N. meningitides infection in the in vivo infant rat model, leading to the identification of the first sRNAs influencing meningococcal bacteremia. Further analysis indicated that one of the sRNAs affecting bacteremia responded to carbon source availability through repression by a GntR-like transcriptional regulator. Both the sRNA and the GntR-like regulator are implicated in the control of gene expression from a common network involved in energy metabolism.


Assuntos
Bacteriemia/sangue , Neisseria meningitidis/isolamento & purificação , RNA Bacteriano/genética , Transcriptoma , Animais , Northern Blotting , Ratos
19.
Infect Immun ; 81(2): 560-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23230289

RESUMO

The NadA adhesin is a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B (MenB) infection. Under in vitro growth conditions, nadA is repressed by the regulator NadR and poorly expressed, resulting in inefficient killing of MenB strains by anti-NadA antibodies. Interestingly, sera from children infected with strains that express low levels of NadA in laboratory growth nevertheless recognize the NadA antigen, suggesting that NadA expression during infection may be different from that observed in vitro. In a strain panel covering a range of NadA levels, repression was relieved through deleting nadR. All nadR knockout strains expressed high levels of NadA and were efficiently killed by sera from subjects immunized with 4CMenB. A selected MenB strain, NGP165, mismatched for other vaccine antigens, is not killed by sera from immunized infants when the strain is grown in vitro. However, in an in vivo passive protection model, the same sera effectively protected infant rats from bacteremia with NGP165. Furthermore, we identify a novel hydroxyphenylacetic acid (HPA) derivative, reported by others to be produced during inflammation, which induces expression of NadA in vitro, leading to efficient antibody-mediated killing. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h postinfection. Our results suggest that during infectious disease, NadR repression is alleviated due to niche-specific signals, resulting in high levels of NadA expression from any nadA-positive (nadA(+)) strain and therefore efficient killing by anti-NadA antibodies elicited by the 4CMenB vaccine.


Assuntos
Adesinas Bacterianas/genética , Vacinas Meningocócicas/administração & dosagem , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/genética , Neisseria meningitidis Sorogrupo B/imunologia , Neisseria meningitidis/genética , Neisseria meningitidis/imunologia , Adesinas Bacterianas/imunologia , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Pré-Escolar , Ensaios Clínicos como Assunto , Feminino , Humanos , Lactente , Recém-Nascido , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/genética , Camundongos , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transcrição Genética
20.
Biochemistry ; 51(34): 6738-52, 2012 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-22834735

RESUMO

Neisserial adhesin A (NadA) is a surface exposed trimeric protein present in most hypervirulent meningococcal strains and involved in epithelial cell adhesion and colonization. The expression of nadA is controlled by Neisserial adhesin regulator (NadR), a member of the MarR family, which binds to the nadA promoter and strongly represses the transcription of nadA. It was recently demonstrated that the DNA-binding activity of NadR was attenuated by 4-hydroxyphenylacetic acid (4-HPA), a natural molecule released in human saliva, thus leading to the de-repression of nadA in vivo. To elucidate the mechanism of regulation of NadR by 4-HPA, we used hydrogen-deuterium exchange mass spectrometry in association with in silico docking and site-directed mutagenesis. We show here that 4-HPA binds at the interface between the dimerization and the DNA-binding domains and stabilizes the homodimeric state of NadR without inducing large conformational changes in the DNA-binding lobes. The residues predicted to be in contact with 4-HPA were further selected for mutagenesis to assess their in vitro and in vivo functions in 4-HPA binding. Our results indicate that Arg(40) is critical for DNA-binding and reveal that Tyr(115) plays a key role in the mechanism of regulation of NadR by 4-HPA. Altogether our data suggest that the mechanism of regulation of NadR by 4-HPA mainly involves the stabilization of the dimer in a configuration incompatible with DNA binding.


Assuntos
Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , Fenilacetatos/metabolismo , Proteínas Repressoras/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dimerização , Regulação Bacteriana da Expressão Gênica , Ligantes , Conformação Molecular , Dados de Sequência Molecular , Neisseria meningitidis/química , Neisseria meningitidis/genética , Fenilacetatos/química , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética
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