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1.
PLoS Genet ; 18(7): e1010305, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35789210

RESUMO

Circadian clocks enable organisms to predict and align their behaviors and physiologies to constant daily day-night environmental cycle. Because the ubiquitin ligase Siah2 has been identified as a potential regulator of circadian clock function in cultured cells, we have used SIAH2-deficient mice to examine its function in vivo. Our experiments demonstrate a striking and unexpected sexually dimorphic effect of SIAH2-deficiency on the regulation of rhythmically expressed genes in the liver. The absence of SIAH2 in females, but not in males, altered the expression of core circadian clock genes and drastically remodeled the rhythmic transcriptome in the liver by increasing the number of day-time expressed genes, and flipping the rhythmic expression from nighttime expressed genes to the daytime. These effects are not readily explained by effects on known sexually dimorphic pathways in females. Moreover, loss of SIAH2 in females, not males, preferentially altered the expression of transcription factors and genes involved in regulating lipid and lipoprotein metabolism. Consequently, SIAH2-deficient females, but not males, displayed disrupted daily lipid and lipoprotein patterns, increased adiposity and impaired metabolic homeostasis. Overall, these data suggest that SIAH2 may be a key component of a female-specific circadian transcriptional output circuit that directs the circadian timing of gene expression to regulate physiological rhythms, at least in the liver. In turn, our findings imply that sex-specific transcriptional mechanisms may closely interact with the circadian clock to tailor overt rhythms for sex-specific needs.


Assuntos
Relógios Circadianos , Ritmo Circadiano , Animais , Relógios Circadianos/genética , Ritmo Circadiano/genética , Feminino , Lipídeos , Lipoproteínas , Masculino , Camundongos , Ubiquitina , Ubiquitina-Proteína Ligases/genética
2.
Hum Mol Genet ; 28(1): 16-30, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30215740

RESUMO

Polycystin-1 (PC1), encoded by the PKD1 gene that is mutated in the autosomal dominant polycystic kidney disease, regulates a number of processes including bone development. Activity of the transcription factor RunX2, which controls osteoblast differentiation, is reduced in Pkd1 mutant mice but the mechanism governing PC1 activation of RunX2 is unclear. PC1 undergoes regulated cleavage that releases its C-terminal tail (CTT), which translocates to the nucleus to modulate transcriptional pathways involved in proliferation and apoptosis. We find that the cleaved CTT of PC1 (PC1-CTT) stimulates the transcriptional coactivator TAZ (Wwtr1), an essential coactivator of RunX2. PC1-CTT physically interacts with TAZ, stimulating RunX2 transcriptional activity in pre-osteoblast cells in a TAZ-dependent manner. The PC1-CTT increases the interaction between TAZ and RunX2 and enhances the recruitment of the p300 transcriptional co-regulatory protein to the TAZ/RunX2/PC1-CTT complex. Zebrafish injected with morpholinos directed against pkd1 manifest severe bone calcification defects and a curly tail phenotype. Injection of messenger RNA (mRNA) encoding the PC1-CTT into pkd1-morphant fish restores bone mineralization and reduces the severity of the curly tail phenotype. These effects are abolished by co-injection of morpholinos directed against TAZ. Injection of mRNA encoding a dominant-active TAZ construct is sufficient to rescue both the curly tail phenotype and the skeletal defects observed in pkd1-morpholino treated fish. Thus, TAZ constitutes a key mechanistic link through which PC1 mediates its physiological functions.


Assuntos
Desenvolvimento Ósseo/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Canais de Cátion TRPP/fisiologia , Animais , Apoptose , Desenvolvimento Ósseo/fisiologia , Diferenciação Celular , Proteína p300 Associada a E1A/fisiologia , Regulação da Expressão Gênica , Genes Reguladores , Células HEK293 , Humanos , Rim/metabolismo , Modelos Animais , Morfolinos , Osteoblastos/metabolismo , Osteogênese/fisiologia , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
3.
J Biol Chem ; 293(15): 5478-5491, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29475951

RESUMO

Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Stx3 (syntaxin 3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform that we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5 (RAN-binding protein 5), targets to the nucleus, and interacts physically and functionally with several transcription factors, including ETV4 (ETS variant 4) and ATF2 (activating transcription factor 2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified, suggesting that nuclear signaling is a conserved, novel function common among these membrane-trafficking proteins.


Assuntos
Proteínas E1A de Adenovirus/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Qa-SNARE/metabolismo , Transdução de Sinais , beta Carioferinas/metabolismo , Proteínas E1A de Adenovirus/genética , Animais , Células COS , Células CACO-2 , Núcleo Celular/genética , Chlorocebus aethiops , Cães , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Proteínas Qa-SNARE/genética , Solubilidade , beta Carioferinas/genética
4.
PLoS Biol ; 13(7): e1002200, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26196739

RESUMO

The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host-pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such "modifiers."


Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Receptores Citoplasmáticos e Nucleares/metabolismo , Fator de Transcrição STAT1/metabolismo , Toxoplasma/imunologia , Animais , Encéfalo/metabolismo , Infecções do Sistema Nervoso Central/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9/metabolismo , Doença Crônica , Interferon gama/metabolismo , Camundongos Endogâmicos CBA
5.
Proc Natl Acad Sci U S A ; 112(40): 12420-5, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26392558

RESUMO

Regulated degradation of proteins by the proteasome is often critical to their function in dynamic cellular pathways. The molecular clock underlying mammalian circadian rhythms relies on the rhythmic expression and degradation of its core components. However, because the tools available for identifying the mechanisms underlying the degradation of a specific protein are limited, the mechanisms regulating clock protein degradation are only beginning to be elucidated. Here we describe a cell-based functional screening approach designed to quickly identify the ubiquitin E3 ligases that induce the degradation of potentially any protein of interest. We screened the nuclear hormone receptor RevErbα (Nr1d1), a key constituent of the mammalian circadian clock, for E3 ligases that regulate its stability and found Seven in absentia2 (Siah2) to be a key regulator of RevErbα stability. Previously implicated in hypoxia signaling, Siah2 overexpression destabilizes RevErbα/ß, and siRNA depletion of Siah2 stabilizes endogenous RevErbα. Moreover, Siah2 depletion delays circadian degradation of RevErbα and lengthens period length. These results demonstrate the utility of functional screening approaches for identifying regulators of protein stability and reveal Siah2 as a previously unidentified circadian clockwork regulator that mediates circadian RevErbα turnover.


Assuntos
Relógios Circadianos/genética , Proteínas Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Ubiquitina-Proteína Ligases/genética , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Camundongos , Microscopia de Fluorescência , Proteínas Nucleares/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Proteólise , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/metabolismo
6.
PLoS Biol ; 12(4): e1001840, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24737000

RESUMO

Over the last decades, researchers have characterized a set of "clock genes" that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Relógios Circadianos/fisiologia , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Inteligência Artificial , Linhagem Celular , Relógios Circadianos/genética , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/biossíntese , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Criptocromos/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Alinhamento de Sequência , Transcrição Gênica/genética
7.
Proc Natl Acad Sci U S A ; 109(38): 15348-53, 2012 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-22949635

RESUMO

Endoderm-mesenchyme cross-talk is a central process in the development of foregut-derived organs. How signaling pathways integrate the activity of multiple ligands to guide organ development is poorly understood. We show that two Wnt ligands, Wnt2 and Wnt7b, cooperatively induce Wnt signaling without affecting the stabilization of the Wnt canonical effector ß-catenin despite it being necessary for Wnt2-Wnt7b cooperativity. Wnt2-Wnt7b cooperation is specific for mesenchymal cell lineages and the combined loss of Wnt2 and Wnt7b leads to more severe developmental defects in the lung than loss of Wnt2 or Wnt7b alone. High-throughput small-molecule screens and biochemical assays reveal that the Pdgf pathway is required for cooperative Wnt2-Wnt7b signaling. Inhibition of Pdgf signaling in cell culture reduces Wnt2-Wnt7b cooperative signaling. Moreover, inhibition of Pdgf signaling in lung explant cultures results in decreased Wnt signaling and lung smooth-muscle development. These data suggest a model in which Pdgf signaling potentiates Wnt2-Wnt7b signaling to promote high levels of Wnt activity in mesenchymal progenitors that is required for proper development of endoderm-derived organs, such as the lung.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt2/metabolismo , Animais , Linhagem Celular , Linhagem da Célula , Epitélio/metabolismo , Humanos , Ligantes , Pulmão/metabolismo , Mesoderma/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo , Organogênese/genética , Transdução de Sinais
8.
Nat Genet ; 38(3): 312-9, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16474406

RESUMO

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica , Transativadores/genética , Células 3T3 , Fatores de Transcrição ARNTL , Animais , Proteínas CLOCK , Linhagem Celular , Retroalimentação , Genes Reporter , Humanos , Luciferases/análise , Luciferases/genética , Luminescência , Camundongos , Plasmídeos , Tempo
9.
Annu Rev Pharmacol Toxicol ; 50: 187-214, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20055702

RESUMO

The physiology of a wide variety of organisms is organized according to periodic environmental changes imposed by the earth's rotation. This way, a large number of physiological processes present diurnal rhythms regulated by an internal timing system called the circadian clock. As part of the rhythmicity in physiology, drug efficacy and toxicity can vary with time. Studies over the past four decades present diurnal oscillations in drug absorption, distribution, metabolism, and excretion. On the other hand, diurnal variations in the availability and sensitivity of drug targets have been correlated with time-dependent changes in drug effectiveness. In this review, we provide evidence supporting the regulation of drug kinetics and dynamics by the circadian clock. We also use the examples of hypertension and cancer to show current achievements and challenges in chronopharmacology.


Assuntos
Relógios Biológicos/genética , Ritmo Circadiano , Farmacocinética , Farmacologia , Fatores de Transcrição ARNTL/genética , Animais , Pressão Sanguínea , Proteínas CLOCK/genética , GMP Cíclico/biossíntese , Humanos , Hipertensão/tratamento farmacológico , Neoplasias/tratamento farmacológico , Óxido Nítrico/biossíntese , Sistema Renina-Angiotensina/efeitos dos fármacos , Núcleo Supraquiasmático/fisiologia
10.
PLoS Biol ; 7(3): e52, 2009 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-19278294

RESUMO

The mammalian circadian clock is a cell-autonomous system that drives oscillations in behavior and physiology in anticipation of daily environmental change. To assess the robustness of a human molecular clock, we systematically depleted known clock components and observed that circadian oscillations are maintained over a wide range of disruptions. We developed a novel strategy termed Gene Dosage Network Analysis (GDNA) in which small interfering RNA (siRNA)-induced dose-dependent changes in gene expression were used to build gene association networks consistent with known biochemical constraints. The use of multiple doses powered the analysis to uncover several novel network features of the circadian clock, including proportional responses and signal propagation through interacting genetic modules. We also observed several examples where a gene is up-regulated following knockdown of its paralog, suggesting the clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. We propose that these network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation.


Assuntos
Relógios Biológicos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Mamíferos/genética , Animais , Duplicação Gênica , Técnicas de Silenciamento de Genes , Humanos , Mamíferos/fisiologia , Camundongos , Camundongos Knockout , Modelos Animais , Modelos Genéticos , RNA Interferente Pequeno , Transdução de Sinais
11.
PLoS Genet ; 5(4): e1000442, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19343201

RESUMO

The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse.


Assuntos
Ritmo Circadiano , Fígado/fisiologia , Mamíferos/genética , Transcrição Gênica , Animais , Linhagem Celular , Células Cultivadas , Expressão Gênica , Humanos , Masculino , Mamíferos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3
12.
Bioinformatics ; 24(23): 2794-5, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18931366

RESUMO

UNLABELLED: Oscillations in mRNA and protein of circadian clock components can be continuously monitored in vitro using synchronized cell lines. These rhythms can be highly variable due to culture conditions and are non-stationary due to baseline trends, damping and drift in period length. We present a technique for characterizing the modal frequencies of oscillation using continuous wavelet decomposition to non-parametrically model changes in amplitude and period while removing baseline effects and noise. AVAILABILITY: The method has been implemented as the package waveclock for the free statistical software program R and is available for download from http://cran.r-project.org/


Assuntos
Ritmo Circadiano/fisiologia , Biologia Computacional/métodos , Algoritmos , Animais , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
13.
Curr Opin Genet Dev ; 15(6): 634-9, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16226457

RESUMO

Comparative genomics approaches are having a remarkable impact on the study of transcriptional regulation in eukaryotes. Many eukaryotic genome sequences are being explored by new computational methods and high-throughput experimental tools such as DNA arrays and genome-wide location analyses. These tools are enabling efficient panning for common regulatory cassettes underlying fundamental biological processes, extending the use of existing techniques for the discovery of response elements to mammals, deciphering the transcriptional regulatory code in eukaryotes and providing the first global insights into a recently described post-transcriptional regulatory mechanism. Collectively, these approaches are rapidly expanding both our knowledge and our definition of transcriptional regulation.


Assuntos
Células Eucarióticas/fisiologia , Regulação da Expressão Gênica/genética , Genômica , Transcrição Gênica/genética , Animais , Regulação da Expressão Gênica/fisiologia , Humanos , Transcrição Gênica/fisiologia
14.
Curr Biol ; 13(3): 189-98, 2003 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-12573214

RESUMO

BACKGROUND: Different types of regulation are utilized to produce a robust circadian clock, including regulation at the transcriptional, posttranscriptional, and translational levels. A screen for rhythmic messages that may be involved in such circadian control identified nocturnin, a novel gene that displays high-amplitude circadian expression in the Xenopus laevis retina, with peak mRNA levels in the early night. Expression of nocturnin mRNA is confined to the clock-containing photoreceptor cell layer within the retina. RESULTS: In these studies, we show that nocturnin removes the poly(A) tail from a synthetic RNA substrate in a process known as deadenylation. Nocturnin nuclease activity is magnesium dependent, as the addition of EDTA or mutation of the residue predicted to bind magnesium disrupts deadenylation. Substrate preference studies show that nocturnin is an exonuclease that specifically degrades the 3' poly(A) tail. While nocturnin is rhythmically expressed in the cytoplasm of the retinal photoreceptor cells, the only other described vertebrate deadenylase, PARN, is constitutively present in most retinal cells, including the photoreceptors. CONCLUSIONS: The distinct spatial and temporal expression of nocturnin and PARN suggests that there may be specific mRNA targets of each deadenylase. Since deadenylation regulates mRNA decay and/or translational silencing, we propose that nocturnin deadenylates clock-related transcripts in a novel mechanism for posttranscriptional regulation in the circadian clock or its outputs.


Assuntos
Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica , Proteínas/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Retina/enzimologia , Animais , Relógios Biológicos/fisiologia , Exorribonucleases/genética , Exorribonucleases/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Magnésio/metabolismo , Proteínas Nucleares , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Fatores de Transcrição , Xenopus laevis
15.
Methods Mol Biol ; 317: 243-54, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16264233

RESUMO

Within the retina there is a circadian clock that controls the 24-h timing of processes such as hormone release, cell movement, and gene transcription. In an effort to better understand the molecular nature of this retinal clock, a differential display (DD) screen was performed to isolate a gene with high amplitude circadian rhythmicity in the Xenopus retina. A novel gene expressed in the early evening in photoreceptor cells was isolated and named nocturnin for night factor. This article outlines the steps we took to study a protein of unknown function, particularly highlighting the analyses one can perform when little more than the primary sequence of a gene is known. In addition, we describe the results of sequence analysis that assisted in predicting the function of nocturnin. We have shown that nocturnin acts as a deadenylase in vitro, removing the poly(A) tail from a mature messenger RNA in a process that either leads to degradation or translational silencing of a message. Although the role of nocturnin in the retina is unknown, future studies to identify target mRNAs that are deadenylated by nocturnin will assist in elucidating its physiological role in this tissue.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Proteínas/genética , Proteínas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ritmo Circadiano , Ácido Edético/química , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Proteínas Nucleares , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas/química , RNA Mensageiro/metabolismo , Retina/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição , Transcrição Gênica , Xenopus
16.
Cell Rep ; 9(5): 1885-1895, 2014 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-25466249

RESUMO

Loss of Pax3, a developmentally regulated transcription factor expressed in premigratory neural crest, results in severe developmental defects and embryonic lethality. Although Pax3 mutations produce profound phenotypes, the intrinsic transcriptional activation exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional coactivators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as Pax3 coactivators. Synergistic coactivation of target genes by Pax3-Taz/Yap65 requires DNA binding by Pax3, is Tead independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 colocalize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryo-lethal neural crest defects and decreased expression of the Pax3 target gene, Mitf. These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Melanócitos/metabolismo , Crista Neural/citologia , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Genes Reporter , Células HEK293 , Via de Sinalização Hippo , Humanos , Luciferases/biossíntese , Luciferases/genética , Camundongos Transgênicos , Fator de Transcrição PAX3 , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Ativação Transcricional
17.
PLoS One ; 8(1): e55782, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383281

RESUMO

Previous studies have demonstrated that certain Wnt ligands can promote high levels of cooperative signaling in a cell type specific manner. To explore the underlying mechanism of this cooperative Wnt signaling, we performed a high-throughput screen of more than 14,000 cDNAs to identify genes that promote cooperative Wnt signaling in the context of a single Wnt ligand, Wnt2. This screen identified several homeobox factors including Msx2, Nkx5.2, and Esx1, in addition to other factors known to promote Wnt signaling including Pias4. Generation of dominant-active or dominant-negative forms of Msx2 indicate that the mechanism by which homeobox factors cooperatively promote Wnt signaling is through their ability to repress gene transcription. These data identify a broad homeobox code, which acts to increase Wnt signaling through transcriptional repression.


Assuntos
Genômica , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genômica/métodos , Ensaios de Triagem em Larga Escala , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Ligantes , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
18.
Dev Cell ; 22(1): 197-210, 2012 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-22178500

RESUMO

Mutations in Pkd1, encoding polycystin-1 (PC1), cause autosomal-dominant polycystic kidney disease (ADPKD). We show that the carboxy-terminal tail (CTT) of PC1 is released by γ-secretase-mediated cleavage and regulates the Wnt and CHOP pathways by binding the transcription factors TCF and CHOP, disrupting their interaction with the common transcriptional coactivator p300. Loss of PC1 causes increased proliferation and apoptosis, while reintroducing PC1-CTT into cultured Pkd1 null cells reestablishes normal growth rate, suppresses apoptosis, and prevents cyst formation. Inhibition of γ-secretase activity impairs the ability of PC1 to suppress growth and apoptosis and leads to cyst formation in cultured renal epithelial cells. Expression of the PC1-CTT is sufficient to rescue the dorsal body curvature phenotype in zebrafish embryos resulting from either γ-secretase inhibition or suppression of Pkd1 expression. Thus, γ-secretase-dependent release of the PC1-CTT creates a protein fragment whose expression is sufficient to suppress ADPKD-related phenotypes in vitro and in vivo.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Apoptose , Fatores de Transcrição TCF/metabolismo , Canais de Cátion TRPP/fisiologia , Fator de Transcrição CHOP/metabolismo , Peixe-Zebra/metabolismo , Fatores de Transcrição de p300-CBP/genética , Animais , Proliferação de Células , Células Cultivadas , Cistos/etiologia , Cistos/metabolismo , Cistos/patologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Rim/metabolismo , Rim/patologia , Fenótipo , Rim Policístico Autossômico Dominante/fisiopatologia , Fatores de Transcrição TCF/genética , Canais de Cátion TRPP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Ativação Transcricional , Via de Sinalização Wnt , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Fatores de Transcrição de p300-CBP/metabolismo
19.
Curr Opin Genet Dev ; 20(6): 581-7, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20926286

RESUMO

The circadian clock is an endogenous oscillator that regulates daily rhythms in behavior and physiology. In recent years, systems biology and genomics approaches re-shaped our view of the clock. Our understanding of outputs that regulate behavior and physiology has been enhanced through gene expression profiling and proteomic analyses. Systems approaches uncovered underlying principles of transcriptional regulation and robustness of the oscillator through perturbation analysis and synthetic methods. Finally, new clock components and modifiers were identified through cell-based screening efforts and proteomics.


Assuntos
Ritmo Circadiano , Genômica , Biologia de Sistemas , Animais , Humanos
20.
Artigo em Inglês | MEDLINE | ID: mdl-20836017

RESUMO

The conventional target centric model of drug discovery is pinned under the weight of prior success and the traditional problems of safety and efficacy for new molecules. An alternative to target centric drug development is to shift focus to the pathways that mediate both biology and pathophysiology. This method has the advantage of not requiring a priori knowledge of the small molecule target, but also comes with it several challenges including target determination. We suggest extending this notion more broadly across the drug discovery process using quantitative network structure-activity relationships (QNSAR), and discuss the steps necessary to test the hypothesis that systems biology approaches can be used to improve the drug discovery process.


Assuntos
Descoberta de Drogas , Biologia de Sistemas , Algoritmos , Química Farmacêutica/métodos , Genoma Humano , Genômica , Humanos , Proteômica , Relação Quantitativa Estrutura-Atividade
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