RESUMO
Currently, microbial conversion of lignocellulose-derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome-scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole-genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations' involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next-generation biofuels.
Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Lignina/metabolismo , Xilose/metabolismoRESUMO
Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.
RESUMO
The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.
Assuntos
Farmacorresistência Bacteriana , Escherichia coli K12 , Proteínas de Escherichia coli , Etanol/farmacologia , Biossíntese de Proteínas , Solventes/farmacologia , Transcrição Gênica , Alelos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Estudo de Associação Genômica Ampla , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Ribossomos/metabolismo , Algoritmos , Imunoprecipitação da Cromatina , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Deleção de Genes , Fases de Leitura Aberta , RNA de Transferência/metabolismo , Ribossomos/química , Fator sigma/química , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismoRESUMO
Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Proteínas de Escherichia coli/genética , Deleção de Genes , Genoma Bacteriano , Fatores de Alongamento de Peptídeos/genética , Ligação Proteica , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (â¼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.
Assuntos
Escherichia coli K12/metabolismo , Etanol/metabolismo , Etanol/toxicidade , Estresse Fisiológico , Zea mays/metabolismo , Aminoácidos/metabolismo , Anaerobiose , Escherichia coli K12/crescimento & desenvolvimento , Fermentação , Perfilação da Expressão Gênica , Glucose/metabolismoRESUMO
The flap domain of multisubunit RNA polymerases (RNAPs), also called the wall, forms one side of the RNA exit channel. In bacterial RNAP, the mobile part of the flap is called the flap tip and makes essential contacts with initiation and elongation factors. Cocrystal structures suggest that the orthologous part of eukaryotic RNAPII, called the flap loop, contacts transcription factor IIB (TFIIB), but the function of the flap loop has not been assessed. We constructed and tested a deletion of the flap loop in human RNAPII (subunit RPB2 Δ873-884) that removes the flap loop interaction interface with TFIIB. Genome-wide analysis of the distribution of the RNAPII with the flap loop deletion expressed in a human embryonic kidney cell line (HEK 293) revealed no effect of the flap loop on global transcription initiation, RNAPII occupancy within genes, or the efficiency of promoter escape and productive elongation. In vitro, the flap loop deletion had no effect on promoter binding, abortive initiation or promoter escape, TFIIS-stimulated transcript cleavage, or inhibition of transcript elongation by the complex of negative elongation factor (NELF) and 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). A modest effect on transcript elongation and pausing was suppressed by TFIIF. Although similar to the flap tip of bacterial RNAP, the RNAPII flap loop is not equivalently essential.
Assuntos
RNA Polimerase II/química , RNA Polimerase II/genética , Transcrição Gênica , Sítios de Ligação/genética , Linhagem Celular , Humanos , Proteínas Nucleares , Regiões Promotoras Genéticas , Engenharia de Proteínas , Estrutura Terciária de Proteína , Deleção de Sequência , Fator de Transcrição TFIIB , Fatores de Transcrição , Fatores de Transcrição TFII , Fatores de Elongação da TranscriçãoRESUMO
Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the -2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring -1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the -2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.
Assuntos
Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Hematopoese/genética , Hematopoese/fisiologia , Motivos de Aminoácidos , Animais , Eritropoese/genética , Eritropoese/fisiologia , Fator de Transcrição GATA2/química , Expressão Gênica , Técnicas de Introdução de Genes , Genes de Troca , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
In development, lineage-restricted transcription factors simultaneously promote differentiation while repressing alternative fates. Molecular dissection of this process has been challenging as transcription factor loci are regulated by many trans-acting factors functioning through dispersed cis elements. It is not understood whether these elements function collectively to confer transcriptional regulation, or individually to control specific aspects of activation or repression, such as initiation versus maintenance. Here, we have analyzed cis element regulation of the critical hematopoietic factor Gata2, which is expressed in early precursors and repressed as GATA-1 levels rise during terminal differentiation. We engineered mice lacking a single cis element -1.8 kb upstream of the Gata2 transcriptional start site. Although Gata2 is normally repressed in late-stage erythroblasts, the -1.8 kb mutation unexpectedly resulted in reactivated Gata2 transcription, blocked differentiation, and an aberrant lineage-specific gene expression pattern. Our findings demonstrate that the -1.8 kb site selectively maintains repression, confers a specific histone modification pattern and expels RNA Polymerase II from the locus. These studies reveal how an individual cis element establishes a normal developmental program via regulating specific steps in the mechanism by which a critical transcription factor is repressed.
Assuntos
Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Animais , Pareamento de Bases/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Células Eritroides/metabolismo , Eritropoese/genética , Fator de Transcrição GATA2/metabolismo , Deleção de Genes , Marcação de Genes , Loci Gênicos/genética , Histonas/metabolismo , Camundongos , Camundongos Mutantes , Nucleoproteínas/metabolismo , Ligação Proteica , RNA Polimerase II/metabolismo , Estresse Fisiológico/genéticaRESUMO
Increased alpha-synuclein gene (SNCA) dosage due to locus multiplication causes autosomal dominant Parkinson's disease (PD). Variation in SNCA expression may be critical in common, genetically complex PD but the underlying regulatory mechanism is unknown. We show that SNCA and the heme metabolism genes ALAS2, FECH, and BLVRB form a block of tightly correlated gene expression in 113 samples of human blood, where SNCA naturally abounds (validated P = 1.6 x 10(-11), 1.8 x 10(-10), and 6.6 x 10(-5)). Genetic complementation analysis revealed that these four genes are co-induced by the transcription factor GATA-1. GATA-1 specifically occupies a conserved region within SNCA intron-1 and directly induces a 6.9-fold increase in alpha-synuclein. Endogenous GATA-2 is highly expressed in substantia nigra vulnerable to PD, occupies intron-1, and modulates SNCA expression in dopaminergic cells. This critical link between GATA factors and SNCA may enable therapies designed to lower alpha-synuclein production.
Assuntos
Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica/fisiologia , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Animais , Northern Blotting , Western Blotting , Células Cultivadas , Biologia Computacional , Ensaio de Imunoadsorção Enzimática , Teste de Complementação Genética , Humanos , Imuno-Histoquímica , Camundongos , Análise em Microsséries , RNA Interferente Pequeno/genéticaRESUMO
Whereas the transcription factors GATA-1 and GATA-2 function both uniquely and redundantly to control blood cell development, the process termed hematopoiesis, mechanisms underlying their unique versus common functions are poorly understood. We used two independent assays to demonstrate that GATA-1 is considerably more stable than GATA-2 in multiple cellular contexts, even though both factors are subject to degradation via the ubiquitin-proteasome system. Studies with GATA factor mutants and novel chimeric GATA factors provided evidence that both GATA-1 and GATA-2 have highly unstable zinc finger core modules. The GATA-1 and GATA-2 N-termini both confer stabilization to their respective zinc finger core modules. In contrast, the GATA-1 and GATA-2 C-termini confer stabilization and destabilization, respectively. As GATA-2 stabilization via proteasome inhibition impairs the capacity of GATA-1 to displace GATA-2 from endogenous chromatin sites, we propose that differential GATA factor stability is an important determinant of chromatin target site occupancy and therefore the establishment of genetic networks that control hematopoiesis.
Assuntos
Cromatina/metabolismo , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA2/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Leupeptinas/farmacologia , Camundongos , Mutação/fisiologia , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
GATA factors are fundamental components of developmentally important transcriptional networks. By contrast to common mechanisms in which transacting factors function directly at promoters, the hematopoietic GATA factors GATA-1 and GATA-2 often assemble dispersed complexes over broad chromosomal regions. For example, GATA-1 and GATA-2 occupy five conserved regions over approximately 100 kb of the Gata2 locus in the transcriptionally repressed and active states, respectively, in erythroid cells. Since it is unknown whether the individual complexes exert qualitatively distinct or identical functions to regulate Gata2 transcription in vivo, we compared the activity of the -3.9 and +9.5 kb sites of the Gata2 locus in transgenic mice. The +9.5 site functioned as an autonomous enhancer in the endothelium and fetal liver of embryonic day 11 embryos, whereas the -3.9 site lacked such activity. Mechanistic studies demonstrated critical requirements for a GATA motif and a neighboring E-box within the +9.5 site for enhancer activity in endothelial and hematopoietic cells. Surprisingly, whereas this GATA-E-box composite motif was sufficient for enhancer activity in an erythroid precursor cell line, its enhancer function in primary human endothelial cells required additional regulatory modules. These results identify the first molecular determinant of Gata2 transcription in vascular endothelium, composed of a core enhancer module active in both endothelial and hematopoietic cells and regulatory modules preferentially required in endothelial cells.
Assuntos
Endotélio Vascular/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica , Sistema Hematopoético/metabolismo , Transcrição Gênica , Animais , Aorta/citologia , Aorta/metabolismo , Sequência de Bases , Células Cultivadas , Endotélio Vascular/citologia , Elementos Facilitadores Genéticos , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Sistema Hematopoético/citologia , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Ativação Transcricional , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at -77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the -77 kb region. The -77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the -77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.
Assuntos
Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Acetilação , Animais , Pareamento de Bases/genética , Sequência de Bases , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Sequência Conservada/genética , Desoxirribonuclease I/metabolismo , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The hematopoietic GATA factors GATA-1 and GATA-2, which have distinct and overlapping roles to regulate blood cell development, are reciprocally expressed during erythropoiesis. GATA-1 directly represses Gata2 transcription, and reduced GATA-2 synthesis promotes red blood cell development. Gata2 repression involves "GATA switches" in which GATA-1 displaces GATA-2 from Gata2 regulatory regions. We show that extragenic GATA switch sites occupied by GATA-2 associate with as much RNA polymerase II (Pol II) and basal transcription factors as present at the active Gata2 promoters. Pol II bound to GATA switch sites in the active locus was phosphorylated on serine 5 of the carboxyl-terminal domain, indicative of elongation competence. GATA-1-mediated displacement of GATA-2 from GATA switch sites reduced Pol II recruitment to all sites except the far upstream -77-kb region. Surprisingly, TFIIB occupancy persisted at most sites upon repression. These results indicate that GATA-2-bound extragenic regulatory elements recruit Pol II, GATA-1 binding expels Pol II, and despite the persistent TFIIB-chromatin complexes, Pol II recruitment is blocked.
Assuntos
Fator de Transcrição GATA1/metabolismo , Fator de Transcrição TFIIB/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , DNA Polimerase II/metabolismo , Primers do DNA/genética , Eritropoese/genética , Eritropoese/fisiologia , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Hematopoese , Humanos , Técnicas In Vitro , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição TFIIB/genética , Transcrição GênicaRESUMO
One of the most common methods for providing postoperative analgesia is via patient-controlled analgesia (PCA). Although the typical approach is to administer opioids via a programmable infusion pump, other drugs and other modes of administration are available. This article reviews the history and practice of many aspects of PCA and provides extensive guidelines for the practice of PCA-administered opioids. In addition, potential adverse effects and recommendations for their monitoring and treatment are reviewed.
Assuntos
Analgesia Controlada pelo Paciente , Analgesia Controlada pelo Paciente/instrumentação , Analgesia Controlada pelo Paciente/métodos , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Humanos , Bombas de Infusão , Infusões Intravenosas/instrumentação , Seleção de PacientesRESUMO
Cis elements that mediate transcription factor binding are abundant within genomes, but the rules governing occupancy of such motifs in chromatin are not understood. The transcription factor GATA-1 that regulates red blood cell development binds with high affinity to GATA motifs, and initial studies suggest that these motifs are often unavailable for occupancy in chromatin. Whereas GATA-2 regulates the differentiation of all blood cell lineages via GATA motif binding, the specificity of GATA-2 chromatin occupancy has not been studied. We found that conditionally active GATA-1 (ER-GATA-1) and GATA-2 occupy only a small subset of the conserved GATA motifs within the murine beta-globin locus. Kinetic analyses in GATA-1-null cells indicated that ER-GATA-1 preferentially occupied GATA motifs at the locus control region (LCR), in which chromatin accessibility is largely GATA-1-independent. Subsequently, ER-GATA-1 increased promoter accessibility and occupied the betamajor promoter. ER-GATA-1 increased erythroid Krüppel-like factor and SWI/SNF chromatin remodeling complex occupancy at restricted LCR sites. These studies revealed three phases of beta-globin locus activation: GATA-1-independent establishment of specific chromatin structure features, GATA-1-dependent LCR complex assembly, and GATA-1-dependent promoter complex assembly. The differential utilization of dispersed GATA motifs therefore establishes spatial/temporal regulation and underlies the multistep activation mechanism.
Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , DNA/metabolismo , Fator de Transcrição GATA1/fisiologia , Animais , Linhagem Celular , Cromatina/química , Sequência Conservada , DNA Helicases , Fator de Transcrição GATA2/metabolismo , Globinas/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologiaRESUMO
The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5' portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.
Assuntos
Alelos , Células Endoteliais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Região 5'-Flanqueadora/genética , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Íntrons/genética , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Receptor Notch1 , Receptor Notch4 , Receptores de Superfície Celular/genética , Receptores Notch , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/genéticaRESUMO
Given the simplicity of the DNA sequence that mediates binding of GATA transcription factors, GATA motifs reside throughout chromosomal DNA. However, chromatin immunoprecipitation analysis has revealed that GATA-1 discriminates exquisitely among these sites. GATA-2 selectively occupies the -2.8-kilobase (kb) region of the GATA-2 locus in the active state despite there being numerous GATA motifs throughout the locus. The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription. We have used high resolution chromatin immunoprecipitation to show that GATA-1 and GATA-2 occupy two additional regions, -3.9 and -1.8 kb of the GATA-2 locus. GATA-1 and GATA-2 had distinct preferences for occupancy at these regions, with GATA-1 and GATA-2 occupancy highest at the -3.9- and -1.8-kb regions, respectively. Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and GATA-2 displacement at the sites. In the transcriptionally active state, DNase I hypersensitive sites (HSs) were detected at the -3.9- and -1.8-kb regions, with a weak HS at the -2.8-kb region. Whereas ER-GATA-1-instigated repression abolished the -1.8-kb HS, the -3.9-kb HS persisted in the repressed state. Transient transfection analysis provided evidence that the -3.9-kb region functions distinctly from the -2.8- and -1.8-kb regions. We propose that GATA-2 transcription is regulated via the collective actions of complexes assembled at the -2.8- and -1.8-kb regions, which share similar properties, and through a qualitatively distinct activity of the -3.9-kb complex.
Assuntos
Proteínas de Ligação a DNA/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Fator de Transcrição GATA2 , Regulação da Expressão Gênica , Camundongos , Células NIH 3T3 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Elucidating mechanisms controlling nuclear processes requires an understanding of the nucleoprotein structure of genes at endogenous chromosomal loci. Traditional approaches to measuring protein-DNA interactions in vitro have often failed to provide insights into physiological mechanisms. Given that most transcription factors interact with simple DNA sequence motifs, which are abundantly distributed throughout a genome, it is essential to pinpoint the small subset of sites bound by factors in vivo. Signaling mechanisms induce the assembly and modulation of complex patterns of histone acetylation, methylation, phosphorylation, and ubiquitination, which are crucial determinants of chromatin accessibility. These seemingly complex issues can be directly addressed by a powerful methodology termed the chromatin immunoprecipitation (ChIP) assay. ChIP analysis involves covalently trapping endogenous proteins at chromatin sites, thereby yielding snapshots of protein-DNA interactions and histone modifications within living cells. The chromatin is sonicated to generate small fragments, and an immunoprecipitation is conducted with an antibody against the desired factor or histone modification. Crosslinks are reversed, and polymerase chain reaction (PCR) is used to assess whether DNA sequences are recovered immune-specifically. Chromatin-domain scanning coupled with quantitative analysis is a powerful means of dissecting mechanisms by which signaling pathways target genes within a complex genome.
Assuntos
Cromatina/química , Proteínas de Ligação a DNA/análise , Testes de Precipitina/métodos , Transdução de Sinais , Fatores de Transcrição/análise , Acetilação , Animais , Núcleo Celular/genética , Cromatina/metabolismo , DNA/análise , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/imunologia , Histonas/metabolismo , Metilação , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismoRESUMO
RNA polymerase II (Pol II) can associate with regulatory elements far from promoters. For the murine beta-globin locus, Pol II binds the beta-globin locus control region (LCR) far upstream of the beta-globin promoters, independent of recruitment to and activation of the betamajor promoter. We describe here an analysis of where Pol II resides within the LCR, how it is recruited to the LCR, and the functional consequences of recruitment. High-resolution analysis of the distribution of Pol II revealed that Pol II binding within the LCR is restricted to the hypersensitive sites. Blocking elongation eliminated the synthesis of genic and extragenic transcripts and eliminated Pol II from the betamajor open reading frame. However, the elongation blockade did not redistribute Pol II at the hypersensitive sites, suggesting that Pol II is recruited to these sites. The distribution of Pol II did not strictly correlate with the distributions of histone acetylation and methylation. As Pol II associates with histone-modifying enzymes, Pol II tracking might be critical for establishing and maintaining broad histone modification patterns. However, blocking elongation did not disrupt the histone modification pattern of the beta-globin locus, indicating that Pol II tracking is not required to maintain the pattern.