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Reduced skeletal muscle mass and oxidative capacity coexist in patients with pulmonary emphysema and are independently associated with higher mortality. If reduced cellular respiration contributes to muscle atrophy in that setting remains unknown. Using a mouse with genetically induced pulmonary emphysema that recapitulates muscle dysfunction, we found that reduced activity of succinate dehydrogenase (SDH) is a hallmark of its myopathic changes. We generated an inducible, muscle-specific SDH knockout mouse that demonstrates lower mitochondrial oxygen consumption, myofiber contractility, and exercise endurance. Respirometry analyses show that in vitro complex I respiration is unaffected by loss of SDH subunit C in muscle mitochondria, which is consistent with the pulmonary emphysema animal data. SDH knockout initially causes succinate accumulation associated with a down-regulated transcriptome but modest proteome effects. Muscle mass, myofiber type composition, and overall body mass constituents remain unaltered in the transgenic mice. Thus, while SDH regulates myofiber respiration in experimental pulmonary emphysema, it does not control muscle mass or other body constituents.
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Respiración de la Célula , Ratones Noqueados , Contracción Muscular , Músculo Esquelético , Enfisema Pulmonar , Succinato Deshidrogenasa , Animales , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/genética , Enfisema Pulmonar/patología , Enfisema Pulmonar/etiología , Succinato Deshidrogenasa/metabolismo , Succinato Deshidrogenasa/genética , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Complejo II de Transporte de Electrones/metabolismo , Complejo II de Transporte de Electrones/genética , Modelos Animales de Enfermedad , Ratones Transgénicos , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Consumo de OxígenoRESUMEN
Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes.
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In this issue of Cell Chemical Biology, Bush et al.1 report an in vitro selection method for optimizing CRISPR-Cas9 single-guide RNAs. This approach may be useful in targeting previously intractable genomic sequences. The results also provide insights into which positions in single-guide RNAs are most amenable to modification.
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Sistemas CRISPR-Cas , Genómica , Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-CasRESUMEN
Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and ß-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. ß-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals ß-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa.
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Neoplasias de la Próstata , Regulador Transcripcional ERG , Proteína p53 Supresora de Tumor , Animales , Humanos , Masculino , Ratones , beta Catenina/genética , beta Catenina/metabolismo , Mutación con Ganancia de Función , Proteínas de Fusión Oncogénica/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proto-Oncogenes , Pirimidinas/biosíntesis , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" that react with phenoxy radicals or biotinoyl-5'-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.
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The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly.
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Chaperonas de Histonas , Nucleosomas , Humanos , Cromatina , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Factores de Elongación Transcripcional/genéticaRESUMEN
The quantitative polymerase chain reaction (qPCR) with detection of duplex DNA yield by intercalator fluorescence is a common and essential technique in nucleic acid analysis. We encountered unexpected results when applying standard qPCR methods to the quantitation of random DNA libraries flanked by regions of fixed sequence, a configuration essential for in vitro selection experiments. Here we describe the results of experiments revealing why conventional qPCR methods can fail to allow automated analysis in such cases, and simple solutions to this problem. In particular we show that renaturation of PCR products containing random regions is incomplete in late PCR cycles when extension fails due to reagent depletion. Intercalator fluorescence can then be lost at standard interrogation temperatures. We show that qPCR analysis of random DNA libraries can be achieved simply by adjusting the step at which intercalator fluorescence is monitored so that the yield of annealed constant regions is detected rather than the yield of full duplex DNA products.
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Aptámeros de Nucleótidos , Aptámeros de Nucleótidos/genética , ADN/análisis , ADN/genética , Biblioteca de Genes , Sustancias Intercalantes , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.
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ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Operón Lac , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Mutations in any of the genes encoding the four subunits of succinate dehydrogenase (SDH), a mitochondrial membrane-bound enzyme complex that is involved in both the tricarboxylic acid cycle and the electron transport chain, can lead to a variety of disorders. Recognized conditions with such mutations include Leigh syndrome and hereditary tumors such as pheochromocytoma and paraganglioma (PPGL), renal cell carcinoma, and gastrointestinal stromal tumor. Tumors appear in SDH mutation carriers with dominant inheritance due to loss of heterozygosity in susceptible cells. Here, we describe a mouse model intended to reproduce hereditary PPGL through Cre-mediated loss of SDHC in cells that express tyrosine hydroxylase (TH), a compartment where PPGL is known to originate. We report that while there is modest expansion of TH+ glomus cells in the carotid body upon SDHC loss, PPGL is not observed in such mice, even in the presence of a conditional dominant negative p53 protein and chronic hypoxia. Instead, we report an unexpected phenotype of nondiabetic obesity beginning at about 20 weeks of age. We hypothesize that this obesity is caused by TH+ cell loss or altered phenotype in key compartments of the central nervous system responsible for regulating feeding behavior, coupled with metabolic changes due to loss of peripheral catecholamine production.
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Neoplasias de las Glándulas Suprarrenales/genética , Modelos Animales de Enfermedad , Síndromes Neoplásicos Hereditarios/genética , Obesidad/genética , Fenotipo , Feocromocitoma/genética , Succinato Deshidrogenasa/genética , Neoplasias de las Glándulas Suprarrenales/patología , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Síndromes Neoplásicos Hereditarios/patología , Obesidad/patología , Feocromocitoma/patología , Succinato Deshidrogenasa/deficienciaRESUMEN
Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H2 O2 is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.
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Ácidos Nucleicos/metabolismo , Fenoles/metabolismo , Biotina/análogos & derivados , Biotina/química , Biotina/metabolismo , Biotinilación , ADN/química , ADN/metabolismo , Estructura Molecular , Ácidos Nucleicos/química , Fenoles/química , Tiramina/análogos & derivados , Tiramina/química , Tiramina/metabolismoRESUMEN
Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the Escherichia coli lac operon switch, in which DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed transcription activator-like effector dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system.
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Proteínas de Escherichia coli , Efectores Tipo Activadores de la Transcripción , ADN Bacteriano , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Operón Lac/genética , Represoras Lac/genética , Represoras Lac/metabolismo , Conformación de Ácido NucleicoRESUMEN
Mitochondrial disorders arise from defects in nuclear genes encoding enzymes of oxidative metabolism. Mutations of metabolic enzymes in somatic tissues can cause cancers due to oncometabolite accumulation. Paraganglioma and pheochromocytoma are examples, whose etiology and therapy are complicated by the absence of representative cell lines or animal models. These tumors can be driven by loss of the tricarboxylic acid cycle enzyme succinate dehydrogenase. We exploit the relationship between succinate accumulation, hypoxic signaling, egg-laying behavior, and morphology in C. elegans to create genetic and pharmacological models of succinate dehydrogenase loss disorders. With optimization, these models may enable future high-throughput screening efforts.
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Neoplasias de las Glándulas Suprarrenales/genética , Proteínas de Caenorhabditis elegans/genética , Paraganglioma/genética , Feocromocitoma/genética , Succinato Deshidrogenasa/genética , Neoplasias de las Glándulas Suprarrenales/tratamiento farmacológico , Neoplasias de las Glándulas Suprarrenales/patología , Aminoácidos Dicarboxílicos/farmacología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Mutación , Paraganglioma/tratamiento farmacológico , Paraganglioma/patología , Feocromocitoma/tratamiento farmacológico , Feocromocitoma/patología , Succinato Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/metabolismo , Ácido Succínico/metabolismoRESUMEN
Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the looped DNA and affinity of the protein that anchors the loop, cellular energetics and DNA negative supercoiling are likely factors determining the stability of the repression loop. E. coli cells undergo numerous highly regulated and dynamic transitions as resources are depleted during bacterial growth. We hypothesized that the probability of DNA looping depends on the growth status of the E. coli culture. We utilized a well-characterized repression loop model assembled from elements of the lac operon to measure loop length-dependent repression at three different culture densities. Remarkably, even with changes in supercoiling, there exists a dynamic compensation in which the contribution of DNA looping to gene repression remains essentially constant.
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BACKGROUND: Succinate dehydrogenase (SDH) loss and mastermind-like 3 (MAML3) translocation are two clinically important genetic alterations that correlate with increased rates of metastasis in subtypes of human paraganglioma and pheochromocytoma (PPGL) neuroendocrine tumors. Although hypotheses propose that succinate accumulation after SDH loss poisons dioxygenases and activates pseudohypoxia and epigenomic hypermethylation, it remains unclear whether these mechanisms account for oncogenic transcriptional patterns. Additionally, MAML3 translocation has recently been identified as a genetic alteration in PPGL, but is poorly understood. We hypothesize that a key to understanding tumorigenesis driven by these genetic alterations is identification of the transcription factors responsible for the observed oncogenic transcriptional changes. METHODS: We leverage publicly-available human tumor gene expression profiling experiments (N = 179) to reconstruct a PPGL tumor-specific transcriptional network. We subsequently use the inferred transcriptional network to perform master regulator analyses nominating transcription factors predicted to control oncogenic transcription in specific PPGL molecular subtypes. Results are validated by analysis of an independent collection of PPGL tumor specimens (N = 188). We then perform a similar master regulator analysis in SDH-loss mouse embryonic fibroblasts (MEFs) to infer aspects of SDH loss master regulator response conserved across species and tissue types. RESULTS: A small number of master regulator transcription factors are predicted to drive the observed subtype-specific gene expression patterns in SDH loss and MAML3 translocation-positive PPGL. Interestingly, although EPAS1 perturbation is detectible in SDH-loss and VHL-loss tumors, it is by no means the most potent factor driving observed patterns of transcriptional dysregulation. Analysis of conserved SDH-loss master regulators in human tumors and MEFs implicated ZNF423, a known modulator of retinoic acid response in neuroblastoma. Subsequent functional analysis revealed a blunted cell death response to retinoic acid in SDH-loss MEFs and blunted differentiation response in SDH-inhibited SH-SY5Y neuroblastoma cells. CONCLUSIONS: The unbiased analyses presented here nominate specific transcription factors that are likely drivers of oncogenic transcription in PPGL tumors. This information has the potential to be exploited for targeted therapy. Additionally, the observation that SDH loss or inhibition results in blunted retinoic acid response suggests a potential developmental etiology for this tumor subtype.
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Neoplasias de las Glándulas Suprarrenales/genética , Proteínas de Unión al ADN/genética , Proteínas de la Membrana/genética , Feocromocitoma/genética , Succinato Deshidrogenasa/genética , Factores de Transcripción/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Algoritmos , Animales , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Bases de Datos Genéticas , Fibroblastos , Humanos , Ratones/embriología , Mutación , Transactivadores , Factores de Transcripción/metabolismo , Transcriptoma , Translocación Genética , Tretinoina/metabolismoRESUMEN
We previously reported the in vitro selection and characterization of a DNA aptamer capable of stimulating remyelination in a mouse model of multiple sclerosis. This aptamer was selected for its ability to bind to suspensions of crude murine myelin in vitro. Our initial studies in vitro and in vivo involved a 40-nucleotide derivative (LJM-3064) of the original 100-nucleotide aptamer. LJM-3064 retained robust myelin-binding properties. Structural characterization of LJM-3064 revealed that the guanosine-rich 5' half of the sequence forms different G-quadruplex-type structures that are variably stable in the presence of physiologically relevant ions. We hypothesized that this structured domain is sufficient for myelin binding. In this study, we confirm that a 20-nucleotide DNA, corresponding to the 5' half of LJM-3064, retains myelin-binding properties. We then optimize this minimal myelin-binding aptamer via systematic evolution of ligands by exponential enrichment after sparse rerandomization. We report a sequence variant (LJM-5708) of the 20-nucleotide myelin-binding aptamer with enhanced myelin-binding properties and the ability to bind cultured human oligodendroglioma cells in vitro, providing the first evidence of cross-species reactivity of this myelin-binding aptamer. As our formulation of DNA aptamers for in vivo remyelination therapy involves conjugation to streptavidin, we verified that the myelin-binding properties of LJM-5708 were retained in conjugates to avidin, streptavidin, and neutravidin. DNA aptamer LJM-5708 is a lead for further preclinical development of remyelinating aptamer technologies.
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Aptámeros de Nucleótidos/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Vaina de Mielina/efectos de los fármacos , Animales , Dicroismo Circular , G-Cuádruplex , Humanos , Ratones , Esclerosis Múltiple/genética , Oligodendroglioma/tratamiento farmacológico , Oligodendroglioma/patología , Unión Proteica/efectos de los fármacos , Técnica SELEX de Producción de Aptámeros , Estreptavidina/químicaRESUMEN
The yeast Nhp6A protein (yNhp6A) is a member of the eukaryotic HMGB family of chromatin factors that enhance apparent DNA flexibility. yNhp6A binds DNA nonspecifically with nM affinity, sharply bending DNA by >60°. It is not known whether the protein binds to unbent DNA and then deforms it, or if bent DNA conformations are 'captured' by protein binding. The former mechanism would be supported by discovery of conditions where unbent DNA is bound by yNhp6A. Here, we employed an array of conformational probes (FRET, fluorescence anisotropy, and circular dichroism) to reveal solution conditions in which an 18-base-pair DNA oligomer indeed remains bound to yNhp6A while unbent. In 100 mM NaCl, yNhp6A-bound DNA unbends as the temperature is raised, with no significant dissociation of the complex detected up to â¼45°C. In 200 mM NaCl, DNA unbending in the intact yNhp6A complex is again detected up to â¼35°C. Microseconds-resolved laser temperature-jump perturbation of the yNhp6a-DNA complex revealed relaxation kinetics that yielded unimolecular DNA bending/unbending rates on timescales of 500 µs-1 ms. These data provide the first direct observation of bending/unbending dynamics of DNA in complex with yNhp6A, suggesting a bind-then-bend mechanism for this protein.
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ADN de Hongos/química , ADN de Hongos/metabolismo , Proteínas HMGN/química , Proteínas HMGN/metabolismo , Conformación de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina/genética , Transferencia Resonante de Energía de Fluorescencia , Proteínas HMGN/fisiología , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. Here we combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to show that high mobility group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new mechanism for regulation of chromatin accessibility. We find that both the double box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins destabilize and unwind DNA from the H2A-H2B dimers. However, unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3-H4)2 tetramer. This difference in nucleosome destabilization may explain why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at highly transcribed ribosomal genes, known to be depleted of histones. In contrast, Nhp6A is found across euchromatin, pointing to a significant difference in cellular function.
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Proteínas HMGN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Microscopía de Fuerza Atómica , Nucleosomas/química , Nucleosomas/ultraestructura , Pinzas ÓpticasRESUMEN
rHIgM22 is a recombinant human monoclonal IgM designed to promote remyelination, and it is currently in Phase I clinical trials in patients with multiple sclerosis (MS). In animal models of demyelination, a single low dose of rHIgM22 stimulates oligodendrocyte maturation, induces remyelination, preserves axons, and slows the decline of locomotor deficits. Natural autoantibodies like rHIgM22 typically bind to multiple antigens with weak affinity. rHIgM22 binds to oligodendrocytes and myelin. Because the antigens for rHIgM22 is prevalent within and exclusive to central nervous system (CNS) myelin, we used CNS myelin particles in combination with surface plasmon resonance to determine the kinetic and affinity constants for the interaction of rHIgM22 to myelin. We found that both the serum and recombinant forms of the antibody bind to myelin with very small dissociation constants in the 100 pM range, which is highly unusual for natural autoantibodies. The extraordinary affinity between rHIgM22 and myelin may explain why such a low effective dose can stimulate CNS repair in animal models of demyelination and underlie the accumulation of rHIgM22 in the CSF in treated MS patients by targeting myelin.
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Anticuerpos Monoclonales/metabolismo , Inmunoglobulina M/metabolismo , Vaina de Mielina/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Cinética , Ratones Endogámicos C57BL , Unión Proteica , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as the Escherichia coli lactose (lac) operon. Here, we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.
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Bacterias/genética , Bacterias/metabolismo , Inmunoprecipitación de Cromatina , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Southern Blotting , Inmunoprecipitación de Cromatina/métodos , División del ADN , Exonucleasas/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
Atomic force microscopy (AFM) is widely used to image and study biological molecules. As an example, we have utilized AFM to investigate how the mechanical properties of DNA polymers depend on electrostatics and the strength of DNA base stacking by studying double-stranded DNA molecules incorporating several different neutral and charged base modifications. Here, we describe ten complementary approaches for determining DNA persistence length by AFM imaging. The combination of different approaches provides increased confidence and statistical reliability over existing methods utilizing only a single approach.