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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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Activating BRAF mutations are found in a small subset of patients with newly diagnosed multiple myeloma, but prevalence increases in late-stage, refractory disease, and the mutations are associated with adverse outcome. This prospective single-arm, open-label, multicenter phase 2 trial assessed the efficacy and safety of combined BRAF/MEK inhibition, using encorafenib and binimetinib, in patients with relapsed/refractory multiple myeloma (RRMM) carrying a BRAFV600E mutation. Patients received 450 mg encorafenib once daily and binimetinib 45 mg twice daily. The primary end point was the overall response rate achieved within the first year after start of treatment according to International Myeloma Working Group criteria. Twelve RRMM patients with a median of 5 prior lines of therapy were enrolled. The overall response rate was 83.3%, with 10 patients achieving at least a partial response. The median progression-free survival was 5.6 months, and overall survival was 55% at 24 months. Emerging resistance to therapy was driven by RAS mutations and structural variants involving the BRAF locus. This is the first prospective clinical trial to demonstrate that combined BRAF/MEK inhibition is highly effective in patients with BRAFV600E-mutated RRMM, and it represents a successful targeted precision medicine approach in this disease. This trial was registered at www.clinicaltrials.gov as #NCT02834364.
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Mieloma Múltiple , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Proteínas Proto-Oncogénicas B-raf/genética , Estudios Prospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Quinasas de Proteína Quinasa Activadas por Mitógenos/uso terapéuticoRESUMEN
After the Zika virus (ZIKV) epidemic in Brazil, ZIKV infections were linked to damage to the central nervous system (CNS) and congenital anomalies. Due to the virus's ability to cross the placenta and reach brain tissue, its effects become severe, leading to Congenital Zika Syndrome (CZS) and resulting in neuroinflammation, microglial activation, and secretion of neurotoxic factors. The presence of ZIKV triggers an inadequate fetal immune response, as the fetus only has the protection of maternal antibodies of the Immunoglobulin G (IgG) class, which are the only antibodies capable of crossing the placenta. Because of limited understanding regarding the long term consequences of ZIKV infection and the involvement of maternal antibodies, this study sought to assess the impact of the ZIKV + IgGâºcomplex on murine microglial cells. The cells were exposed to ZIKV, IgG antibodies, and the ZIKV + IgGâºcomplex for 24 and 72 h. Treatment-induced cytotoxic effects were evaluated using the cell viability assay, oxidative stress, and mitochondrial membrane potential. The findings indicated that IgG antibodies exhibit cytotoxic effects on microglia, whether alone or in the presence of ZIKV, leading to compromised cell viability, disrupted mitochondrial membrane potential, and heightened oxidative damage. Our conclusion is that IgG antibodies exert detrimental effects on microglia, triggering their activation and potentially disrupting the creation of a neurotoxic environment. Moreover, the presence of antibodies may correlate with an elevated risk of ZIKV-induced neuroinflammation, contributing to long-term CNS damage.
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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
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
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
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
Genetic switches must alternate between states whose probabilities are dependent on regulatory signals. Classical examples of transcriptional control in bacteria depend on repressive DNA loops anchored by proteins whose structures are sensitive to small molecule inducers or co-repressors. We are interested in exploiting these natural principles to engineer artificial switches for transcriptional control of bacterial genes. Here, we implement designed homodimeric DNA looping proteins ('Transcription Activator-Like Effector Dimers'; TALEDs) for this purpose in living bacteria. Using well-studied FKBP dimerization domains, we build switches that mimic regulatory characteristics of classical Escherichia coli lactose, galactose and tryptophan operon promoters, including induction or co-repression by small molecules. Engineered DNA looping using TALEDs is thus a new approach to tuning gene expression in bacteria. Similar principles may also be applicable for gene control in eukaryotes.
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Regulación Bacteriana de la Expresión Génica , Efectores Tipo Activadores de la Transcripción/genética , ADN/química , Proteínas de Escherichia coli/metabolismo , Operón Lac , Represoras Lac/metabolismo , Modelos Genéticos , Ingeniería de Proteínas , Multimerización de Proteína , Efectores Tipo Activadores de la Transcripción/química , Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.
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ADN Bacteriano/metabolismo , Escherichia coli/genética , Proteínas HMGN/metabolismo , Represoras Lac/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Inmunoprecipitación de Cromatina , ADN Bacteriano/química , Proteínas HMGN/química , Represoras Lac/genética , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The Escherichia coli lactose operon provides a paradigm for understanding gene control by DNA looping where the lac repressor (LacI) protein competes with RNA polymerase for DNA binding. Not all promoter loops involve direct competition between repressor and RNA polymerase. This raises the possibility that positioning a promoter within a tightly constrained DNA loop is repressive per se, an idea that has previously only been considered in vitro. Here, we engineer living E. coli bacteria to measure repression due to promoter positioning within such a tightly constrained DNA loop in the absence of protein-protein binding competition. We show that promoters held within such DNA loops are repressed â¼100-fold, with up to an additional â¼10-fold repression (â¼1000-fold total) dependent on topological positioning of the promoter on the inner or outer face of the DNA loop. Chromatin immunoprecipitation data suggest that repression involves inhibition of both RNA polymerase initiation and elongation. These in vivo results show that gene repression can result from tightly looping promoter DNA even in the absence of direct competition between repressor and RNA polymerase binding.
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Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Operón Lac , Regiones Promotoras Genéticas , Unión Competitiva , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Silenciador del Gen , Genes Reporteros , Represoras Lac/genética , Represoras Lac/metabolismo , Modelos Genéticos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.
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ADN Bacteriano/química , Regulación Bacteriana de la Expresión Génica , Represoras Lac/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Genéticos , Regiones Operadoras GenéticasRESUMEN
UNLABELLED: Dancers often view injuries as a necessary sacrifice for participating in their art form. The purpose of this research was to determine the frequency and patterns of injury in a non-conservatory, liberal arts dance environment. These data may enable dance departments to provide more effective health resources. METHODS: Dancers registered in technique courses in a liberal arts dance department (including ballet, modern, tap, and jazz) completed an injury questionnaire immediately following the occurrence of any dance-related injury over the course of one semester. RESULTS: Out of 168 students registered in the department, 46 injuries were reported throughout the semester. The greatest rate of injury was in September and December with 0.95 and 0.65 injuries reported per day, respectively. 89.1% of participants indicated that they would use a direct-access, on-campus physical therapist or athletic trainer if available, though 45.7% of injured participants indicated that they would seek treatment off campus. CONCLUSIONS: Dancers in a liberal arts collegiate program may train at a higher intensity during the semester than summer break, which likely accounts for the high incidence of injury in September. Of those injured, most planned on self-treating, but none planned on missing class. Pre-semester screening and post-semester cross-training education should be implemented in liberal arts dance programs to help decrease the rate of injury seen when returning to dance following prolonged time off from dancing. Additionally, direct access to physical therapy or athletic training treatment would likely be utilized by these students if available.
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Traumatismos en Atletas/diagnóstico , Traumatismos en Atletas/epidemiología , Baile/lesiones , Estado de Salud , Autoinforme , Estudiantes/estadística & datos numéricos , Femenino , Humanos , Incidencia , Masculino , Factores de Riesgo , Adulto JovenRESUMEN
LacI/GalR transcription regulators have extensive, non-conserved interfaces between their regulatory domains and the 18 amino acids that serve as 'linkers' to their DNA-binding domains. These non-conserved interfaces might contribute to functional differences between paralogs. Previously, two chimeras created by domain recombination displayed novel functional properties. Here, we present a synthetic protein family, which was created by joining the LacI DNA-binding domain/linker to seven additional regulatory domains. Despite 'mismatched' interfaces, chimeras maintained allosteric response to their cognate effectors. Therefore, allostery in many LacI/GalR proteins does not require interfaces with precisely matched interactions. Nevertheless, the chimeric interfaces were not silent to mutagenesis, and preliminary comparisons suggest that the chimeras provide an ideal context for systematically exploring functional contributions of non-conserved positions. DNA looping experiments revealed higher order (dimer-dimer) oligomerization in several chimeras, which might be possible for the natural paralogs. Finally, the biological significance of repression differences was determined by measuring bacterial growth rates on lactose minimal media. Unexpectedly, moderate and strong repressors showed an apparent induction phase, even though inducers were not provided; therefore, an unknown mechanism might contribute to regulation of the lac operon. Nevertheless, altered growth correlated with altered repression, which indicates that observed functional modifications are significant.
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Proteínas de Escherichia coli/química , Regulación Bacteriana de la Expresión Génica , Represoras Lac/química , Proteínas Represoras/química , Transcripción Genética , Regulación Alostérica , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Operón Lac , Represoras Lac/genética , Represoras Lac/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Regulación hacia ArribaRESUMEN
The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of 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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Inmunoprecipitación de Cromatina , ADN Bacteriano , Escherichia coli , ADN Bacteriano/metabolismo , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Inmunoprecipitación de Cromatina/métodos , Unión Proteica , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Operón Lac , Reacción en Cadena de la Polimerasa/métodos , Southern Blotting , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismoRESUMEN
CONTEXT.: Although the basic principles of intraoperative diagnosis in surgical neuropathology have not changed in the last century, the last several decades have seen dramatic changes in tumor classification, terminology, molecular classification, and modalities used for intraoperative diagnosis. As many neuropathologic intraoperative diagnoses are performed by general surgical pathologists, awareness of these recent changes is important for the most accurate intraoperative diagnosis. OBJECTIVE.: To describe recent changes in the practice of intraoperative surgical neuropathology, with an emphasis on new entities, tumor classification, and anticipated ancillary tests, including molecular testing. DATA SOURCES.: The sources for this review include the fifth edition of the World Health Organization Classification of Tumours of the Central Nervous System, primary literature on intraoperative diagnosis and newly described tumor entities, and the authors' clinical experience. CONCLUSIONS.: A significant majority of neuropathologic diagnoses require ancillary testing, including molecular analysis, for appropriate classification. Therefore, the primary goal for any neurosurgical intraoperative diagnosis is the identification of diagnostic tissue and the preservation of the appropriate tissue for molecular testing. The intraoperative pathologist should seek to place a tumor in the most accurate diagnostic category possible, but specific diagnosis at the time of an intraoperative diagnosis is often not possible. Many entities have seen adjustments to grading criteria, including the incorporation of molecular features into grading. Awareness of these changes can help to avoid overgrading or undergrading at the time of intraoperative evaluation.
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Crooke cell adenoma (CCA) is a rare and aggressive subtype of a corticotroph adenoma, which requires lifetime surveillance. There have been 106 cases of CCAs reported in the English literature. We describe 2 cases of CCA, a 48-year-old man and an 84-year-old woman who both presented with binocular diplopia and temple pain. Neither case had clinical Cushing syndrome. Laboratory values for the 48-year-old man revealed, adrenocorticotropin (ACTH) 103â pg/mL (22â pmol/L) (RR: 7-63â pg/mL) and evening cortisol 14â µg/dL (386â nmol/L) (RR: 2.7-10.5â µg/dL). Computed tomography imaging demonstrated a mass adjacent to the right cavernous sinus extending into the sphenoid sinus. He underwent tumor resection with adjuvant radiation and has had a stable residual tumor for 4 years. Preoperative laboratory values for the 84-year-old woman revealed, ACTH 69â pg/mL (15 pmol/L) (RR: 7-63â pg/mL) and evening cortisol 16.2â µg/dL (447â nmol/L) (RR: 2.7-10.5 µg/dL). Brain magnetic resonance imaging revealed, a mass compressing the optic chiasm. She underwent resection and has had a stable residual tumor for 2 years. Surgical pathology in both cases revealed cytoplasmic hyaline deposits of more than 50% of the tumor cells, consistent with CCA. The CCA although rare, should be considered when evaluating cases with subclinical Cushing disease and visual symptoms.
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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
The inflexibility of double-stranded DNA with respect to bending and twisting is well established in vitro. Understanding apparent DNA physical properties in vivo is a greater challenge. Here, we exploit repression looping with components of the Escherichia coli lac operon to monitor DNA flexibility in living cells. We create a minimal system for testing the shortest possible DNA repression loops that contain an E. coli promoter, and compare the results to prior experiments. Our data reveal that loop-independent repression occurs for certain tight operator/promoter spacings. When only loop-dependent repression is considered, fits to a thermodynamic model show that DNA twisting limits looping in vivo, although the apparent DNA twist flexibility is 2- to 4-fold higher than in vitro. In contrast, length-dependent resistance to DNA bending is not observed in these experiments, even for the shortest loops constraining <0.4 persistence lengths of DNA. As observed previously for other looping configurations, loss of the nucleoid protein heat unstable (HU) markedly disables DNA looping in vivo. Length-independent DNA bending energy may reflect the activities of architectural proteins and the structure of the DNA topological domain. We suggest that the shortest loops are formed in apical loops rather than along the DNA plectonemic superhelix.
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ADN Bacteriano/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Operón Lac , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/genética , Represoras Lac/metabolismo , Conformación de Ácido Nucleico , Regiones Operadoras Genéticas , Regiones Promotoras GenéticasRESUMEN
The descriptions of muscle pathology in dysferlinopathy patients have classically included an inflammatory infiltrate that can mimic inflammatory myopathies. Based on over 20 years of institutional experience in evaluating dystrophic and inflammatory myopathy muscle biopsies at the University of Iowa, we hypothesized the inflammatory histopathology of dysferlinopathy is more similar to limb-girdle pattern muscular dystrophies such as calpainopathy and Becker muscular dystrophy, and distinct from true inflammatory myopathies. Muscle biopsies from 32 dysferlinopathy, 30 calpainopathy, 30 Becker muscular dystrophy, and 30 inflammatory myopathies (15 each of dermatomyositis and inclusion body myositis) were analyzed through digital quantitation of CD3, CD4, CD8, CD20, and PU.1 immunostaining. The expression of MHC class I and deposition of complement C5b-9 was also evaluated. Dysferlinopathy, calpainopathy, and Becker muscular dystrophy muscle biopsies had similar numbers of inflammatory cell infiltrates and significantly fewer CD3+ T-lymphocytes than dermatomyositis (p = 0.05) and inclusion body myositis (p < 0.0001) biopsies. There was no statistically significant difference in the number of PU.1+ macrophages identified in any diagnostic group. MHC class I expression was significantly lower in the limb-girdle pattern muscular dystrophies compared to the inflammatory myopathies (p < 0.0001). In contrast, complement C5b-9 deposition was similar among dysferlinopathy, dermatomyositis, and inclusion body myositis biopsies but significantly greater than calpainopathy and Becker muscular dystrophy biopsies (p = 0.05). Compared to calpainopathy, Becker muscular dystrophy, and inflammatory myopathies, the unique profile of minimal inflammatory cell infiltrates, absent to focal MHC class I, and diffuse myofiber complement C5b-9 deposition is the pathologic signature of dysferlinopathy muscle biopsies.