RESUMEN

BACKGROUND: Protein interactions participate in many molecular mechanisms involved in cellular processes. The human TATA box binding protein (hTBP) interacts with Antennapedia (Antp) through its N-terminal region, specifically via its glutamine homopeptides. This PolyQ region acts as a binding site for other transcription factors under normal conditions, but when it expands, it generates spinocerebellar ataxia 17 (SCA17), whose protein aggregates in the brain prevent its correct functioning. OBJECTIVE: To determine whether the hTBP glutamine-rich region is involved in its interaction with homeoproteins and the role it plays in the formation of protein aggregates in SCA17. MATERIAL AND METHODS: We characterized hTBP interaction with other homeoproteins using BiFC, and modeled SCA17 in Drosophila melanogaster by targeting hTBPQ80 to the fly brain using UAS/GAL4. RESULTS: There was hTBP interaction with homeoproteins through its glutamine-rich region, and hTBP protein aggregates with expanded glutamines were found to affect the locomotor capacity of flies. CONCLUSIONS: The study of hTBP interactions opens the possibility for the search for new therapeutic strategies in neurodegenerative pathologies such as SCA17.

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ANTECEDENTES: Las interacciones proteicas participan en una gran cantidad de mecanismos moleculares que rigen los procesos celulares. La proteína de unión a la caja TATA humana (hTBP) interacciona con Antennapedia (Antp) a través de su extremo N-terminal, específicamente a través de sus homopéptidos de glutaminas. Esta región PolyQ sirve como sitio de unión a factores de transcripción en condiciones normales, pero cuando se expande genera la ataxia espinal cerebelosa 17 (SCA17), cuyos agregados proteicos en el cerebro impiden su funcionamiento correcto. OBJETIVO: Determinar si la región rica en glutaminas de hTBP interviene en su interacción con homeoproteínas y el papel que tiene en la formación de agregados proteicos en SCA17. MATERIAL Y MÉTODOS: Se caracterizó la interacción de hTBP con otras homeoproteínas usando BiFC y se modeló SCA17 en Drosophila melanogaster dirigiendo hTBPQ80 al cerebro de las moscas usando UAS/GAL4. RESULTADOS: Existió interacción de hTBP con homeoproteínas a través de su región rica en glutaminas. Los agregados proteicos de hTBP con las glutaminas expandidas afectaron la capacidad locomotriz de las moscas. CONCLUSIONES: El estudio de las interacciones de hTBP abre la posibilidad para la búsqueda de nuevas estrategias terapéuticas en patologías neurodegenerativas como SCA17.

Asunto(s)

Drosophila melanogaster , Ataxias Espinocerebelosas , Proteína de Unión a TATA-Box , Animales , Humanos , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Glutamina/metabolismo , Péptidos/metabolismo , Agregado de Proteínas/fisiología , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/genética , Proteína de Unión a TATA-Box/metabolismo , Proteína de Unión a TATA-Box/genética

RESUMEN

The understanding of the mechanisms that regulate gene expression to establish differentiation programs and determine cell lineages, is one of the major challenges in Developmental Biology. Besides the participation of tissue-specific transcription factors and epigenetic processes, the role of general transcription factors has been ignored. Only in recent years, there have been scarce studies that address this issue. Here, we review the studies on the biological activity of some TATA-box binding protein (TBP)-associated factors (TAFs) during the proliferation of stem/progenitor cells and their involvement in cell differentiation. Particularly, the accumulated evidence suggests that TAF4, TAF4b, TAF7L, TAF8, TAF9, and TAF10, among others, participate in nervous system development, adipogenesis, myogenesis, and epidermal differentiation; while TAF1, TAF7, TAF15 may be involved in the regulation of stem cell proliferative abilities and cell cycle progression. On the other hand, evidence suggests that TBP variants such as TBPL1 and TBPL2 might be regulating some developmental processes such as germ cell maturation and differentiation, myogenesis, or ventral specification during development. Our analysis shows that it is necessary to study in greater depth the biological function of these factors and its participation in the assembly of specific transcription complexes that contribute to the differential gene expression that gives rise to the great diversity of cell types existing in an organism. The understanding of TAFs' regulation might lead to the development of new therapies for patients which suffer from mutations, alterations, and dysregulation of these essential elements of the transcriptional machinery.

Asunto(s)

Proteína de Unión a TATA-Box , Humanos , Diferenciación Celular/genética , Mutación , Proteínas Nucleares/genética , Proteínas Similares a la Proteína de Unión a TATA-Box/química , Proteínas Similares a la Proteína de Unión a TATA-Box/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/genética , Animales

RESUMEN

Spinocerebellar ataxia type 17 or ATX-TBP is a CAG/CAA repeat expansion disorder characterized by marked clinical heterogeneity. Reports of affected carriers with subthreshold repeat expansions and of patients with Parkinson's disease (PD) with expanded repeats have cast doubt on the established cutoff values of the expansions and the phenotypic spectrum of this disorder. The objective of this systematic review was to explore the genotype-phenotype relationships for repeat expansions in TBP to delineate the ATX-TBP phenotype and reevaluate the pathological range of repeat expansions. The International Parkinson and Movement Disorder Society Genetic Mutation Database (MDSGene) standardized data extraction protocol was followed. Clinically affected carriers of reported ATX-TBP expansions were included. Publications that contained repeat sizes in screened cohorts of patients with PD and/or healthy individuals were included for a separate evaluation of cutoff values. Phenotypic and genotypic data for 346 ATX-TBP patients were curated. Overall, 97.7% of the patients had ≥41 repeats, while 99.6% of patients with PD and 99.9% of healthy individuals had ≤42 repeats, with a gray zone of reduced penetrance between 41 and 45 repeats. Pure parkinsonism was more common in ATX-TBP patients with 41 to 45 repeats than in the group with ≥46 repeats, which conversely more often presented with a complex phenotype with mixed movement disorders. An updated genotype-phenotype assessment for ATX-TBP is provided, and new repeat expansion cutoff values of reduced penetrance (41-45 expanded repeats) and full penetrance (46-66 expanded repeats) are proposed. These adjusted cutoff values will have diagnostic and counseling implications and may guide future clinical trial protocol. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Asunto(s)

Enfermedad de Parkinson , Ataxias Espinocerebelosas , Humanos , Estudios de Asociación Genética , Enfermedad de Parkinson/genética , Ataxias Espinocerebelosas/genética , Proteína de Unión a TATA-Box/genética , Expansión de Repetición de Trinucleótido

RESUMEN

The assembly of transcription complexes on eukaryotic promoters involves a series of steps, including chromatin remodeling, recruitment of TATA-binding protein (TBP)-containing complexes, the RNA polymerase II holoenzyme, and additional basal transcription factors. This review describes the transcriptional regulation by TBP and its corresponding homologs that constitute the TBP family and their interactions with promoter DNA. The C-terminal core domain of TBP is highly conserved and contains two structural repeats that fold into a saddle-like structure, essential for the interaction with the TATA-box on DNA. Based on the TBP C-terminal core domain similarity, three TBP-related factors (TRFs) or TBP-like factors (TBPLs) have been discovered in metazoans, TRF1, TBPL1, and TBPL2. TBP is autoregulated, and once bound to DNA, repressors such as Mot1 induce TBP to dissociate, while other factors such as NC2 and the NOT complex convert the active TBP/DNA complex into inactive, negatively regulating TBP. TFIIA antagonizes the TBP repressors but may be effective only in conjunction with the RNA polymerase II holoenzyme recruitment to the promoter by promoter-bound activators. TRF1 has been discovered inDrosophila melanogasterandAnophelesbut found absent in vertebrates and yeast. TBPL1 cannot bind to the TATA-box; instead, TBPL1 prefers binding to TATA-less promoters. However, TBPL1 shows a stronger association with TFIIA than TBP. The TCT core promoter element is present in most ribosomal protein genes inDrosophilaand humans, and TBPL1 is required for the transcription of these genes. TBP directly participates in the DNA repair mechanism, and TBPL1 mediates cell cycle arrest and apoptosis. TBPL2 is closely related to its TBP paralog, showing 95% sequence similarity with the TBP core domain. Like TBP, TBPL2 also binds to the TATA-box and shows interactions with TFIIA, TFIIB, and other basal transcription factors. Despite these advances, much remains to be explored in this family of transcription factors.

Asunto(s)

ARN Polimerasa II , Proteína de Unión a TATA-Box , Factores de Transcripción , Transcripción Genética , Adenosina Trifosfatasas/genética , Animales , ADN/genética , Drosophila , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Proteínas Nucleares/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , TATA Box/genética , Proteínas Similares a la Proteína de Unión a TATA-Box/química , Proteínas Similares a la Proteína de Unión a TATA-Box/genética , Proteínas Similares a la Proteína de Unión a TATA-Box/metabolismo , Factores Asociados con la Proteína de Unión a TATA , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIA/genética , Factor de Transcripción TFIIA/metabolismo , Factores de Transcripción/genética

RESUMEN

Transcription is the first step of gene expression regulation and is a fundamental mechanism for establishing the viability and development of a cell. The TATA box-binding protein (TBP) interaction with a TATA box in a promoter is one of the best studied mechanisms in transcription initiation. TBP is a transcription factor that is highly conserved from archaea to humans and is essential for the transcription initiated by each of the three RNA polymerases. In addition, the discovery of TBP-related factor 1 (TRF1) and other factors related to TBP shed light on the variability among transcription initiation complexes, thus demonstrating that the compositions of these complexes are, in fact, more complicated than originally believed. Despite these facts, the majority of studies on transcription have been performed on animal, plant and fungal cells, which serve as canonical models, and information regarding protist cells is relatively scarce. The aim of this work is to review the diversity of the TBPs that have been documented in protists and describe some of the specific features that differentiate them from their counterparts in higher eukaryotes.

Asunto(s)

Eucariontes/genética , TATA Box , Proteína de Unión a TATA-Box , Transcripción Genética , Eucariontes/metabolismo , Genes Protozoarios , Variación Genética , Giardia/genética , Giardia/metabolismo , Leishmania/genética , Leishmania/metabolismo , Filogenia , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Trypanosoma/genética , Trypanosoma/metabolismo

RESUMEN

The protozoan parasite Trichomonas vaginalis is a common human pathogen from one of the earliest-diverging eukaryotic lineages. At the transcriptional level, the highly conserved Inr element of RNA pol II-transcribed genes surrounds the transcription start site and is recognised by IBP39, a protein exclusive of T. vaginalis. Typical TATA boxes have not been identified in this organism but, in contrast, BLAST analyses of the T. vaginalis genome identified two genes encoding putative TATA-binding proteins (herein referred to as TvTBP1 and TvTBP2). The goal of this work was to characterise these two proteins at the molecular level. Our results show that both TvTBPs theoretically adopt the saddle-shaped structure distinctive to TBPs and both Tvtbp genes are expressed in T. vaginalis. TvTBP1 did not complement a Saccharomyces cerevisiae mutant lacking TBP; however, TvTBP1 and TvTBP2 proteins bound T. vaginalis DNA promoter sequences in EMSA assays. We propose that TvTBP1 may be part of the preinitiation transcription complex in T. vaginalis since TvTBP1 recombinant protein was able to bind IBP39 in vitro. This work represents the first approach towards the characterisation of general transcription factors in this early divergent organism.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Protozoarias/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Trichomonas vaginalis/metabolismo , Modelos Moleculares , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/genética , Transcripción Genética , Trichomonas vaginalis/genética

RESUMEN

qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb, Gapdh, Tbp, Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative ΔCq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16-18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha, Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh, Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development.

Asunto(s)

Corazón/embriología , Prosencéfalo/embriología , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Somitos/embriología , Animales , Perfilación de la Expresión Génica/normas , Regulación del Desarrollo de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Ratones , Prosencéfalo/química , Estándares de Referencia , Programas Informáticos , Somitos/química , Proteína de Unión a TATA-Box/genética , Distribución Tisular

RESUMEN

BACKGROUND: Entamoeba histolytica is the protozoan parasite responsible for human amebiasis. It causes up to 100,000 deaths worldwide each year. This parasite has two closely related basal transcription factors, the TATA-box binding protein (EhTBP) and the TBP-related factor 1 (EhTRF1). TBP binds to the canonical TATTTAAA-box, as well as to different TATA variants. TRF1 also binds to the TATTTAAA-box. However, their binding capacity to diverse core promoter elements, including the GAAC-element, and their role in gene regulation in this parasite remains unknown. METHODS: EMSA experiments were performed to determine the binding capacity of recombinant TBP and TRF1 to TATA variants, GAAC and GAAC-like boxes. For the functional analysis under different stress stimuli (e.g. growth curve, serum depletion, heat-shock, and UV-irradiation) and during the interaction with mammalian cells (erythrocytes, MDCK cell monolayers, and hepatocytes of hamsters), RT-qPCR, and gene knockdown were performed. RESULTS: Both transcription factors bound to the different TATA variants tested, as well as to the GAAC-boxes, suggesting that they are GAAC-box-binding proteins. The K D values determined for TBP and TRF1 for the different TATA variants and GAAC-box were in the range of 10-12 M to 10-11 M. During the death phase of growth or in serum depletion, Ehtbp mRNA levels significantly increased, whereas the mRNA level of Ehtrf1 did not change under these conditions. Ehtrf1 gene expression was negatively regulated by UV-irradiation and heat-shock stress, with no changes in Ehtbp gene expression. Moreover, Ehtrf1 gene also showed a negative regulation during erythrophagocytosis, liver abscess formation, and a transient expression level increase at the initial phase of MDCK cell destruction. Finally, the Ehtbp gene knockdown displayed a drastic decrease in the efficiency of erythrophagocytosis in G3 trophozoites. CONCLUSIONS: To our knowledge, this study reveals that these basal transcription factors are able to bind multiple core promoter elements. However, their immediate change in gene expression level in response to different stimuli, as well as during the interaction with mammalian cells, and the diminishing of erythrophagocytosis by silencing the Ehtbp gene indicate the different physiological roles of these transcription factors in E. histolytica.

Asunto(s)

Proteínas Portadoras/genética , Regulación de la Expresión Génica , Proteína de Unión a TATA-Box/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Factores de Transcripción/genética , Animales , Proteínas Portadoras/metabolismo , Clonación Molecular , Cricetinae , Perros , Entamoeba histolytica/genética , Técnicas de Silenciamiento del Gen , Hepatocitos/parasitología , Interacciones Huésped-Parásitos/genética , Células de Riñón Canino Madin Darby , Regiones Promotoras Genéticas , Reacción en Cadena en Tiempo Real de la Polimerasa , Estrés Fisiológico/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Transcripción Genética

RESUMEN

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.

Asunto(s)

Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas Ribosómicas/genética , Schizosaccharomyces/genética , TATA Box , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Expresión Génica , Complejo Mediador/genética , Complejo Mediador/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/metabolismo , Schizosaccharomyces/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIA/genética , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismo , Transcripción Genética

RESUMEN

We previously reported that rats and mice that have been raised for more than 30 generations in La Paz, Bolivia (3600 m), display divergent physiological responses to high altitude, including improved respiratory and metabolic control in mice. In the present study, we asked whether these traits would also be present in response to hypoxia at sea level. To answer this question, we exposed rats (Sprague Dawley) and mice (FVB) to normoxia (21% O2) or hypoxia (15 and 12% O2) for 6 h and measured ventilation and metabolic rate (whole-body plethysmography), and expression of the transcription factor HIF-1α (ELISA and mass spectrometry) and other proteins whose expression are regulated by hypoxia (glucose transporter 1, pyruvate dehydrogenase kinase 1 and angiopoietin 2; mass spectrometry) in the brainstem. In response to hypoxia, compared with rats, mice had higher minute ventilation, lower metabolic rate and higher expression of HIF-1α in the brainstem. In mice, the expression level of HIF-1α was positively correlated with ventilation and negatively correlated with metabolic rate. In rats, the concentration of brainstem cytosolic protein decreased by 38% at 12% O2, while expression of the glucose transporter 1 increased. We conclude that mice and rats raised at sea level have divergent physiological and molecular responses to hypoxia, supporting the hypothesis that mice have innate traits that favor adaptation to altitude.

Asunto(s)

Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Hipoxia/fisiopatología , Altitud , Animales , Metabolismo Basal , Bolivia , Tronco Encefálico/metabolismo , Tronco Encefálico/fisiopatología , Núcleo Celular/metabolismo , Citosol/metabolismo , Geografía , Transportador de Glucosa de Tipo 1/metabolismo , Masculino , Ratones , Ratas Sprague-Dawley , Respiración , Fracciones Subcelulares/metabolismo , Proteína de Unión a TATA-Box/metabolismo

RESUMEN

Different members of the HD-Zip I family of transcription factors exhibit differential AHA-like activation motifs, able to interact with proteins of the basal transcriptional machinery. Homeodomain-leucine zipper proteins are transcription factors unique to plants, classified in four subfamilies. Subfamily I members have been mainly associated to abiotic stress responses. Several ones have been characterized using knockout or overexpressors plants, indicating that they take part in different signal transduction pathways even when their expression patterns are similar and they bind the same DNA sequence. A bioinformatic analysis has revealed the existence of conserved motifs outside the HD-Zip domain, including transactivation AHA motifs. Here, we demonstrate that these putative activation motifs are functional. Four members of the Arabidopsis family were chosen: AtHB1, AtHB7, AtHB12 and AtHB13. All of them exhibited activation activity in yeast and in plants but with different degrees. The protein segment necessary for such activation was different for these four transcription factors as well as the role of the tryptophans they present. When interaction with components of the basal transcription machinery was tested, AtHB1 was able to interact with TBP, AtHB12 interacted with TFIIB, AtHB7 interacted with both, TBP and TFIIB while AtHB13 showed weak interactions with any of them, in yeast two-hybrid as well as in pull-down assays. Transient transformation of Arabidopsis seedlings confirmed the activation capacity and specificity of these transcription factors and showed some differences with the results obtained in yeast. In conclusion, the differential activation functionality of these transcription factors adds an important level of functional divergence of these proteins, and together with their expression patterns, these differences could explain, at least in part, their functional divergence.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción/genética , Activación Transcripcional , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Leucina Zippers , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plantones/genética , Plantones/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos Híbridos

RESUMEN

There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.

Asunto(s)

Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Hipoxantina Fosforribosiltransferasa/genética , Ovario/efectos de los fármacos , Estándares de Referencia , Proteína de Unión a TATA-Box/genética , Animales , Cebus , Crioprotectores/farmacología , Femenino , Hipotermia , Ovario/citología , Ovario/metabolismo , Oxígeno/farmacología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Immediate-early 3 (IE3) gene products are required to activate early (E)-stage gene expression of murine cytomegaloviruses (MCMV). The first early gene activated by IE3 is the M112-113 gene (also called E1), although a complete understanding of the activation mechanism is still lacking. In this paper, we identify a 10-bp cis-regulating motif upstream of the M112-113 TATA box as important for IE3 activation of M112-113 expression. Results from DNA affinity assays and chromatin immunoprecipitation assays show that the association of IE3 with the M112-113 gene promoter was eliminated by deletion of the 10-bp DNA sequence, now named IE3AM (for IE3 activating motif). In addition, IE3 interacts with TATA box binding protein (TBP), a core protein of TFIID (transcription initiation) complexes. Finally, we created an IE3AM-deleted MCMV (MCMVdIE3AM) using a bacterial artificial chromosome system. The mutant virus can still replicate in NIH 3T3 cells but at a significantly lower level. The defectiveness of the MCMVdIE3AM infection can be rescued in an M112-113-complemented cell line. Our results suggest that the interactions of IE3 with IE3AM and with TBP stabilize the TFIID complex at the M112-113 promoter such that M112-113 gene expression can be activated and/or enhanced.

Asunto(s)

Regulación Viral de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Muromegalovirus/fisiología , Regiones Promotoras Genéticas , Proteínas Virales/genética , Replicación Viral , Animales , Línea Celular , ADN Viral/genética , ADN Viral/metabolismo , Expresión Génica , Infecciones por Herpesviridae/virología , Ratones , Muromegalovirus/genética , Muromegalovirus/metabolismo , Células 3T3 NIH , Eliminación de Secuencia , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Proteínas Virales/metabolismo

RESUMEN

Accurate estimation of gene expression differences during development requires sensitive techniques combined with gold-standard normalization procedures. This is particularly true in the case of quantitative traits, where expression changes might be small. Nevertheless, systematic selection and validation of reference genes has been overlooked, even in Drosophila studies. Here, we tested the stability of six traditional reference genes across samples of imaginal wing disks from morphologically divergent strains of Drosophila melanogaster, in a two-class comparison: quantitative or qualitative variation in wing morphology. Overall, we identified and validated a pair of genes (RpL32 and Tbp) as being stably expressed in both experimental comparisons. These genes might be considered as a bona fide pair of reference genes for gene expression analyses of morphological divergence in D. melanogaster wings. They might also be taken as good candidates for experimental identification of stable reference genes in other morphological comparisons using Drosophila or other insect species. Besides, we found that some genes traditionally used as reference in qPCR experiments were not stably expressed in wing disks from the different fly strains. In fact, a significant bias was observed when the expression of three genes of interest, which are involved in the regulation of growth and patterning during imaginal wing development, was normalized with such putative reference genes. Our results demonstrate how inaccurate findings and opposite conclusions might be drawn if traditional reference genes are arbitrarily used for internal normalization without proper validation in the given experimental condition, a practice still common in qPCR experiments.

Asunto(s)

Proteínas de Drosophila , Perfilación de la Expresión Génica/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas , Proteína de Unión a TATA-Box , Alas de Animales/crecimiento & desarrollo , Animales , ADN Complementario/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , ARN/genética , Estándares de Referencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo

RESUMEN

Entamoeba histolytica is the protozoan parasite which causes human amoebiasis. In this parasite, few encoding genes for transcription factors have been cloned and characterized. The E. histolytica TATA-box binding protein (EhTBP) is the first basal transcription factor that has been studied. To continue with the identification of other members of the basal transcription machinery, we performed an in silico analysis of the E. histolytica genome and found three loci encoding for polypeptides with similarity to EhTBP. One locus has a 100% identity to the previously Ehtbp gene reported by our group. The second locus encodes for a 212 aa polypeptide that is 100% identical to residues 23-234 from EhTBP. The third one encodes for a 216 aa polypeptide of 24kDa that showed 42.6% identity and 73.7% similarity to EhTBP. This protein was named E. histolytica TBP-related factor 1 (EhTRF1). Ehtrf1 gene was expressed in bacteria and the purified 28kDa recombinant polypeptide showed the capacity to bind to TATTTAAA-box by electrophoretic mobility shift assays. K(D) values for rEhTBP and rEhTRF1 were (1.71+/-2.90)x10(-12)M and (1.12+/-0.160)x10(-11)M, respectively. Homology modeling of EhTRF1 and EhTBP revealed that, although they were very similar, they showed some differences on their surfaces. Thus, E. histolytica is a unicellular organism having two members of the TBP family.

Asunto(s)

Entamoeba histolytica/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Similares a la Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/genética , Secuencia de Aminoácidos , Clonación Molecular , Ensayo de Cambio de Movilidad Electroforética , Entamoeba histolytica/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas Protozoarias/metabolismo , Proteínas Similares a la Proteína de Unión a TATA-Box/genética , Proteínas Similares a la Proteína de Unión a TATA-Box/metabolismo , Proteína de Unión a TATA-Box/metabolismo

RESUMEN

Recent findings associate transcription start in trypanosomatids with chromatin regions containing modified and variant histones. TATA-binding protein (TBP) and other fundamental transcription factors have been also found at these Transcription Start Sites (TSS). Results of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments show that Trypanosoma cruzi TBP (TcTBP) has an in vitro binding preference for G-rich sequences. This finding correlates with the presence of G-rich stretches at the Strand Switch Regions (SSR) and at some putative internal TSS in Trypanosoma brucei and Leishmania. A scanning study of partially assembled T. cruzi genomic contigs determined the presence of G-rich stretches in the coding strands. TcTBP affinity for G-rich sequences suggests that this factor could play a role in locating the initiation complex in the right TSS, probably by "sensing" the G-content on the strand to be transcribed.

Asunto(s)

Secuencia Rica en GC/fisiología , Proteína de Unión a TATA-Box/metabolismo , Trypanosoma cruzi/metabolismo , Secuencia de Consenso , Trypanosoma cruzi/genética

RESUMEN

BACKGROUND: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. RESULTS: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. CONCLUSION: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

Asunto(s)

Neoplasias Encefálicas/genética , Expresión Génica , Glioblastoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Genes Esenciales/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Neoplasias Pulmonares/genética , Modelos Biológicos , Estándares de Referencia , Proteína de Unión a TATA-Box/genética

RESUMEN

The spliced leader (SL) RNA gene promoter is the only RNA polymerase II-dependent promoter characterized to date in trypanosomatids. Transcription of this small nuclear RNA is critical for trypanosomatid cell life because it is needed for polycistronic primary transcripts processing into individual translatable mRNAs. In recent years, a set of divergent fundamental transcription factors required for SL RNA gene transcription have been identified in different trypanosomatids. By means of a yeast two-hybrid system, we analyzed the protein-protein interactions between components of the SL RNA gene promoter binding complex. We also studied the interactions of already described motifs of TATA-binding protein (TBP) and transcription factor II B (TFIIB) orthologs separately. This was followed by investigations of DNA-protein interactions within the SL RNA gene promoter binding complex using one-hybrid analysis. Our results suggest that the complex has two "cores" which contact the promoter DNA, trypanosomal small nuclear RNA activating protein complex (tSNAPc), which has strong interactions between its subunits and a more labile TBP-TFIIA sub-complex.

Asunto(s)

Regiones Promotoras Genéticas/genética , ARN Protozoario/genética , ARN Lider Empalmado/genética , Trypanosoma cruzi/genética , Animales , Unión Proteica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , ARN Protozoario/metabolismo , ARN Nuclear Pequeño/genética , ARN Lider Empalmado/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/metabolismo , Trypanosoma cruzi/metabolismo

RESUMEN

Spinocerebellar ataxia type 17 (SCA17) is caused by expansion of a CAG/CAA repeat in the TBP gene. Most pathogenic alleles are interrupted and are stably transmitted from parent to offspring without anticipation. We identified three SCA17 families with expansion of uninterrupted alleles, thus greatly increasing the number of known intergenerational transmissions of such alleles. We found that uninterrupted SCA17 alleles are unstable, associated with anticipation, and show a paternal expansion bias that increases with age. Even small increments in repeat length resulted in inordinate increases in anticipation. Anticipation was also associated with childhood presentation. Sequencing of all SCA17 alleles is required for effective genetic counseling.

Asunto(s)

Anticipación Genética , Ataxias Espinocerebelosas/genética , Proteína de Unión a TATA-Box/genética , Expansión de Repetición de Trinucleótido/genética , Adolescente , Adulto , Edad de Inicio , Alelos , Niño , Progresión de la Enfermedad , Femenino , Humanos , Masculino , México , Persona de Mediana Edad , Linaje , Distribución por Sexo , Ataxias Espinocerebelosas/diagnóstico

RESUMEN

To gain a better understanding on the function of the potato Solanum tuberosum Multiprotein Bridging Factor 1 protein (StMBF1) its interaction with the TATA box binding protein (TBP) was demonstrated. In addition we reported that StMBF1 rescues the yeast mbf1 mutant phenotype, indicating its role as a plant co-activator. These data reinforce the hypothesis that MBF1 function is also conserved among non closely related plant species. In addition, measurement of StMBF1 protein level by Western blot using anti-StMBF1 antibodies indicated that the protein level increased upon H(2)O(2) and heat shock treatments. However, the potato beta-1,3-glucanase protein level was not changed under the same experimental conditions. These data indicate that StMBF1 participates in the cell stress response against oxidative stress allowing us to suggest that MBF1 genes from different plant groups may share similar functions.

Asunto(s)

Estrés Oxidativo , Solanum tuberosum/genética , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Respuesta al Choque Térmico , Peróxido de Hidrógeno/toxicidad , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Transactivadores/genética , Regulación hacia Arriba , Levaduras