RESUMEN
UNLABELLED: Brucella suis, facultative intracellular bacterial pathogen of mammals, and Agrobacterium tumefaciens, a plant pathogen, both use a VirB type IV secretion system (T4SS) to translocate effector molecules into host cells. HspL, an α-crystalline-type small heat-shock protein, acts as a chaperone for the Agrobacterium VirB8 protein, an essential component of the VirB system. An Agrobacterium mutant lacking hspL is attenuated due to a misfunctional T4SS. We have investigated whether IbpA (BRA0051), the Brucella HspL homologue, plays a similar role. Unlike HspL, IbpA does not interact with VirB8, and an IbpA mutant shows full virulence and no defect in VirB expression. These data show that the Brucella α-crystalline-type small heat-shock protein IbpA is not required for Brucella virulence. SIGNIFICANCE AND IMPACT OF STUDY: Many bacteria use type IV secretion systems (T4SS), multi-protein machines, to translocate DNA and protein substrates across their envelope. Understanding how T4SS function is important as they play major roles in the spread of plasmids carrying antibiotic resistance and in pathogenicity. In the plant pathogen Agrobacterium tumefaciens, HspL, an α-crystalline-type small heat-shock protein, acts as a chaperone for the essential type IV secretion system component VirB8. Here, we show that this is not the case for all T4SS; in the zoonotic pathogen Brucella suis, IbpA, the protein most related to HspL, does not play this role.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella suis/genética , Chaperonas Moleculares/genética , Factores de Virulencia/metabolismo , Agrobacterium tumefaciens/genética , Animales , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos , Brucella suis/metabolismo , Brucella suis/patogenicidad , Línea Celular , Expresión Génica , Macrófagos/microbiología , Ratones , Viabilidad Microbiana , Chaperonas Moleculares/metabolismo , Plásmidos , Transporte de Proteínas , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
The involvement of the Sox family of transcription factors in the development of the central nervous system (CNS) appears to be conserved in invertebrates and vertebrates. In Drosophila, SoxNeuro (SoxN) was recently shown to be involved in the formation of neuroblasts [Development 129 (2002) 4193; Development 129 (2002) 4219]. Through a yeast two-hybrid assay searching for proteins interacting with SoxN, we have isolated a novel protein in Drosophila, SoxNeuro Co-Factor (SNCF). The expression of the SNCF gene was detected during early embryogenesis at the blastoderm stages, and stopped just at the beginning of gastrulation. In transfected cells, the protein localised to nuclei, and strongly accumulated in nucleoli. SNCF was able to enhance SoxN mediated transcriptional activity in transfected cells, suggesting that SNCF might act as a SoxN co-activator. Finally, data are presented showing the existence in Drosophila of several proteins with a domain of homology to SNCF, which are all expressed early in embryogenesis at the blastoderm stage.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Nucléolo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción SOX , Homología de Secuencia de Aminoácido , Activación Transcripcional , TransfecciónRESUMEN
Genes of the Sox family encode evolutionarily conserved HMG box containing transcription factors, which play key roles in various events of cell determination/differentiation during development. The total number of Sox genes in Drosophila melanogaster was estimated to be eight, after classical molecular cloning approaches and exhaustive screening of the complete Drosophila genome. Here we report the embryonic and larval expression pattern of four previously uncharacterized Sox genes, through antibody staining and in situ hybridization experiments.
Asunto(s)
Drosophila melanogaster/genética , Genoma , Factores de Transcripción/genética , Animales , Clonación Molecular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Bases de Datos como Asunto , Proteínas de Drosophila/biosíntesis , Proteínas del Grupo de Alta Movilidad/biosíntesis , Hibridación in Situ , Familia de Multigenes , Sistemas de Lectura Abierta , Factores de Transcripción SOX , Factores de Tiempo , Distribución Tisular , Factores de Transcripción/biosíntesisRESUMEN
The Sox gene family consists of several genes related by encoding a 79 amino acid DNA-binding domain known as the HMG box. This box shares strong sequence similarity to that of the testis determining protein SRY. SOX proteins are transcription factors having critical roles in the regulation of diverse developmental processes in the animal kingdom. We have characterised the human SOX7 gene and compared it to its mouse orthologue. Chromosomal mapping analyses localised mouse Sox7 on band D of mouse chromosome 14, and assigned human SOX7 in a region of shared synteny on human chromosome 8 (8p22). A detailed expression analysis was performed in both species. Sox7 mRNA was detected during embryonic development in many tissues, most abundantly in brain, heart, lung, kidney, prostate, colon and spleen, suggesting a role in their respective differentiation and development. In addition, mouse Sox7 expression was shown to parallel mouse Sox18 mRNA localisation in diverse situations. Our studies also demonstrate the presence of a functional transactivation domain in SOX7 protein C-terminus, as well as the ability of SOX7 protein to significantly reduce Wnt/beta-catenin-stimulated transcription. In view of these and other findings, we suggest different modes of action for SOX7 inside the cell including repression of Wnt signalling.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Mapeo Físico de Cromosoma , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Transactivadores , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Línea Celular , Cromosomas Humanos Par 8/genética , Clonación Molecular , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Etiquetas de Secuencia Expresada , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/química , Humanos , Factor de Unión 1 al Potenciador Linfoide , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXF , Alineación de Secuencia , Transducción de Señal , Sintenía , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Factores de Transcripción/genética , Proteínas Wnt , beta CateninaRESUMEN
In mammals, male sex determination is initiated by SRY (sex-determining region of the Y chromosome) gene expression and followed by testicular development. This study describes specific down-regulation of the human SRY gene transcription by cAMP stimulation using reverse transcription-polymerase chain reaction experiments. Using transfection experiments, conserved nuclear hormone receptor (NHR1) and Sp1 consensus binding sites were identified as essential for this cAMP transcriptional response. Steroidogenic factor-1 (SF-1), a component of the sex-determination cascade, binds specifically to the NHR1 site and activates the SRY promoter. Activation of SF-1 was abolished by cAMP pretreatment of the cells, suggesting a possible effect of cAMP on the SF-1 protein itself. Indeed, human SF-1 protein contains at least two in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites, leading after phosphorylation to a modification of both DNA-binding activity and interaction with general transcription factors such as Sp1. Taken together, these data suggest that cAMP responsiveness of human SRY promoter involves both SF-1 and Sp1 sites and could act via PKA phosphorylation of the SF-1 protein itself.
Asunto(s)
AMP Cíclico/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares , Factores de Transcripción/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Cartilla de ADN/química , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Factores de Transcripción Fushi Tarazu , Células HeLa , Proteínas de Homeodominio , Humanos , Fosforilación , Plásmidos , Pruebas de Precipitina , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , ARN/química , ARN/aislamiento & purificación , Receptor Cross-Talk/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de la Región Y Determinante del Sexo , Factor de Transcripción Sp1/metabolismo , Factor Esteroidogénico 1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , TransfecciónRESUMEN
We have identified a novel Sox gene in zebrafish (Danio rerio), Sox31, closely related to mammalian group B Sox genes. The gene is maternally expressed. Zygotic transcription starts at gastrulation, in the presumptive neuroectoderm. Later, expression is restricted to the developing central nervous system, including forebrain, midbrain, hindbrain and spinal cord.
Asunto(s)
Embrión no Mamífero/metabolismo , Proteínas del Grupo de Alta Movilidad/biosíntesis , Proteínas del Grupo de Alta Movilidad/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Sistema Nervioso Central/embriología , Ectodermo/metabolismo , Femenino , Gástrula/metabolismo , Hibridación in Situ , Mesencéfalo/embriología , Datos de Secuencia Molecular , Madres , Prosencéfalo/embriología , Rombencéfalo/embriología , Factores de Transcripción SOX , Homología de Secuencia de Aminoácido , Médula Espinal/embriología , Factores de Tiempo , Pez CebraRESUMEN
A cDNA encoding a new human actin-related protein (ARP) was cloned. The corresponding protein is highly conserved with the previously described ARP3 protein, suggesting that it represents a second isoform of the human ARP3 subfamily. This new actin-related protein was subsequently named ARP3beta and represents the second example of multiple isoforms of an actin-related protein in a single organism. The ARP3beta gene was mapped to chromosome band 7q34, centromeric to Sonic Hedgehog. Gene structure analysis revealed that at least part of the observed ARP3beta mRNA heterogeneity is caused by alternative splicing resulting in exon skipping. Transcripts produced after exon 2 skipping are predicted to encode truncated products, whose functionality is still unclear. An ARP3beta pseudogene was detected on chromosome 2p11 by database searching. Several ARP3beta mRNA species were detected by Northern blotting and their abundance varied importantly among tissues: the highest expression levels were detected in fetal and adult brain, whereas lower levels were observed in liver, muscle and pancreas. In contrast, ARP3 mRNAs were detected in all tissues tested. Using in situ hybridization, the expression of ARP3beta in brain was shown to be restricted to neurons and epithelial cells from choroid plexus. This suggests a specific function for ARP3beta in the physiology of the development and/or maintenance of distinct subsets of nerve cells.
Asunto(s)
Actinas/biosíntesis , Actinas/genética , Empalme Alternativo , Encéfalo/metabolismo , Proteínas del Citoesqueleto , Proteína 3 Relacionada con la Actina , Actinas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/embriología , Sistema Nervioso Central/metabolismo , Cromosomas Humanos Par 7 , ADN Complementario/metabolismo , Exones , Humanos , Hibridación in Situ , Hibridación Fluorescente in Situ , Intrones , Datos de Secuencia Molecular , Neuronas/metabolismo , Seudogenes , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
SOX proteins belong to a multigenic family characterized by a unique DNA binding domain, known as the high mobility group box, that is related to that of the testis determining gene SRY. cDNA sequences for more than 30 SOX genes have been identified, and some are known to have diverse roles in vertebrate differentiation and development. Here, we report the isolation and characterization of mouse Sox15 that was uncovered during a screen for high mobility group box containing transcription factors that are expressed at different levels during skeletal muscle differentiation. Sox15 cDNAs were found at a much higher frequency in myoblasts prior to their differentiation into myotubes. Electrophoretic mobility shift assays indicated that recombinant SOX15 protein was capable of binding to a consensus DNA binding site for SOX proteins. When overexpressed in C2C12 myoblasts, wild type SOX15, but not a C-terminal truncated form or the related protein SOX11, specifically inhibited activation of muscle-specific genes and expression of the basic helix-loop-helix myogenic factors myogenin and MyoD, resulting in a failure of the cells to differentiate into myotubes. These results suggest a specific and repressive role for SOX15, requiring the C-terminal domain, during myogenesis.
Asunto(s)
Diferenciación Celular , Proteínas del Grupo de Alta Movilidad/genética , Músculos/embriología , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación , Proteína MioD/genética , Proteína MioD/metabolismo , Miogenina/genética , Miogenina/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes , Factores de Transcripción SOX , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismoRESUMEN
We describe the identification and detailed expression pattern of a second Drosophila Sox gene, SoxNeuro (SoxN), highly related to mammalian group B Sox1, 2, 3 genes. SoxN is expressed in a highly dynamic pattern during embyogenesis, being associated with the development of the central nervous system (CNS), from the early steps onwards. We present strong evidence that the early SoxN neuroectoderm expression is controlled by the zygotic dorso-ventral patterning genes (dpp, sog, brk, twi).
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Proteínas del Grupo de Alta Movilidad/genética , Proteínas de Insectos/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Drosophila/genética , Expresión Génica , Humanos , Datos de Secuencia Molecular , Factores de Transcripción SOXRESUMEN
Many transcription factors have been identified and implicated in male sex determination pathway. Specifically involved in Sertoli cell differentiation and subsequent anti-Müllerian hormone (AMH) secretion in eutherian mammals, they include steroidogenic factor-1 (SF-1), SOX9 (SRY HMG box related gene 9), WT1 (Wilms' tumor 1), and GATA-4 (a zinc finger transcription factor). These factors have been described to execute their function in the male sex determination pathway by controlling AMH transcriptional expression. To understand the hierarchies of these factors and their involvement in the developing testis, for the first time we show the expression and subcellular localization of these factors by immunohistochemistry in the early human testis during embryogenesis compared with AMH expression. If these studies do not refute their possible synergistic interaction to control AMH expression in human embryo, they also reveal a new sexual dimorphism in SOX9 expression during the sex determination process. We show that SOX9 sex specifically shifts from the cytoplasmic to the nuclear compartment at the time of testis differentiation and AMH expression. Putative models for this subcellular distribution are discussed.
Asunto(s)
Proteínas de Unión al ADN/análisis , Glicoproteínas , Inhibidores de Crecimiento/análisis , Proteínas del Grupo de Alta Movilidad/análisis , Hormonas Testiculares/análisis , Testículo/embriología , Factores de Transcripción/análisis , Hormona Antimülleriana , Factores de Transcripción Fushi Tarazu , Proteínas de Homeodominio , Humanos , Masculino , Receptores Citoplasmáticos y Nucleares , Factor de Transcripción SOX9 , Factor Esteroidogénico 1 , Fracciones Subcelulares , Testículo/química , Proteínas WT1RESUMEN
Sex determination in mammals is controlled by various transcription factors. Following the identification of SRY on the Y chromosome, several other factors have been identified. They can normally be identified as being involved in sex determination by the identification of sex reversal mutations or deletions, functional studies, and also by male-specific expression patterns in embryos. Here, it is shown that DMRT1, recently demonstrated to be deleted in 9p monosomies associated with sex reversal, is specifically expressed during sex determination in the genital ridge of human male, but not female, embryos, similar to SRY.
Asunto(s)
Procesos de Determinación del Sexo , Factores de Transcripción/genética , Femenino , Humanos , MasculinoRESUMEN
Sex determination relies on the translation of chromosomal sex established at fertilisation into gonadal sex (testis or ovary), and later into somatic sex (male or female) under the control of gonadal hormone secretions. The aim of the current review will be to highlight our knowledge of the key events which, in the presence of a Y chromosome, induce the organisation of the developing epithelial cells located inside the genital ridges into testicular cords. Many groups have tried to define the molecules relevant to this process, with a double goal: unravelling a molecular pathway which leads to cell fate decision (Sertoli cell in this particular case) during development; improving the establishment of a diagnosis and subsequent medical management in cases where chromosomal, gonadal and then somatic sexes are discordant. Recent progress made in this area will be depicted, with the introduction of several pieces to this developmental jigsaw puzzle.
Asunto(s)
Mamíferos/genética , Procesos de Determinación del Sexo , Animales , Femenino , Humanos , MasculinoRESUMEN
From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans.
Asunto(s)
Evolución Molecular , Proteínas del Grupo de Alta Movilidad/genética , Animales , Proteínas de Unión al ADN/genética , Bases de Datos Factuales , Variación Genética , Humanos , Familia de Multigenes , Filogenia , Factores de Transcripción/genéticaRESUMEN
SOX proteins contain a conserved HMG-related DNA-binding domain. They fulfill essential functions during the development of animals. Mutations in several SOX genes have been implicated in human diseases. We present here a new set of PCR primers designed to amplify a broad range of SOX HMG-box sequences. These primers facilitated the cloning of several new SOX HMG boxes from human genomic DNA, revealing unexpected complexity of the SOX gene family.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Familia de Multigenes , Proteínas Nucleares , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Proteínas de Unión al ADN/química , Proteínas del Grupo de Alta Movilidad/biosíntesis , Proteínas del Grupo de Alta Movilidad/química , Humanos , Mamíferos , Ratones , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína de la Región Y Determinante del SexoRESUMEN
For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Glicoproteínas , Gónadas/crecimiento & desarrollo , Inhibidores de Crecimiento/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Hormonas Testiculares/genética , Factores de Transcripción/metabolismo , Animales , Hormona Antimülleriana , Células COS , Secuencia de Consenso/genética , Huella de ADN , Factores de Transcripción Fushi Tarazu , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio , Humanos , Inmunohistoquímica , Masculino , Proteínas Nucleares/metabolismo , Unión Proteica/fisiología , Receptores Citoplasmáticos y Nucleares , Proteínas Recombinantes/genética , Factor de Transcripción SOX9 , Eliminación de Secuencia/genética , Homología de Secuencia de Ácido Nucleico , Diferenciación Sexual/genética , Factor Esteroidogénico 1 , Activación Transcripcional/genética , Transfección/genéticaRESUMEN
Anti-müllerian hormone type II receptor (AMHRII) is a serine/threonine receptor and a member of type II receptors of the transforming growth factor beta superfamily. AMHRII has been recently identified in humans, mice, rats, and rabbits. In the male embryo, the AMHRII gene has been shown to be expressed in Sertoli's cells, in Leydig's cells and in the mesenchymal cells surrounding the müllerian duct. To determine the functional region of the AMHRII promoter as well as the factors controlling AMHRII gene expression, we used a 1.1-kilobase DNA fragment from the 5'-flanking region of the human AMHRII gene to generate a series of deletion or mutation and analyzed the resulting transcriptional activities after transfection of the NT2/D1 teratocarcinoma cell line. Our results indicate that maximal expression of the AMHRII promoter in this particular cell line, a cell line positive for endogenous AMHRII expression, requires a conserved estrogen receptor half-site element (AGGTCA) identical to the binding element for steroidogenic factor-1 (SF-1). Studies of this SF-1 binding element using gel mobility shift, antibody supershift assays, and transient transfections of reporter constructs indicate that SF-1 can bind and transactivate the AMHRII promoter. Finally, SF-1 protein expression in human male embryos was shown to display a good coincidence with the previously reported AMHRII expression profile. We then propose that SF-1 may be a key transcriptional regulator of AMHRII gene expression during early human development.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Receptores de Péptidos/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa , Proteínas Quinasas S6 Ribosómicas/metabolismo , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Secuencia de Bases , ADN , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Técnica del Anticuerpo Fluorescente , Factores de Transcripción Fushi Tarazu , Proteínas de Homeodominio , Humanos , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares , Receptores de Factores de Crecimiento Transformadores beta , Proteínas Quinasas S6 Ribosómicas/genética , Factor Esteroidogénico 1 , Factores de Transcripción/metabolismo , Células Tumorales CultivadasAsunto(s)
Proteínas de Unión al ADN/genética , Muridae/genética , Proteínas Nucleares , Procesos de Determinación del Sexo , Diferenciación Sexual/genética , Factores de Transcripción , Animales , Femenino , Masculino , Muridae/clasificación , Proteína de la Región Y Determinante del Sexo , Cromosoma XRESUMEN
To investigate the molecular basis of the human SRY gene regulation, we have examined the significance of two potential binding sites for the transcription factor Sp1 (Sp1A: -124 to -131 and Sp1B: -147 to -154) by DNase I footprinting and gel mobility shift assays. Cotransfection experiments in Drosophila SL2 cells implicated Sp1 protein in the transcriptional activation of the SRY promoter.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Nucleares , Regiones Promotoras Genéticas , Procesos de Determinación del Sexo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , ADN/genética , ADN/metabolismo , Huella de ADN , Desoxirribonucleasa I , Drosophila , Femenino , Humanos , Masculino , Proteína de la Región Y Determinante del Sexo , Activación Transcripcional , TransfecciónRESUMEN
The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function. Homozygous mutations of WT1 can be found in approximately 15% of Wilms' tumors. Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males. A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene. Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors. Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide. Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele. In contrast to Denys-Drash syndrome, no mutant protein is produced. The splice mutation leads to an imbalance of WT1 isoforms in vivo , as detected by RT-PCR on streak gonadal tissue. Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform.
Asunto(s)
Proteínas de Unión al ADN/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Disgenesia Gonadal/genética , Insuficiencia Renal/genética , Factores de Transcripción/genética , Anomalías Urogenitales/genética , Empalme Alternativo , Femenino , Humanos , Intrones/genética , Cariotipificación , Masculino , Mutación Puntual , Reacción en Cadena de la Polimerasa , Síndrome , Proteínas WT1 , Dedos de Zinc/genéticaRESUMEN
Of the several strategies that eukaryotes have evolved to modulate transcription factor activity, phosphorylation is regarded as one of the major mechanisms in signal-dependent transcriptional control. To conclusively demonstrate that the human sex-determining gene SRY is affected by such a post-translational control mechanism, we have analyzed its phosphorylation status in living cells. In the present study, we show that the cyclic AMP-dependent protein kinase (PKA) phosphorylates the human SRY protein in vitro as well as in vivo on serine residues located in the N-terminal part of the protein. This phosphorylation event was shown to positively regulate SRY DNA-binding activity and to enhance the ability of SRY to inhibit a basal promoter activity located downstream of an SRY DNA-binding site concatamer. Together these results strongly support the hypothesis that human SRY is a natural substrate for PKA in vivo and that this phosphorylation significantly modulates its major activity, DNA-binding, thereby possibly altering its biological function.