RESUMEN
Inverted CCAAT box-binding protein of 90 kDa (ICBP90) is over-expressed in several types of cancer, including breast, prostate and lung cancers. In search for proteins that interact with the set and ring-associated (SRA) domain of ICBP90, we used the two-hybrid system and screened a placental cDNA library. Several clones coding for a new domain of DNMT1 were found. The interaction, between the ICBP90 SRA domain and the DNMT1 domain, has been confirmed with purified proteins by glutathione-S-transferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macro-molecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Co-immunoprecipitation experiments showed that ICBP90 and DNMT1 are present in the same molecular complex, which was further confirmed by co-localization experiments as assessed by immunocytochemistry. Downregulation of ICBP90 and DNMT1 decreased VEGF gene expression, a major pro-angiogenic factor, whereas those of p16(INK4A) gene and RB1 gene were significantly enhanced. Together, these results indicate that DNMT1 and ICBP90 are involved in VEGF gene expression, possibly via an interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 and an upregulation of p16(INK4A). They further suggest a new role of ICBP90 in the relationship between histone ubiquitination and DNA methylation in the context of tumoral angiogenesis and tumour suppressor genes silencing.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/fisiología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/fisiología , Regulación de la Expresión Génica , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas Potenciadoras de Unión a CCAAT/química , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1 , Genes Supresores de Tumor , Histonas/metabolismo , Humanos , Inmunoprecipitación , Células Jurkat , Modelos Biológicos , Neovascularización Patológica/genética , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Interferencia de ARN , Ubiquitina-Proteína Ligasas , Ubiquitinación/genéticaRESUMEN
Preinitiation complex assembly is nucleated by the binding of TFIID to the promoters of protein coding genes transcribed by RNA polymerase II. TFIID is comprised of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s). We investigated the transcription properties of TBP and TFIID on chromatin templates. On naked templates both TBP and purified TFIID are able to initiate basal transcription. However, on chromatin templates only TBP mediates transcription initiation in a heat-treated extract, whereas TFIID does not. Moreover, TBP-mediated chromatin transcription is blocked in a nontreated extract. These observations suggest that a chromatin-targeted repressor is present in crude extracts and that chromatin per se is not refractory to transcription mediated by TBP. As TBP can function through TAF(II)-independent and TAF(II)-dependent pathways, the repression of TBP-mediated basal transcription may be an additional level to the control of Pol II transcription initiation on chromatin.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , TATA Box , Factores de Transcripción TFII/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , ADN Polimerasa II/metabolismo , Drosophila/embriología , Embrión no Mamífero , Células HeLa , Humanos , Proteínas de Insectos/metabolismo , Cinética , Proteína de Unión a TATA-Box , Moldes Genéticos , Factor de Transcripción TFIIDRESUMEN
Initiation of transcription of a gene from a core promoter region by RNA polymerase II requires the assembly of several initiation factors to form a preinitiation complex. Assembly of this complex is thought to be nucleated exclusively by the sequence-specific binding of the TFIID transcription factor complex, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s), to the different promoters. Here we isolate and characterize a new multiprotein complex that does not contain either TBP or a TBP-like factor but is composed of several TAF(II)s and other proteins. This complex can replace TFIID on both TATA-containing and TATA-lacking promoters in in vitro transcription assays. Moreover, an anti-TBP antibody that inhibits TBP- and TFIID-dependent transcription does not inhibit activity of this new complex. These results indicate that TBP-free RNA polymerase II mediated transcription may be able to occur in mammalian cells and that multiple preinitiation complexes may play an important role in regulating gene expression.
Asunto(s)
Proteínas de Unión al ADN/fisiología , ARN Polimerasa II/metabolismo , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción TFII/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Huella de ADN , Células HeLa , Humanos , Proteína de Unión a TATA-Box , Factor de Transcripción TFIID , Activación TranscripcionalRESUMEN
The basal RNA polymerase II transcription factor, TFIID, is composed of the TATA binding protein (TBP) and 8-13 TBP-associated factors (TAFs) ranging from 250 to 17 kDa. The structure of the human gene encoding the 30-kDa subunit of TFIID, TAF2H, has been determined. The gene consists of five exons (ranging from 66 to 248 bp) and four introns (ranging from 83 to 211 bp). The transcription start site of the mRNA was mapped, and it shares a weak homology to the consensus of known initiator elements. Using in situ hybridization on human metaphase chromosomes, the TAF2H gene has been localized in the 11p15.2-p15.5 region of the human genome.
Asunto(s)
Cromosomas Humanos Par 11 , Hominidae/genética , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Exones , Genoma Humano , Células HeLa , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Factor de Transcripción TFIID , Factores de Transcripción/biosíntesis , Transcripción GenéticaRESUMEN
We have cloned cDNAs encoding three novel TAFIIs [TATA-binding protein (TBP)-associated factors] from the human (h) HeLa cell TFIID complexes hTAFII28, hTAFII20 and hTAFII18. hTAFII28 is a core hTAFII present in both of the previously described hTFIID species which either lack or contain hTAFII30 (hTFIID alpha and hTFIID beta respectively), and is the homologue of Drosophila (d)TAFII30 beta. hTAFII18 is a novel hTAFII which shows homology to the N-terminal region of the yeast TAFIISPT3, but has no known Drosophila counterpart. In contrast to hTAFII28, hTAFII18 is a TFIID beta-specific hTAFII. hTAFII20 is the homologue of p22, an alternatively spliced form of dTAFII30 alpha (p32). Using a combination of protein affinity chromatography and cotransfection and immunoprecipitation assays, we have identified a series of in vitro and intracellular interactions among the novel hTAFIIs and between the novel hTAFIIs and hTAFII30 or TBP. We show that hTAFII28 interacts with hTAFII18 both in vitro and intracellularly; in contrast to its Drosophila homologue, hTAFII28 also interacts directly with TBP. Deletion analysis indicates that TBP and hTAFII18 bind to distinct domains of hTAFII28. hTAFII18 also interacts with TBP, but it interacts more strongly with hTAFII28 and hTAFII30. The binding of hTAFII28 and hTAFII30 requires distinct domains of hTAFII18. As observed with the homologous Drosophila proteins, hTAFII20 interacts directly with TBP; however, additional interactions between hTAFII20 and hTAFII28 or hTAFII30 were detected. These results reveal differences not only in subunit composition, but also in the organization of dTFIID and hTFIID complexes.
Asunto(s)
Factores de Transcripción/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Cromatografía de Afinidad , Clonación Molecular , ADN Complementario/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Humanos , Sustancias Macromoleculares , Modelos Estructurales , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Biosíntesis de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TATA Box , Proteína de Unión a TATA-Box , Factor de Transcripción TFIID , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , TransfecciónRESUMEN
We showed previously that coactivators mediating stimulation by different activators were associated with the TATA-binding protein (TBP) in distinct TFIID complexes. We have characterized a human TBP-associated factor (TAF), hTAFII30, associated with a subset of TFIID complexes. hTAFII30 interacts with the AF-2-containing region E of the human estrogen receptor (ER), but not with ER AF-1 or VP16. An antibody against hTAFII30 inhibited transcriptional stimulation by the ER AF-2 without affecting basal or VP16-activated transcription and allowed the separation of TFIID complex(es) containing hTAFII30 from complexes mediating the activity of VP16. These results directly demonstrate the existence of functionally distinct TFIID populations that share common TAFIIs but differ in specific TAFIIs.
Asunto(s)
Proteínas de Unión al ADN/genética , Receptores de Estrógenos/metabolismo , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Células HeLa , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Histona Acetiltransferasas , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia , TATA Box , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIDRESUMEN
In eukaryotes, the TATA box binding protein (TBP) is an integral component of the transcription initiation complexes of all three classes of nuclear RNA polymerases. In this study we have investigated the role of the N-terminal region of human TBP in transcription initiation from RNA polymerase (Pol) I, II and III promoters by using three monoclonal antibodies (mAbs). Each antibody recognizes a distinct epitope in the N-terminal domain of human TBP. We demonstrate that these antibodies differentially affect transcription from distinct classes of promoters. One antibody, mAb1C2, and a synthetic peptide comprising its epitope selectively inhibited in vitro transcription from TATA-containing, but not from TATA-less promoters, irrespective of whether they were transcribed by Pol II or Pol III. Transcription by Pol I, on the other hand, was not affected. Two other antibodies and their respective epitope peptides did not affect transcription from any of the promoters tested. Order of addition experiments indicate that mAb1C2 did not prevent binding of TBP to the TATA box or the formation of the TBP-TFIIA-TFIIB complex but rather inhibited a subsequent step of preinitiation complex formation. These data suggest that a defined region within the N-terminal domain of human TBP may be involved in specific protein-protein interactions required for the assembly of functional preinitiation complexes on TATA-containing, but not on TATA-less promoters.