RESUMEN
Zinc transporter 2 (ZnT2) is one of the cellular factors responsible for Zn homeostasis. Upon Zn overload, ZnT2 reduces cellular Zn by transporting it into excretory vesicles. We investigated the molecular mechanism that regulates human ZnT2 (hZnT2) gene expression. Zn induces hZnT2 expression in dose- and time-dependent manners. Overexpression of metal-responsive transcription factor 1 (MTF-1) increases hZnT2 transcription, whereas depletion of MTF-1 reduces hZnT2 expression. There are five putative metal response elements (MREs) within 1kb upstream of the hZnT2 gene. A serial deletion of the hZnT2 promoter region (from 5' to 3') shows that the two MREs proximal to the gene are essential for Zn-induced promoter activity. Further mutation analysis concludes that the penultimate MRE (MREb) supports the metal-induced promoter activity. The hZnT2 promoter has also a zinc finger E-box binding homeobox (ZEB) binding element. Mutation or deletion of this ZEB binding element elevates the basal and Zn-induced hZnT2 promoter activities. Knockdown of ZEB1 mRNA enhances the hZnT2 transcript level in HEK-293 cells. In MCF-7 (ZEB-deficient) cells, expression of ZEB proteins attenuates the Zn-induced hZnT2 expression. However, expressions of MTF-1 target genes such as human ZnT1 and metallothionein IIA were not affected. Our study shows the expression of the hZnT2 gene is coordinately regulated via active and suppressive modulators.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Animales , Células CHO , Cadmio/metabolismo , Proteínas de Transporte de Catión/genética , Cricetulus , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Genes Reporteros , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células MCF-7 , Metalotioneína/genética , Metalotioneína/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Transcripción Genética , Transfección , Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Factor de Transcripción MTF-1RESUMEN
Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Escherichia coli/genética , Femenino , Ratones , Mutación , Mapas de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Elementos de Respuesta , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Factor de Transcripción MTF-1RESUMEN
MTF-1 (metal-responsive transcription factor 1) is an essential mammalian protein for embryonic development and modulates the expression of genes involving in zinc homoeostasis and responding to oxidative stress. We report in the present paper that PTEN (phosphatase and tensin homologue deleted on chromosome 10) associates with MTF-1 in the cells. These two proteins interact via the acidic domain of MTF-1 and the phosphatase/C2 domain of PTEN. Depletion of PTEN reduced MT (metallothionein) gene expression and increased cellular sensitivity to cadmium toxicity. PTEN did not alter the nuclear translocation, protein stability or DNA-binding activity of MTF-1. Zinc increased MTF-1-PTEN interaction in a dose-dependent manner. The interaction elevated within 2 h of zinc addition and declined afterwards in the cells. The enhanced binding activity occurred mainly in the cytoplasm and reduced after translocating the MTF-1 into the nucleus. Blocking signalling through the PI3K (phosphoinositide 3-kinase) pathway did not alter the zinc-induced MT expression. Analysis of enzymatically inactive PTEN mutants demonstrated that protein but not lipid phosphatase activity of PTEN was involved in the regulation of MTF-1 activity. The same regulatory role of PTEN was also noted in the regulation of ZnT1 (zinc transporter 1), another target gene of MTF-1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fosfohidrolasa PTEN/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Animales , Células CHO , Cadmio , Proteínas de Transporte de Catión , Cricetinae , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Homeostasis , Humanos , Metalotioneína/genética , Metalotioneína/metabolismo , Ratones , Fosfohidrolasa PTEN/genética , Factores de Transcripción/genética , Zinc/metabolismo , Factor de Transcripción MTF-1RESUMEN
Metal-responsive transcription factor 1 (MTF-1) is an essential protein required for mouse embryonic development. We report here the occurrence of sumoylation on MTF-1. Mutational studies demonstrated that sumoylation occurs on the lysine residue at position 627 (Lys(627)) of mouse MTF-1. Small ubiquitin-like modifier (SUMO)-1 was fused to the C terminus of MTF-1 to mimic the sumoylated form of the protein and it suppressed the transcriptional activity of MTF-1. The nuclear translocation activity, DNA-binding activity, and protein stability of SUMO-fused MTF-1 are similar to that of wild type MTF-1. The level of sumoylation was reduced by metal in a dose- and time-dependent manner. The fact that zinc reduces MTF-1 sumoylation makes the suppressive role of sumoylated MTF-1 in transcription physiologically less significant because the SUMO moiety of MTF-1 is removed when MTF-1 translocates into nucleus. We further identified a SUMO-interacting motif (SIM) on MTF-1. Remarkably, MTF-1 binds sumoylated MTF-1 and/or other cellular factors in a SIM-dependent manner. This interaction was disrupted by treating cells with zinc. Gel permeation chromatography demonstrated that MTF-1 forms SIM-dependent complexes. This cross-interaction transpires in the cytoplasm and markedly reduces upon nuclear translocation. It can therefore be concluded that SUMO conjugation and the SIM on MTF-1 do not play a critical role in suppressing transcriptional activity. Instead, MTF-1 forms complexes with cellular factors through SIM and SUMO moiety in the cytoplasm. The result explores a new understanding for the mode of MTF-1 assembly and regulation in cells.