RESUMEN
Signal transducer and activator of transcription 5 (STAT5) is activated by numerous cytokines that control blood cell development. STAT5 was also shown to actively participate in leukemogenesis. Among the target genes involved in cell growth, STAT5 had been shown to activate cyclin D1 gene expression. We now show that thrombopoietin-dependent activation of the cyclin D1 promoter depends on the integrity of a new bipartite proximal element that specifically binds STAT5A and -B transcription factors. We demonstrate that the stable recruitment of STAT5 to this element in vitro requires the integrity of an adjacent octamer element that constitutively binds the ubiquitous POU homeodomain protein Oct-1. We observe that cytokine-activated STAT5 and Oct-1 form a unique complex with the cyclin D1 promoter sequence. We find that STAT5 interacts with Oct-1 in vivo, following activation by different cytokines in various cellular contexts. This interaction involves a small motif in the carboxy-terminal region of STAT5 which, remarkably, is similar to an Oct-1 POU-interacting motif present in two well-known partners of Oct-1, namely, OBF-1/Bob and SNAP190. Our data offer new insights into the transcriptional regulation of the key cell cycle regulator cyclin D1 and emphasize the active roles of both STAT5 and Oct-1 in this process.
Asunto(s)
Ciclina D1/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de la Leche , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Ciclina D1/metabolismo , Citocinas/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes bcl-1 , Factor C1 de la Célula Huésped , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Ratones , Factor 1 de Transcripción de Unión a Octámeros , Regiones Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT5 , Trombopoyetina/farmacología , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Proteínas Supresoras de TumorRESUMEN
mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5' cap structure and the 3' poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.
Asunto(s)
Diferenciación Celular/fisiología , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , Humanos , Megacariocitos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Trombopoyetina/metabolismoRESUMEN
OBJECTIVE: p21(WAF1/Cip/kip) and p27(Kip1) are cyclin-dependant kinase inhibitors controlling cell-cycle exit and differentiation of numerous cell types. Among hematopoietic cells, megakaryocytes express high levels of p21, while in erythroid cells, p27(Kip1) is predominant. As p21 and p27 could display overlapping functions and as megakaryocytes and erythroid cells derive from a bipotent progenitor, we developed an in vivo model to determine the specific role of p21 in controlling the proliferation/differentiation balance of erythroid and megakaryocytic progenitors. METHODS: Transgenic mice that overexpressed p21 under the control of the human GPIIb promoter in early progenitors and along megakaryocytic differentiation were generated. Different subsets of hematopoietic progenitors (BFU and CFU) and primitive cells (CAFC, LTC-IC) were analyzed by methylcellulose assay. Phenotypic evolution and clonogenic properties of the lin(-) population were analyzed along erythroid and megakaryocytic differentiation. RESULTS: We observed p21 ectopic expression in early hematopoietic progenitors (lin(-)Sca(+)), megakaryocytes, and, to a lesser extent, erythroid cells. This expression induced an important decrease in the number of CFU-MK, BFU-E, CFU-E, primitive progenitors (CAFC day 35), and LTC-IC, but did not affect the maturation process of these cells and the blood cell count. CONCLUSIONS: We show that variation of p21 expression level changes the fate of hematopoietic cells by favoring either proliferation or differentiation pathways. This effect of p21 is exerted not only at the level of primitive progenitors but also in more mature progenitors. However, in vivo, a systemic compensation mechanism is most likely activated in response to variations of the flow of progenitor production.
Asunto(s)
Ciclinas/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Glicoproteína IIb de Membrana Plaquetaria/genética , Regiones Promotoras Genéticas , Animales , Recuento de Células Sanguíneas , Diferenciación Celular , División Celular , Linaje de la Célula , Ensayo de Unidades Formadoras de Colonias , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , ADN Complementario/genética , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Regulación de la Expresión Génica , Genes Sintéticos , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Homeostasis , Humanos , Megacariocitos/citología , Megacariocitos/metabolismo , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Class I MHC complexes (MHC(I)) are essential in mediating immune response. The transport of antigenic peptides (TAP) to MHC(I) and the stable expression of MHC(I) on the cell surface require the presence of a dedicated TAP. In this study we report that IFN-gamma and thrombopoietin (TPO) strongly increase TAP1 protein expression in megakaryocytes, followed by an enhanced expression of MHC(I) on the cell surface. This expression parallels the enhanced TAP1 promoter activity and TAP1 mRNA expression, which are independent of protein synthesis. We also show that this cytokine-dependent expression of TAP1 transcripts depends on STAT1 and IFN regulatory factor-2 (IRF-2), but not on IRF-1, and provide evidence that IRF-2 constitutively binds to the TAP1 gene promoter and enhances TAP1 promoter activity. We show that IRF-2 forms a complex with STAT1 and the cytokine-responsive region of the TAP1 promoter in any TPO or IFN-gamma target cells tested. Interaction of IRF-2 and STAT1 on the promoter depends on the DNA-binding domain of IRF-2. Overall, our data indicate that TPO and IFN-gamma activate the expression of TAP1 via a new mechanism that involves functional cooperation between STAT1 and IRF-2 on the TAP1 promoter.