RESUMEN
The ETS family member Tel is rearranged in human leukemia of both myeloid and lymphoid origin while the ETS member Fli-1 is insertionally activated in Friend erythroleukemia in mice and is translocated to the EWS locus in Ewing's sarcoma. In previous studies we demonstrated that Tel binds to Fli-1 and blocks transactivation of megakaryocytic promoters by Fli-1. In this study we demonstrate that expression of Fli-1 in the leukemia cell line K562 induces a megakaryocytic phenotype and the expression of the platelet markers GPIX, GP1balpha, and GPIIb. Introduction of Tel blocked the megakaryocytic phenotype induced by Fli-1, suggesting a biological correlation to the biochemical interaction of Tel and Fli-1 reported previously.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/farmacología , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas , Proteínas Represoras , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/farmacología , Biomarcadores , Western Blotting , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Megacariocitos/inmunología , Familia de Multigenes , Fenotipo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/biosíntesis , Complejo GPIb-IX de Glicoproteína Plaquetaria/biosíntesis , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas c-ets , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas/química , Proteína ETS de Variante de Translocación 6RESUMEN
Glycoprotein (GP) IX is a subunit of the von Willebrand receptor, GPIb-V-IX, which mediates adhesion of platelets to the subendothelium of damaged blood vessels. Previous characterization of the GPIX promoter identified a functional Ets site that, when disrupted, reduced promoter activity. However, the Ets protein(s) that regulated GPIX promoter expression was unknown. In this study, transient cotransfection of several GPIX promoter/reporter constructs into 293T kidney fibroblasts with a Fli-1 expression vector shows that the oncogenic protein Fli-1 can transactivate the GPIX promoter when an intact GPIX Ets site is present. In addition, Fli-1 binding of the GPIX Ets site was identified in antibody supershift experiments in nuclear extracts derived from hematopoietic human erythroleukemia cells. Comparative studies showed that Fli-1 was also able to transactivate the GPIbalpha and, to a lesser extent, the GPIIb promoter. Immunoblot analysis identified Fli-1 protein in lysates derived from platelets. In addition, expression of Fli-1 was identified immunohistochemically in megakaryocytes derived from CD34(+) cells treated with the megakaryocyte differentiation and proliferation factor, thrombopoietin. These results suggest that Fli-1 is likely to regulate lineage-specific genes during megakaryocytopoiesis.
Asunto(s)
Plaquetas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Megacariocitos/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas , Transactivadores/metabolismo , Activación Transcripcional , Sitios de Unión , Línea Celular , Humanos , Células K562 , Riñón , Luciferasas/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/biosíntesis , Proteína Proto-Oncogénica c-fli-1 , Proteínas Recombinantes de Fusión/biosíntesis , TransfecciónRESUMEN
The tel gene, recently shown to be translocated in a spectrum of acute and chronic human leukemias, belongs to the ets family of sequence-specific transcription factors. To determine the role of Tel in normal hematopoietic development, we used the tel gene as the bait in the yeast two-hybrid system to screen a hematopoietic stem cell library. Two partners were identified: Tel binds to itself, and Tel binds to the ets family member Fli-1. In vitro and in vivo assays confirmed these interactions. In transient transfection assays, Fli-1 transactivates megakaryocytic specific promoters, and Tel inhibits this effect of Fli-1. Transactivation studies using deletion mutants of Tel, and the Tel-AML-1 fusion protein, indicate that the helix-loop-helix domain of Tel only partially inhibits transactivation and that complete inhibition requires the full-length Tel molecule, including the DNA binding domain. The Tel and Fli-1 proteins are expressed early in hematopoiesis, and the inability of Tel fusion proteins such as Tel-AML-1 to counteract Fli-1 mediated transactivation may contribute to the malignant phenotype in human leukemias where this fusion protein is present.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas , Proteínas Represoras , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Bases , Diferenciación Celular , Cartilla de ADN , Proteínas de Unión al ADN/química , Secuencias Hélice-Asa-Hélice , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Sistemas de Lectura Abierta , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas c-ets , Factores de Transcripción/química , Células Tumorales Cultivadas , Proteína ETS de Variante de Translocación 6RESUMEN
The human leukocyte integrin CD18 molecule exists on the leukocyte surface in heterodimeric complexes with individual CD11 subunits, which mediate important leukocyte adhesion reactions. The CD18 subunit is developmentally regulated with the highest levels present on mature leukocytes of all lineages. To identify the regulatory sequences responsible for the tissue- and stage-specific expression of the CD18 subunit, we used 3.5 kb of regulatory sequence upstream from the human CD18 gene transcription start site to drive expression of a modified human CD4 reporter gene in transgenic mice. Despite the inclusion of Sp1 and PU.1 sites in the construct, and the generation of founder lines possessing multiple copies of the transgene, the reporter gene was expressed in low levels in the leukocytes of the transgenic mice. These studies indicate that although PU.1 and Sp1 sites are required for CD18 promoter activity in vitro, additional regulatory regions appear to be required for high levels of copy number dependent expression in vivo.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos CD18/genética , Antígenos CD4/biosíntesis , Expresión Génica , Regiones Promotoras Genéticas , Animales , Antígenos CD/genética , Antígenos CD4/genética , Citometría de Flujo , Hormona del Crecimiento/biosíntesis , Humanos , Antígeno-1 Asociado a Función de Linfocito/genética , Ratones , Ratones Transgénicos , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Factores de TiempoRESUMEN
Two cDNAs encoding calnexin (Cln)-like and calreticulin (Crl)-like proteins have been isolated by immunoscreening of a maize leaf cDNA library. In the deduced amino acid (aa) sequences, several regions that are conserved for Cln and Crl proteins from all sources have been identified. These regions can be classified into two distinct motifs which are repeated four times each in Cln and three times each in Crl sequences. One of these motifs, containing a highly acidic 17-aa sequence, has high homology to a Ca(2+)-binding domain previously characterized in both Cln and Crl from mammalian tissues. Motifs for retention in endoplasmic reticulum (Crl) and for an integral membrane-spanning sequence (Cln) have also been identified.