RESUMEN
The NTF-like family of transcription factors have been implicated in developmental regulation in organisms as diverse as Drosophila and man. The two mammalian members of this family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related proteins sharing an overall amino acid identity of 72%. CP2, the best characterized of these factors, is a ubiquitously expressed 66-kDa protein that binds the regulatory regions of many diverse genes. Consequently, a role for CP2 has been proposed in globin gene expression, T-cell responses to mitogenic stimulation, and several other cellular processes. To elucidate the in vivo role of CP2, we have generated mice nullizygous for the CP2 allele. These animals were born in a normal Mendelian distribution and displayed no defects in growth, behavior, fertility, or development. Specifically, no perturbation of hematopoietic differentiation, globin gene expression, or immunological responses to T- and B-cell mitogenic stimulation was observed. RNA and protein analysis confirmed that the nullizygous mice expressed no full-length or truncated version of CP2. Electrophoretic mobility shift assays with nuclear extracts from multiple tissues demonstrated loss of CP2 DNA binding activity in the -/- lines. However, a slower migrating complex that was ablated with antiserum to NF2d9, the murine homologue of LBP-1a, was observed with these extracts. Furthermore, we demonstrate that recombinant LBP-1a can bind to known CP2 consensus sites and form protein complexes with previously defined heteromeric partners of CP2. These results suggest that LBP-1a/NF2d9 may compensate for loss of CP2 expression in vivo and that further analysis of the role of the NTF family of proteins requires the targeting of the NF2d9 gene.
Asunto(s)
Proteínas de Unión al ADN/genética , Marcación de Gen , Factores de Transcripción/genética , Animales , ADN/metabolismo , Embrión de Mamíferos/metabolismo , Hematopoyesis , Ratones , Ratones Transgénicos , Proteínas de Unión al ARNRESUMEN
The stage selector protein (SSP) is a heteromeric complex involved in preferential expression of the human gamma-globin genes in fetal-erythroid cells. We have previously identified the ubiquitous transcription factor CP2 as a component of this complex. Using the protein dimerization domain of CP2 in a yeast two-hybrid screen, we have cloned a novel gene, NF-E4, encoding the tissue-restricted component of the SSP. NF-E4 and CP2 coimmunoprecipitate from extract derived from a fetal-erythroid cell line, and antiserum to NF-E4 ablates binding of the SSP to the gamma promoter. NF-E4 is expressed in fetal liver, cord blood, and bone marrow and in the K562 and HEL cell lines, which constitutively express the fetal globin genes. Enforced expression of NF-E4 in K562 cells and primary erythroid progenitors induces endogenous fetal globin gene expression, suggesting a possible strategy for therapeutic intervention in the hemoglobinopathies.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Sangre Fetal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Globinas/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Secuencia de Bases , Codón Iniciador/genética , ADN/genética , ADN/metabolismo , Dimerización , Perfilación de la Expresión Génica , Globinas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Células K562 , Sustancias Macromoleculares , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión , Factores de Transcripción/química , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
We have recently described the action of Oncostatin M (OSM) to inhibit the proliferation of breast cancer cells. In this study we examined the action of OSM on 2 breast cancer cell lines to further characterize the nature of OSM inhibition of cellular proliferation. Treatment with OSM for 6 days resulted in an approximately 2- to 5-fold decrease in cell number, which was independent of estrogen receptor status. Consistent with this, colony formation was reduced to approximately 50% when cells were exposed to OSM in primary agar cultures. Clonogenicity was further inhibited following 7 days treatment with OSM in monolayer cultures: the total number of clonogenic cells was suppressed approximately 10-fold. Analysis of cell cycle status in OSM-treated cells demonstrated a 40% reduction in the proportion of cells in S phase within 12 hr, with an increase in cells in G0/G1. After 6 days, there was a 10-fold reduction in the absolute number of cells in S phase in OSM-treated cultures. These changes were associated with striking changes in cellular morphology, including disruption of intercellular junctions and the production of lipid droplets. There was a 5-fold increase of c-fos and c-myc mRNA within 30 min of commencing treatment with OSM. In addition, in the ER positive cells there was a decrease in ER mRNA (evident within approximately 2 hr) and ER protein expression following treatment with OSM. Conversely, there was a 5-fold increase in epidermal growth factor receptor (EGFR) mRNA within 4 hr, and a 2.5-fold rise in mRNA for transforming growth factor alpha (TGF alpha). Thus, the inhibition of breast cancer cells by OSM was associated with decreased clonogenicity, a decrease in S phase cells and a variety of phenotypic changes, all consistent with the induction of differentiation.
