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1.
Mol Cell Biol ; 16(8): 4024-34, 1996 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8754800

RESUMEN

The Gfi-1 proto-oncogene encodes a zinc finger protein with six C2H2-type, C-terminal zinc finger motifs and is activated by provirus integration in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. In this report, we show that Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence-specific manner. The Gfi-1 binding site, TAAATCAC(A/T)GCA, was defined via random oligonucleotide selection utilizing a bacterially expressed glutathione S-transferase-Gfi-1 fusion protein. Binding to this site was confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oliginucleotides defined the relative importance of specific bases at the consensus binding site. Deletion of individual zinc fingers demonstrated that only zinc fingers 3, 4, and 5 are required for sequence-specific DNA binding. Potential Gfi-1 binding sites were detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early-chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3 fibroblasts, were repressed by Gfi-1, and the repression was abrogated by mutation of critical residues in the two Gfi-1 binding sites. These results suggest that Gfi-1 may play a role in HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Proteínas Represoras , Factores de Transcripción , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Regulación de la Expresión Génica , Células HL-60 , Humanos , Metilación , Ratones , Datos de Secuencia Molecular , Proto-Oncogenes Mas , ARN Mensajero/genética , Alineación de Secuencia , Relación Estructura-Actividad , Dedos de Zinc
2.
J Virol ; 68(12): 7709-16, 1994 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-7966560

RESUMEN

In earlier studies, we have shown that superinfection of an interleukin-2 (IL-2)-dependent, Moloney murine leukemia virus (MoMuLV)-induced rat T-cell lymphoma line (4437A) with mink cell focus-forming (also called polytropic) murine retroviruses induces rapid progression to IL-2-independent growth. In this report, we present evidence that the vast majority (> 90%) of the IL-2-independent lines established from polytropic or xenotropic virus-infected 4437A cells carry provirus insertions in the 3' untranslated region of the IL-9 receptor gene (Gfi-2 [for growth factor independence-2]/IL-9R). Prior to superinfection, the cells express neither IL-9 nor IL-9R. Following superinfection and provirus insertion in the Gfi-2/IL-9R locus, the cells express high levels of mRNA transcripts with a truncated 3' untranslated region which are predicted to encode the normal IL-9R protein product. The same IL-2-independent cells also express IL-9 which is induced by an insertional mutagenesis-independent mechanism. The establishment of an IL-9-dependent autocrine loop was sufficient to render the cells IL-2 independent, as suggested by the finding that 4437A cells, expressing a stably transfected Gfi-2/IL-9R construct, do not require IL-2 when maintained in IL-9-containing media. Additional experiments designed on the basis of these results showed that IL-9 gene expression is induced rapidly following the infection of 4437A cells by polytropic or xenotropic viruses and occurs in the absence of selection for IL-2-independent growth. Taken together, these data suggest that infection of 4437A cells by mink cell focus-forming or xenotropic viruses induces the expression of IL-9, which in turn rapidly selects the cells expressing the IL-9 receptor through an insertional mutagenesis-dependent mechanism. Given that both the polytropic and xenotropic viruses can induce the IL-9-dependent autocrine loop, the reduced ability of the xenotropic viruses to rapidly induce IL-2 independence in culture and tumors in animals is likely to be the result of their lower growth rates.


Asunto(s)
Expresión Génica , Interleucina-2/farmacología , Interleucina-2/fisiología , Virus Inductores de Focos en Células del Visón/inmunología , Virus de la Leucemia Murina de Moloney/inmunología , Receptores de Interleucina-2/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Humanos , Interleucina-2/biosíntesis , Hígado/inmunología , Linfoma de Células T/inmunología , Ratones , Datos de Secuencia Molecular , Provirus/inmunología , Ratas , Receptores de Interleucina-2/biosíntesis , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transducción de Señal , Bazo/inmunología , Timo/inmunología , Transfección , Células Tumorales Cultivadas
3.
Am J Anat ; 191(3): 312-20, 1991 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-1927975

RESUMEN

HL-60 cells, a human promyelocytic leukemia cell line, contain amplified c-myc DNA sequences and mRNA transcripts. These cells can be induced to undergo macrophage differentiation by phorbol esters, which results in suppression of c-myc expression and cessation of cell proliferation. The nuclear matrix (NM), a nuclear skeleton resistant to DNase I digestion and high salt extraction, is proposed to be involved in DNA replication, gene regulation, and the correct distribution of DNA at mitosis. We have previously identified a nuclear-matrix-associated region (MAR) of the c-myc protooncogene to reside in a 1.4-kb region between Cla I and Eco RI restriction sites at the 3'-end of the gene. A 172-bp Dra I/Dra I subfragment of the 1.4-kb region was shown to be a major component of the MAR (myc-MAR), and this subfragment was demonstrated to be recognized by a nuclear protein (p25). In this report we demonstrate that phi X174 DNA, or the synthetic copolymers poly[d(G.C)] and poly[d(A.T)], are not effective suppressors of the binding of the myc-MAR to isolated NM, indicating that the binding sequence(s) are unique. We find that the addition of partially purified protein p25 increases the relative affinity of the myc-MAR for HL-60 NM in an in vitro assay system. NM isolated from HL-60 macrophages induced by phorbol esters retains significantly more myc-MAR DNA fragment in the presence of an excess amount of competitor DNA than does NM from untreated HL-60 cells. These data suggest that a change of the myc-MAR association with the NM occurs after monocytic differentiation of HL-60 cells.


Asunto(s)
Diferenciación Celular , Genes myc , Matriz Nuclear/metabolismo , Composición de Base , Sitios de Unión/efectos de los fármacos , Unión Competitiva , Diferenciación Celular/efectos de los fármacos , Línea Celular , Humanos , Ésteres del Forbol/farmacología , Mapeo Restrictivo
4.
Cancer Res ; 50(11): 3199-206, 1990 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-2185883

RESUMEN

A nuclear matrix (NM)-associated region (MAR) of the protooncogene c-myc is identified in a human leukemia cell line (HL-60). A binding assay between isolated NM and 32P-end-labeled c-myc fragments in the presence of unlabeled competitors was used, and a 3'-end DraI/DraI fragment of 172 base pairs containing the first of the two polyadenylation [poly(A)] signals was identified as an in vitro MAR. Direct detection of endogenous c-myc fragments remaining NM bound after restriction digestion was used, and an in vivo MAR has been identified as the ClaI/EcoRI 1.4-kilobase pair fragment containing the 172-base pair in vitro MAR fragment. In addition, a nuclear protein (Mr = 25,000, p25) demonstrating preferential binding to the 172-base pair c-myc MAR has been identified and partially purified. This protein is diminished in the nuclei of the cells induced by phorbol ester to undergo macrophage differentiation. Footprint analysis shows that p25 binds to two regions of the 172-base pair fragment. One contains the first of two poly(A) addition signals and a topo II box-like sequence, and the other (AATTTCAATCCTAGTA) is 17 base pairs downstream of the first poly(A) signal.


Asunto(s)
ADN de Neoplasias/análisis , Leucemia Promielocítica Aguda/genética , Proteínas Nucleares/análisis , Proteínas Proto-Oncogénicas/análisis , Autorradiografía , Secuencia de Bases , Southern Blotting , Western Blotting , Línea Celular , Sondas de ADN , Proteínas de Unión al ADN/análisis , Electroforesis en Gel de Poliacrilamida , Humanos , Leucemia Promielocítica Aguda/patología , Datos de Secuencia Molecular , Matriz Nuclear/análisis , Proteínas Proto-Oncogénicas c-myc
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