RESUMO
The budding yeast RENT complex, consisting of at least three proteins (Net1, Cdc14, Sir2), is anchored to the nucleolus by Net1. RENT controls mitotic exit, nucleolar silencing, and nucleolar localization of Nop1. Here, we report two new functions of Net1. First, Net1 directly binds Pol I and stimulates rRNA synthesis both in vitro and in vivo. Second, Net1 modulates nucleolar structure by regulating rDNA morphology and proper localization of multiple nucleolar antigens, including Pol I. Importantly, we show that the nucleolar and previously described cell cycle functions of the RENT complex can be uncoupled by a dominant mutant allele of CDC14. The independent functions of Net1 link a key event in the cell cycle to nucleolar processes that are fundamental to cell growth.
Assuntos
Nucléolo Celular/fisiologia , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição , Proteínas Tirosina Fosfatases , RNA Polimerase I/metabolismo , Ribonucleoproteínas Nucleolares Pequenas , Proteínas de Saccharomyces cerevisiae , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transcrição Gênica , Animais , Northern Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/ultraestrutura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Proteínas Nucleares/genética , Conformação de Ácido Nucleico , Fenótipo , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/fisiologia , Sirtuína 2 , Sirtuínas , Esporos Fúngicos/fisiologia , Temperatura , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Interferons (IFNs) are a family of multifunctional proteins involved in immune activation, regulation of cell growth and antiviral response. They exert their functions by induction of several IFN-stimulated genes, including IFN regulatory factors (IRFs), a family of transcriptional regulators. One of these factors, IRF-2, was initially cloned as an antagonistic counterpart to IRF-1 with oncogenic potential. Here we describe a second isoform of IRF-2, termed IRF-2s, cloned from human and murine cells. This isoform lacks two amino acids located C-terminal of the DNA-binding domain, which is conserved in all IRF family members, leading to a change in the predicted secondary structure. Both isoforms have similar binding affinities to known target sequences in electrophoretic mobility shift assays. Using reporter gene constructs with the type IV promoter region of the MHC class II transactivator (CIITA), which is the essential factor for IFN-gamma-induced MHC class II expression, we show that the short isoform IRF-2s exhibits a weaker activation ability compared to IRF-2. Thus, our data present the first evidence of two IRF-2 isoforms with different regulatory ability.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares , Proteínas Repressoras , Fatores de Transcrição , Ativação Transcricional , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , DNA/genética , Proteínas de Ligação a DNA/genética , Genes MHC da Classe II/genética , Genes Reporter , Humanos , Fator Regulador 2 de Interferon , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/genética , Alinhamento de Sequência , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , TransfecçãoRESUMO
PURPOSE: Mice experiments have established an important role for interferon regulatory factor (IRF) family members in hematopoiesis. We wanted to study the expression of interferon regulatory factor 4 (IRF4) in various hematologic disorders, especially chronic myeloid leukemia (CML), and its association with response to interferon alfa (IFN-alpha) treatment in CML. MATERIALS AND METHODS: Blood samples from various hematopoietic cell lines, different leukemia patients (70 CML, 29 acute myeloid leukemia [AML], 10 chronic myelomonocytic leukemia [CMMoL], 10 acute lymphoblastic leukemia, and 10 chronic lymphoid leukemia patients), and 33 healthy volunteers were monitored for IRF4 expression by reverse transcriptase polymerase chain reaction. Then, with a focus on CML, the IRF4 level was determined in sorted cell subpopulations from CML patients and healthy volunteers and in in vitro-stimulated CML cells. Furthermore, IRF4 expression was compared in the CML samples taken before IFN-alpha therapy and in 47 additional CML samples taken during IFN-alpha therapy. IRF4 expression was then correlated with cytogenetic response to IFN-alpha. RESULTS: IRF4 expression was significantly impaired in CML, AML, and CMMoL samples. The downregulation of IRF4 in CML samples was predominantly found in T cells. In CML patients during IFN-alpha therapy, a significant increase in IRF4 levels was detected, and this was also observed in sorted T cells from CML patients. The increase seen during IFN-alpha therapy was not due to different blood counts. In regard to the cytogenetic response with IFN-alpha, a good response was associated with high IRF4 expression. CONCLUSION: IRF4 expression is downregulated in T cells of CML patients, and its increase is associated with a good response to IFN-alpha therapy. These data suggest IRF4 expression as a useful marker to monitor, if not predict, response to IFN-alpha in CML.
