RESUMO
Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate a wide array of developmental processes in many cell types, including cell fate specification, differentiation and morphogenesis. Our studies describe the cloning of a gene from the nematode Caenorhabditis elegans that is closely related to the vertebrate-activated B-cell factor (ABF) gene. The nematode gene product CeABF-1 was detected by northern blot analysis from RNA isolated from pooled nematodes representing different developmental stages. The developmental expression profile of CeABF-1 was shown by RT-PCR analysis to be predominantly expressed in the larval stages L3 and L4, with lower levels observed in the L2 larval stage and adult. We also show that CeABF-1 is capable of forming heterodimers with E2A proteins and binding E-box target sites. Mammalian cells transfected with CeABF-1 expression plasmids were capable of blocking E2A-mediated gene transcription, but full repression activity required the presence of two conserved amino acid residues found within the first helix of the CeABF-1 bHLH domain. These results suggest a conserved mechanism of gene repression between certain class II bHLH and class I bHLH proteins found in vertebrates and invertebrates.
Assuntos
Proteínas de Caenorhabditis elegans/isolamento & purificação , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Sequência Conservada , Proteínas de Ligação a DNA , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Clonagem Molecular , DNA/genética , DNA/metabolismo , Dimerização , Elementos E-Box/genética , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Sequências Hélice-Alça-Hélice , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
The activated B-cell factor (ABF)-1 cDNA was initially isolated from Epstein-Barr virus (EBV)-infected B cells and codes for a DNA-binding protein belonging to the basic helix-loop-helix (bHLH) family of transcription factors. In this study, we characterized the nuclear localization signal of ABF-1, mapped two distinct transcriptional repression domains, and identified one ABF-1-interacting protein, Id-2. By examining the subcellular location of deletion mutants of ABF-1 fused to green fluorescent protein (GFP), critical regions involved in nuclear localization were determined. Analysis of GFP-tagged ABF-1 deletion mutants revealed two separate regions capable of directing nuclear localization. One region mapped to the N-terminal amino acids 71 to 103, whereas the second region localized to the C-terminal bHLH domain. Transient transfection of ABF-1 deletion mutants demonstrated that the N-terminal amino acids 1 to 40 and the bHLH domain function together to achieve maximum repression of E2A activity. Taken together, these results indicate that ABF-1 is a nuclear transcriptional repressor with two distinct regions that function in a synergistic fashion to attenuate E2A-mediated gene activation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Alça-Hélice , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Proteína 2 Inibidora de Diferenciação , Sinais de Localização Nuclear/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The human ABF-1 gene is expressed in activated B-cells and Epstein-Barr virus-immortalized lymphoblastoid cell lines. ABF-1 represents the only member belonging to the basic helix-loop-helix (bHLH) family of transcription factors whose expression pattern is restricted to B-cells. ABF-1 forms heterodimeric complexes with E2A to modulate gene transcription. We report the cloning and characterization of the human ABF-1 gene and the promoter region. The gene spans more than 3 kb and contains two exons. Exon 1 contains 274 bp of a 5'-untranslated sequence (UTR) while exon 2 contains 1097 bp of 3'-UTR. Promoter analysis of the 5'-flanking region revealed no apparent B-cell-restricted control elements within approximately 700 bp, but clearly demonstrated the presence of a functional minimal promoter residing immediately upstream of the transcription start site. Analysis of the region containing the minimal promoter activity identified no CCAAT or TATA sequence. Lastly, we have assigned the ABF-1 gene to human chromosome 8q21.1 using fluorescence in situ hybridization (FISH). The cloning of the human ABF-1 gene will facilitate further biochemical and genetic studies of its function in the regulation of B-cell differentiation.
