RESUMO
The CCAAT/enhancer binding protein family (C/EBP) are transcription factors that play integral roles in the development and function of many organ systems, including hematopoietic cells, adipose tissues, and liver. We have identified and characterized putative zebrafish orthologs of mammalian C/EBP alpha, beta, gamma, and delta using low-stringency hybridization screening and computer searches of the GenBank EST database. c/ebpa and g were mapped within 1 cM of each other on linkage group (LG) 7, syntenic with human CEBPA and G genes on chromosome 19. c/ebpb was mapped to LG8, and c/ebpd was mapped to LG24, on the same LG as a recently identified unique c/ebp in zebrafish, c/ebp1. The mapping of these genes established new syntenic relationships between LG8 and human chromosome 20, extended existing synteny between LG7 and human chromosome 19, and confirmed the synteny between LG24 and human chromosome 8. In addition, these syntenies between zebrafish and human chromosomes are also conserved in the mouse genome. To characterize the expression of these genes, RNA in situ hybridization in embryos of wild type and a hematopoietic mutant, cloche, was performed. The results showed that zebrafish c/ebpa, b, g, and d were expressed in many embryonic tissues. c/ebpa and b were expressed in a subset of hematopoietic cells in a region consistent with myeloid expression. In addition, there was expression of c/ebpa and b in the liver and c/ebpa, b, and d in regions of the gastrointestinal tract. The expression of the c/ebps may serve as important markers for analysis of myelopoiesis, hepatic development, and other developmental processes in the future.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Fatores de Transcrição , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Embrião não Mamífero/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Hibridização In Situ , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologiaRESUMO
The CCAAT/enhancer-binding protein (C/EBP) family consists of transcription factors essential for hematopoiesis. The defining feature of the C/EBPs is a highly conserved carboxy-terminal bZIP domain that is necessary and sufficient for dimerization and DNA binding, whereas their amino-terminal domains are unique. This study reports a novel c/ebp gene (c/ebp1) from zebrafish that encodes a protein homologous to mammalian C/EBPs within the bZIP domain, but with an amino terminus lacking homology to any C/EBP or to any known sequence. In zebrafish embryos, c/ebp1 expression was initially observed in cells within the yolk sac circulation valley at approximately the 16-to 18-somite stage, and at 24 hours postfertilization (hpf), also in circulating cells. Most c/ebp1(+) cells also expressed a known early macrophage marker, leukocyte-specific plastin (l-plastin). Expression of both markers was lost in cloche, a mutant affecting hematopoiesis at the level of the hemangioblast. Expression of both markers was retained in m683 and spadetail, mutants affecting erythropoiesis, but not myelopoiesis. Further, c/ebp1 expression was lost in a mutant with defective myelopoiesis, but intact erythropoiesis. These data suggest that c/ebp1 is expressed exclusively in myeloid cells. In electrophoretic mobility shift assays, c/ebp1 was able to bind a C/EBP consensus DNA site. Further, a chimeric protein containing the amino-terminal domain of c/ebp1 fused to the DNA-binding domain of GAL4 induced a GAL4 reporter 4000-fold in NIH3T3 cells. These results suggest that c/ebp1 is a novel member of the C/EBP family that may function as a potent transcriptional activator in myeloid cells.