RESUMO
B-Myb is a highly conserved vertebrate member of the Myb transcription factor family, which is expressed in virtually all proliferating cells. A large body of evidence suggests that B-Myb plays an important role in cell cycle regulation; however, the exact nature of its function has not yet been clarified. We have used gene targeting in chicken DT40 cells, a cell line exhibiting very high rates of homologous recombination, to create cells expressing endogenous B-myb in a doxycyclin-dependent manner. We find that the cells proliferate well in the absence of B-Myb, suggesting that B-Myb is not essential for cell proliferation per se. However, cells lacking B-Myb are more sensitive to DNA-damage induced by UV-irradiation and alkylation. Our work provides the first direct evidence for a novel function of B-Myb in the response to DNA-damage. The cells described here should be a useful model to characterize this function in more detail.
Assuntos
Galinhas/genética , Dano ao DNA , Genes myb , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Animais , Apoptose , Ciclo Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Clonais , Doxiciclina/farmacologia , Regulação da Expressão Gênica , Marcação de Genes , Engenharia Genética , Análise em Microsséries , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/deficiência , RNA Mensageiro/metabolismo , Recombinação Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
The c-myb proto-oncogene is highly expressed in a wide variety of immature hematopoietic cells and plays a key role in the development of the hematopoietic system. c-myb and its retroviral counterpart v-myb encode transcription factors which have been implicated in the regulation of certain target genes. Targeting of c-myb in mouse embryonic stem cells by homologous recombination has provided clear evidence that c-myb is necessary for the proper development of most myeloid lineages of the hematopoietic system as well as of T-lymphocytes. Here we have explored the function of c-myb in the B-lymphoid lineage. We have used the chicken DT40 cells, a pre B-cell line which shows extremely high efficiencies of homologous recombination, as a model system to disrupt c-myb. DT40 cells lacking a functional c-myb gene are viable and show only minor perturbations of their growth parameters, indicating that c-myb is not an essential gene in these cells. We have used the c-myb null DT40 cells to analyse the expression of genes which have been previously been identified as myb target genes. Neither c-myc nor bcl-2, two putative myb targets, showed altered expression in the cells lacking c-myb. However, expression of the Pdcd4 gene, a myb target gene originally identified in a myelomonocytic cell line expressing a conditional form of v-myb, was diminished in the absence of c-myb. The c-myb knock-out cells described here should provide a useful model system for the identification and characterization of c-myb target genes in B-lymphoid cells.