RESUMO
To understand the function of basic Krüppel-like factor (BKLF), it was confirmed by direct fluorescence and indirect fluorescence observation that hBKLF was localized in nucleus, and distributed throughout nucleoplasm in a speckled pattern, except the nucleoli. This pattern is similar to many but not all transcription factors. To clarify the specific sequence responsible for its nuclear localization, a series of deletion mutants of GFP/hBKLF were constructed. By observing their subcellular localization, it was found that the three zinc fingers of hBKLF and the N-terminal aside from the fingers all served as nuclear localization signals (NLS); the sub-NLS of hBKLF was located in the N-terminus, including the CtBP-binding motif and the proline rich domain. These results provided a basis for further clarifying the function of BKLF.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Animais , Células COS , Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde , Humanos , Fatores de Transcrição Kruppel-Like , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Dedos de Zinco/genéticaRESUMO
To study the transcription regulatory function of basic Krüppel-like factor (BKLF)on gamma- and epsilon-globin genes, recombinant expression vectors containing the full-length human BKLF gene, and a deletion mutant that lost N-terminal 40 amino acids, were constructed and used, respectively, to transiently transfect COS7 cells in order to assay their reporter activities. Results showed that hBKLF was able to repress the activity of gamma- and epsilon-globin gene promoters, while the antisense nucleic acid specific for hBKLF activated the transcription of these promoters. Deleting 40 amino acids from N-terminus did not influence the transcriptional repression of hBKLF. The stimulatory function of FKLF on gamma- and epsilon-globin gene promoters was also significantly reduced by hBKLF. In addition, BKLF bound the CACCC element in the SHP1 (SH2-containing protein tyrosine phosphatase 1) gene promoter. These results suggest that gamma- and epsilon-globin genes may be transcriptional targets of BKLF, providing evidence for further studies on the role of BKLF in participating the transcriptional regulation of haemocyte-specific genes.
Assuntos
Proteínas de Ligação a DNA/genética , Globinas/genética , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação/genética , Células COS , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fatores de Transcrição Kruppel-Like , Camundongos , Mutação , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/genética , Deleção de Sequência , Transcrição Gênica/genética , TransfecçãoRESUMO
The FLD4585 clone from the cDNA library of human fetal liver may encode a hematopoietic related transcription factor. Here we tried to clone its full-length cDNA from the 22 weeks-gestation human fetal liver and study its functional domains, genomic structure, chromosomal localization, subcellular site and expression pattern. To obtain the full-length cDNA of FLD4585 clone, 5' RACE technique was used. Bioinformatics was used to analyze its genomic structure, chromosomal localization and potential functional domains. Its subcellular localization was shown by GFP fusion technique. The expression pattern was studied by Northern blot, RT-PCR and Western blot. The results show the full-length cDNA encoded by FLD4585 clone is 1810 bp long and encodes a 345 amino acids protein with high homology to mouse BKLF (basic Krüppel-like factor). Its characteristic C-terminal three contiguous C2H2 zinc fingers place it within the family of Krüppel-like factors. Bioinformatics studies show hBKLF gene spans over 33 kb on chromosome 4p15.2-4p16.1 and contains 6 exons and 5 introns. GFP-hBKLF fusion technique showed hBKLF was present in the nuclei of COS-7 cells in a punctate pattern, whereas it was absent in the nucleoli. By Northern blot, hBKLF has two transcripts, one between 4.4 kb-7.5 kb and the other between 1.35 kb-2.4 kb. The larger transcript exists widely in human tissues. However, the smaller transcript was more restricted in blood leukocytes, liver and bone marrow. RT-PCR showed erythrocytes and granulocytes could both express hBKLF and its level increased as they matured. The expression level in fetal liver decreased as it developed towards adult liver and its hematopoietic function gradually lost. Taken together, in this paper we have successfully cloned the full length cDNA of hBKLF. Expression study suggests it may have broad functions in vivo, especially the functions in hematopoietic tissues.