RESUMO
GATA transcription factors bind the consensus sequence WGATAR, present in the flanking regions of most erythroid specific genes. GATA-1 and GATA-2, coexpressed in erythroid cells, are important for expression of erythroid genes. To elucidate the role of specific GATA transcription factors on globin gene expression, we examined the human alpha- and beta-globin gene clusters for all GATA sites. Conserved GATA sites were found in each of the hypersensitive sites in both beta-and alpha clusters and in proximal regulatory regions of the zeta-, epsilon- and gamma-globin but not the alpha, delta or beta-globin genes. We then tested the effect of increasing levels of GATA-1 and GATA-2 on the expression of endogenous globin genes in human erythroid cells. Increasing GATA-1 levels in K562 cells decreased the levels of epsilon-globin mRNA but had no effect on the levels of expression of gamma, zeta or alpha-globin genes. Increasing GATA-2 levels increased epsilon-globin and gamma-globin transcripts. Increasing levels of GATA-1 also caused a decrease in the expression of endogenous GATA-2, while increased levels of GATA-2 had no effect on GATA-1 mRNA. Our results indicate a differential role of GATA-1 and -2 transcription factors on globin transcripts and suggest a correlation between the conservation of GATA sites in the regulatory regions and the ability of endogenous globin genes to respond to GATA transcription factors. They also suggest that quantitative changes in the levels of GATA-1 or GATA-2 can result in alterations of globin target gene expression and may participate in the ontogenic control of the globin genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Hemoglobinas/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Sequência Conservada , DNA Recombinante , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Fator de Transcrição GATA2 , Regulação da Expressão Gênica , Globinas/genética , Globinas/metabolismo , Hemoglobinas/metabolismo , Humanos , Células K562 , Camundongos , Família Multigênica , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Nickel-dependent recognition and oxidation of guanine have been linked in part through the paramagnetic effects of nickel on the NMR of model oligonucleotide duplexes. Direct interaction between nickel and guanine N7 had originally been postulated from correlations between the efficiency of guanine oxidation and the environment surrounding its N7 position. (1)H and (31)P NMR spectra of DNA containing a single, isolated extrahelical guanine are consistent with selective binding of nickel to the N7 of this unique base over a background of nonspecific association to the phosphate backbone. The presence of a macrocyclic complex or simple salt of nickel did not detectably alter the structure of the duplex or extrahelical residue. Accordingly, nickel appeared to bind the extrahelical guanine N7 within the major groove as indicated by paramagnetic effects on the proton signals of nucleotides on the 5' but not 3' side of the nickel binding site. Similar (1)H NMR analysis of DNA containing a dynamic equilibrium of extrahelical guanine residues also suggested that the nickel complex did not affect the native distribution of structures. Oxidation of these sites by a nickel-mediated pathway consequently reflected their solvent accessibility in a general and metal-independent manner. The close proximity of the extrahelical guanines produced a composite of paramagnetic effects on each adjacent nucleotide resulting from both direct and proximal coordination of nickel.
