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Increased erythroid-specific expression of a mutated HPFH gamma-globin promoter requires the erythroid factor NFE-1. 总被引:7,自引:3,他引:4
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S Nicolis A Ronchi N Malgaretti R Mantovani B Giglioni S Ottolenghi 《Nucleic acids research》1989,17(14):5509-5516
The -175 T greater than C mutation in the promoter of the A gamma- or G gamma-globin gene causes a 50-100 fold increase of the expression of the respective gene in adult erythroid cells (Hereditary Persistence of Fetal Hemoglobin). We show here that this mutation increases 3-9 fold the expression of a gamma-CAT reporter plasmid transfected into the erythroid cells K562, but not that of the same plasmid in non erythroid cells. The overexpression of the mutant is abolished by the mutation of the binding site for the erythroid specific factor NFE1; inactivation of the adjacent binding site for the ubiquitous factor OTF1 does not cause overexpression of the normal gamma-globin promoter. Previous results demonstrated that the -175 mutation slightly increases the in vitro binding of NFE1 and almost abolishes that of OTF1; the present functional data indicate that altered binding of NFE1, but not of OTF1, is responsible for the observed overexpression of the mutated promoter. 相似文献
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The -117 mutation in Greek HPFH affects the binding of three nuclear factors to the CCAAT region of the gamma-globin gene. 总被引:27,自引:4,他引:23
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The Greek form of hereditary persistence of fetal hemoglobin (HPFH) is associated with a point mutation immediately upstream of the distal of the two CCAAT elements of the A gamma-globin gene. Three proteins present in nuclear extracts of erythroleukemia cells bind to this CCAAT region and contact the nucleotide mutated in Greek HPFH. The ubiquitous CCAAT-binding factor CP1 interacts preferentially with the proximal CCAAT sequence. An erythroid cell-specific factor, referred to as NF-E, binds with a higher affinity to the distal CCAAT region and interacts only with sequences flanking the CCAAT motif. The third protein is the vertebrate homologue of the sea urchin CCAAT displacement protein and recognizes sequences in both CCAAT elements and their flanking sequences. While the point mutation in Greek HPFH slightly strengthens the binding of CP1 and the CCAAT displacement protein, the same base change strongly reduces the binding of NF-E to the distal CCAAT region, suggesting a possible role of NF-E in the repression of gamma-globin genes in adult erythroid cells. 相似文献
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The deletion of the distal CCAAT box region of the A gamma-globin gene in black HPFH abolishes the binding of the erythroid specific protein NFE3 and of the CCAAT displacement protein. 总被引:9,自引:4,他引:5
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Non-deletion Hereditary Persistence of Fetal Hemoglobin (HPFH) is characterized by great elevation of the synthesis, in adult age, of fetal hemoglobin (HbF), of either the A gamma or G gamma type. Strong genetic evidence indicates point mutations in the G gamma- or A gamma-globin promoter as responsible for overexpression of the mutated gene. Here we report that a 13 nucleotides deletion in the CCAAT box region of the A gamma-globin promoter, associated with greater than 100 fold overexpression of the gene, abolishes the in vitro binding of the ubiquitous factors CP1 and CDP (CCAAT displacement protein) and of the erythroid specific protein NFE3. Loss of NFE3 binding is consistent with a similar effect of the -117 G greater than A HPFH mutation, suggesting a possible role of NFE3 as a negatively acting factor. In addition, loss of CDP binding indicates that this alteration might also contribute to the HPFH phenotype in this particular case, suggesting possible heterogeneity of the mechanisms causing HPFH. 相似文献
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Purified gamma-globin specific complementary DNA has been used to demonstrate the presence of the gene for gamma-globin in DNA from human adult red blood cells. This finding sheds doubt on any theory involving looping-out excision of genes to explain the switch over from synthesis of gamma-globin to beta-globin at birth. 相似文献
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Inducibility of the HS II enhancer depends on binding of an erythroid specific nuclear protein. 总被引:16,自引:5,他引:16
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An erythroid specific, inducible enhancer associated with hypersensitive site II (HS II) plays a central role in the function of the human beta globin dominant control region. The HS II enhancer consists of tandem AP-1 binding sites and has been shown to bind members of the ubiquitous jun and fos families of proteins. The same sites are now shown to bind the erythroid specific protein, NF-E2. Inducibility of the HS II enhancer depends on NF-E2 binding, even in the presence of another hypersensitive site. Further, increased activity of the enhancer in induced K562 cells correlates with the presence of NF-E2, which appears to be present in a modified form. NF-E2 is distinct from some enhancer binding proteins in K562 nuclear extracts, in that it does not contain Fos or Fra-1 protein. Thus, binding by NF-E2 may be the mechanism, whereby tandem AP-1 binding sites confer erythroid specificity on the HS II enhancer. 相似文献
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Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin. 总被引:17,自引:9,他引:17
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D L Gumucio K L Rood T A Gray M F Riordan C I Sartor F S Collins 《Molecular and cellular biology》1988,8(12):5310-5322
The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation. 相似文献
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Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene. 总被引:54,自引:15,他引:39
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We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprinting, gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation, contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor, which we name NF-E2. NF-E1 is a factor that also binds the promoter and the enhancer (present in the 3' flanking region) of the human beta-globin gene. NF-E2 has not yet been described and although it binds to a sequence containing the Ap1 consensus, it appears to be different from Ap1. 相似文献
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Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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We performed a systematic functional analysis of the human gamma-globin promoter to identify its activator domains. We used a panel of truncation and scanning mutants as well as transfection in human K562 fetal erythroid cells. The various mutations produced relatively small changes in promoter function in both transient and stable transfection assays. The CACCC region and the region containing the binding sites for protein GATA-1 behaved as activator domains. We also obtained evidence for a minor activator site in the - 200 to - 190 region. The results are consistent with the interpretation that gamma-globin gene regulation may occur in part through multiple small effects of promoter elements. 相似文献
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Quantitation of specific binding of erythropoietin to human erythroid colony-forming cells 总被引:5,自引:0,他引:5
Highly purified human erythroid colony-forming cells (ECFC), which consist predominately of colony-forming units-erythroid (CFU-E), were prepared from human blood and used to study the binding and processing of erythropoietin (Ep). When radioiodinated human recombinant Ep (125I-rEp) was incubated with these cells, binding was specific and saturable. Specific binding was directly proportional to cell concentration and did not occur with other human cells. Saturation of specific binding at 3 degrees C occurred at 1 nM (3.9/U/ml), and Scatchard analysis revealed two classes of binding sites on the cell surface. Of a total of 1,050 binding sites per ECFC, one-fifth had a Kd of 0.10 nM, while the remainder had a Kd of 0.57 nM. Specific binding was twofold greater at 37 degrees C than at 3 degrees C, and removal of surface-bound Ep with acid indicated that 125I-rEp was internalized into the cells after incubation at 37 degrees C. Further incubation at this temperature showed a decline of cellular radioactivity, with a release of small molecular weight degradation fragments into the medium. These studies demonstrate two classes of receptors for Ep on normal human ECFC. Internalization and degradation of EP occur, and the biologic effect of the hormone is produced by a small number of Ep molecules, as demonstrated in murine erythroid progenitor cells. 相似文献