RESUMO
A new megathrombocytopenic syndrome with giant platelets in peripheral blood and severe thrombocytopenia was diagnosed in a 4-month-old boy. His clinical course included repeated hemorrhagic incidents leading to death at age 37 months. Bone marrow ultrastructural analysis revealed numerous dystrophic megakaryocytes with giant membrane complexes. Although these features were similar to those described for megakaryocytes in mice lacking the gene for transcription factor p45-NF-E2, no abnormalities in the p45-NF-E2 gene could be documented. Platelet membrane analysis showed a reduction in glycoprotein (GP) Ib, but normal content of GPIIb and GPIIIa. Analysis of genes encoding for GPIb alpha and beta, GPV, and GPIX ruled out the possibility that the observed platelet abnormality is a variant of Bernard-Soulier syndrome. A moderate neutropenia was associated with a complete lack of expression of sialyl-Lewis-X on the surface of polymorphonuclear neutrophils. A common defect in posttranslational modification of glycoproteins could account for the diverse cellular abnormalities.
Assuntos
Megacariócitos/ultraestrutura , Neutropenia/diagnóstico , Oligossacarídeos/metabolismo , Trombocitopenia/diagnóstico , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Humanos , Lactente , Contagem de Leucócitos , Masculino , Neutropenia/patologia , Neutrófilos/metabolismo , Contagem de Plaquetas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Antígeno Sialil Lewis X , Síndrome , Trombocitopenia/metabolismo , Trombocitopenia/patologiaRESUMO
The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Genes de Troca , Globinas/genética , Hemoglobinas/genética , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides , Animais , Sequência de Bases , Fatores de Transcrição COUP , Linhagem Celular , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário e Fetal/genética , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
Transcription factor p45 NF-E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF-E2. To find target genes for p45 NF-E2 in megakaryopoiesis, we used an in vivo immunoselection assay: genomic fragments bound to p45 NF-E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti-p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). We demonstrate that the TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF-E2. First, its promoter contains a functional NF-E2 binding site; second, the intronic NF-E2 binding site is located within a chromatin-dependent enhancer element; third, p45-null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Megacariócitos/enzimologia , Tromboxano-A Sintase/biossíntese , Tromboxano-A Sintase/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Cromatina/fisiologia , Clonagem Molecular , Primers do DNA , Elementos Facilitadores Genéticos , Fatores de Ligação de DNA Eritroide Específicos , Hematopoese , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Sequências Reguladoras de Ácido Nucleico , Transfecção , Dedos de ZincoRESUMO
The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. To understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the promoter of this gene. A 200 bp fragment is sufficient for high-level specific expression. This fragment can bind several trans-acting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in HEL cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. Furthermore, GATA-1 and two Ets proteins, Ets-1 and Fli-1, can trans-activate the MPL promoter in heterologous cells. The effects of GATA-1 and these two Ets proteins are additive. Together with our previous results on the glycoprotein IIb (GpIIb) promoter, this study indicates a molecular basis for the coregulation of early markers of megakaryocyte differentiation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Megacariócitos/metabolismo , Proteínas de Neoplasias , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Citocinas , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Bases , Cromossomos Humanos Par 11/genética , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína Proto-Oncogênica c-ets-1 , Proteína Proto-Oncogênica c-fli-1 , Proteínas Proto-Oncogênicas c-ets , Receptores de Trombopoetina , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transativadores/genética , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The MPL gene codes for the thrombopoietin receptor, whose ligand specifically controls megakaryocytic differentiation. In order to understand the molecular basis for the megakaryocyte-specific expression of MPL, we analyzed the regulatory elements of this gene. Two regions are hypersensitive to DNase I in nuclei of cells that express MPL: the promoter and a portion of intron 6. The latter behaves as a chromatin-dependent enhancer. A 200 bp fragment of the promoter is sufficient for high-level specific expression. This fragment can bind several transacting factors in vitro, including GATA-1 and members of the Ets family. GATA-1 binds with low affinity to a unique GATA motif at -70 in the MPL promoter, and destruction of this site yields only a modest decrease in expression level in human erythroleukemia (HEL) cells. Ets proteins also bind with low affinity to two sites. One is located at position -15 and its destruction reduces expression to 50%; the other is located immediately downstream of the GATA motif and plays a crucial role in expression of the promoter in HEL cells, as its inactivation reduces expression to 15%. This study indicates a molecular basis for the coregulation of markers of megakaryocyte differentiation. Finally, we describe other nuclear factor binding sites that may be involved in the cell-type-specific expression of MPL.
Assuntos
Regulação da Expressão Gênica , Megacariócitos/metabolismo , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/genética , Receptores de Citocinas , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Células HeLa , Humanos , Íntrons , Células Jurkat , Células K562 , Megacariócitos/fisiologia , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Receptores de Trombopoetina , Fator de Transcrição Sp1/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The human c-mpl proto-oncogene encodes a member of the cytokine receptor superfamily, expressed mainly in CD 34-positive hematopoietic progenitors and in the megakaryocytic lineage. To investigate the elements required for this tissue-specific expression, we cloned the human c-mpl gene (MPL) as well as the 5' end of the mouse gene. The human c-mpl gene contains 12 exons distributed over 17 kb of DNA. Each of the two "cytokine receptor domains" of Mpl is encoded by a set of four exons, the transmembrane domain by a single exon and the cytoplasmic domain by two exons. We also describe how three types of mRNA, encoding different proteins, are generated. The major species contains all 12 exons; mRNAs encoding a protein with a smaller cytoplasmic domain are produced by termination of the transcript within intron 10, and mRNAs encoding a putative soluble form of the c-Mpl protein lack exons 9 and 10. The promoter regions of the human and mouse genes were characterized. These promoters are GC-rich and contain putative binding sites for proteins of the Ets and GATA families. Finally, we show that a 700-bp fragment of the human c-mpl promoter is active in the HEL and K562 cell lines, which express erythroid and megakaryocytic markers, but is inactive in the nonhematopoietic HeLa cell line and the Jurkat T lymphoid cell line.
Assuntos
Proteínas de Neoplasias , Proto-Oncogenes , Receptores de Citocinas , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Éxons , Expressão Gênica , Hematopoese/genética , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Receptores Imunológicos/genética , Receptores de Trombopoetina , Transcrição GênicaRESUMO
The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Megacariócitos/fisiologia , Glicoproteínas da Membrana de Plaquetas/genética , Regiões Promotoras Genéticas , Proteínas Repressoras , Transativadores , Fatores de Transcrição/metabolismo , Sequência de Bases , Elementos Facilitadores Genéticos , Células HeLa , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Proteína Proto-Oncogênica c-ets-1 , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , RNA Mensageiro/genética , Ativação TranscricionalRESUMO
We have studied the elements involved in the tetradecanoylphorbol acetate (TPA)-mediated extinction of erythroid-specific genes. We show that transcription driven by a -714/+78-base pair DNA fragment of the erythroid promoter of the human porphobilinogen deaminase gene is down-regulated upon TPA treatment of erythroleukemic cells. Examination of the DNA binding activity of trans-acting factors involved in the expression of the porphobilinogen deaminase erythroid promoter showed (i) a constitutive expression of the CACC binding proteins and (ii) a decrease in DNA binding activity of two tissue-specific factors, NF-E1 and NF-E2. Kinetics experiments indicated that NF-E2 was down-regulated after 1 h of TPA treatment whereas NF-E1 was down-regulated at the protein and mRNA levels only after 5 h of TPA treatment. These results suggest that different pathways, acting via different transcription factors, are involved in the TPA-mediated extinction of erythroid-specific genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Hidroximetilbilano Sintase/genética , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Dedos de Zinco , Sequência de Bases , Linhagem Celular , Fatores de Ligação de DNA Eritroide Específicos , Humanos , Cinética , Leucemia Eritroblástica Aguda , Dados de Sequência Molecular , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Sondas de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genéticaRESUMO
Erythroid-specific genes contain binding sites for NF-E1 (also called GF-1 and Eryf-1; refs 1-3 respectively), the principal DNA-binding protein of the erythrocytic lineage. NF-E1 expression seems to be restricted to the erythrocytic lineage. A closely related (if not identical) protein is found in both a human megakaryocytic cell line and purified human megakaryocytes; it binds to promoter regions of two megakaryocytic-specific genes. The binding sites and partial proteolysis profile of this protein are indistinguishable from those of the erythroid protein; also, NF-E1 messenger RNA is the same size in both the megakaryocytic and erythroid cell lines. Furthermore, point mutations that abolish binding of NF-E1 result in a 70% decrease in the transcriptional activity of a megakaryocytic-specific promoter. We also find that NF-E2, another trans-acting factor of the erythrocytic lineage, is present in megakaryocytes. Transcriptional effects in both lineages might then be mediated in part by the same specific trans-acting factors. Our data strengthen the idea of a close association between the erythrocytic and the megakaryocytic lineages and could also explain the expression of markers specific to the erythrocytic and megakaryocytic lineages in most erythroblastic and megakaryoblastic permanent cell lines.
Assuntos
Proteínas de Ligação a DNA/genética , Eritrócitos/análise , Células-Tronco Hematopoéticas/análise , Megacariócitos/análise , Fatores de Transcrição , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Endopeptidase K , Fatores de Ligação de DNA Eritroide Específicos , Expressão Gênica , Humanos , Mutação , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Serina Endopeptidases/metabolismo , Transativadores , Transcrição GênicaRESUMO
Porphobilinogen deaminase, the third enzyme of the haem biosynthetic pathway, is encoded by two distinct mRNA species expressed in a tissue-specific manner from a single gene. These two mRNAs are transcribed from two promoters and only differ in their first exon. An inherited deficiency or porphobilinogen deaminase in man is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. In the present report, we describe the molecular abnormality responsible for a variant form of acute intermittent porphyria where the enzyme defect is restricted to non-erythroid cells. Upon cloning and sequencing the mutant allele of a patient from a large Finnish kindred, a single-base substitution within the 5'-splice donor sequence of intron 1 was found at the last position of exon 1 (CG----CT). The identification of this mutation allowed us to detect asymptomatic gene carriers among family members using in vitro amplification of DNA and hybridization of the target sequence to allele-specific oligonucleotides.
Assuntos
Amônia-Liases/genética , Eritrócitos/enzimologia , Hidroximetilbilano Sintase/genética , Porfirias/genética , Sequência de Bases , Clonagem Molecular , DNA/genética , Feminino , Finlândia , Triagem de Portadores Genéticos , Humanos , Hidroximetilbilano Sintase/sangue , Masculino , Dados de Sequência Molecular , Mutação , Porfirias/enzimologiaRESUMO
Two cis-acting sequences, recognized by two erythroid-specific trans-acting factors, are involved in the regulation of the erythroid promoter of the human gene coding for porphobilinogen deaminase (PBGD). The first region, located at -70, binds the erythroid factor NF-E1, and point mutations within this region abolish the induction of transcription of this promoter during murine erythroleukemia (MEL) cell differentiation. The second region, located at -160, binds the erythroid-specific factor NF-E2 and the ubiquitous factor AP1. Using UV cross-linking, we show that NF-E2 has a higher molecular weight than AP1, demonstrating that NF-E2 is not an erythroid-specific degradation product of AP1. By point mutagenesis of the NF-E2/AP1 binding site, we define mutations that abolish binding of either NF-E2 alone or AP1 and NF-E2 together. Regulation of transcription of the PBGD erythroid promoter is abolished by those mutations, suggesting that NF-E2 but not AP1 is necessary for correct regulation of this promoter in erythroid cells.
Assuntos
Amônia-Liases/genética , Genes Reguladores , Genes , Hidroximetilbilano Sintase/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/genética , Camundongos , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Transcrição GênicaRESUMO
The human gene coding for porphobilinogen deaminase (PBG-D) is transcribed into two distinct transcription units giving two mRNAs. These units originate from two adjacent promoters distant of 3 kilobase pairs. The upstream promoter is active in all cell types, whereas the downstream promoter is active only in erythroid cells. We have studied the expression of this gene either after introduction of the corresponding human chromosome into murine erythroid cells using somatic hybrids or after transfection into both erythroid and non-erythroid cells. Using somatic hybrids, we showed that activation of the erythroid-specific promoter of the PBG-D gene did not reduce the rate of initiation of the ubiquitous promoter. Transfection experiments in erythroid cells showed that the PBG-D erythroid transcription unit, controlled by the PBG-D erythroid promoter, was correctly transcribed and regulated. Furthermore, we found that the PBG-D erythroid promoter alone was sufficient for correct expression and regulation of a reporter gene during erythroid differentiation. When the human PBG-D gene was transfected into non-erythroid cells, only the ubiquitous promoter was active. Deletion of the ubiquitous promoter did not lead to any activation of the erythroid promoter, suggesting that its inactivity in non-erythroid cells was not due to promoter occlusion but to a strict erythroid specificity.
Assuntos
Amônia-Liases/genética , Genes , Hidroximetilbilano Sintase/genética , Transcrição Gênica , Transfecção , Animais , Linhagem Celular , Células Cultivadas , Elementos Facilitadores Genéticos , Globinas/genética , Humanos , Leucemia Eritroblástica Aguda/enzimologia , Camundongos , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Mapeamento por Restrição , Vírus 40 dos Símios/genéticaAssuntos
Amônia-Liases/genética , Hidroximetilbilano Sintase/genética , Regiões Promotoras Genéticas , Animais , Sítios de Ligação , Diferenciação Celular , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática , Homologia de Genes , Humanos , Hidroximetilbilano Sintase/biossíntese , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patologia , Camundongos , Especificidade de Órgãos , Proteínas Recombinantes de Fusão/biossíntese , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
An inherited deficiency of porphobilinogen deaminase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] in humans is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. It was previously demonstrated that porphobilinogen deaminase is encoded by two distinct mRNA species expressed in a tissue-specific manner. Analysis of the genomic sequences indicated that these two mRNAs are transcribed from two promoters and only differ in their first exon. The first mutation identified in the human porphobilinogen deaminase gene is a single-base substitution (G----A) in the canonical 5' splice donor site of intron 1. This mutation leads to a particular subtype of acute intermittent porphyria characterized by the restriction of the enzymatic defect to nonerythropoietic tissues. Hybridization analysis using oligonucleotide probes after in vitro amplification of genomic DNA offers another possibility of detecting asymptomatic carriers of the mutation in affected families.
Assuntos
Amônia-Liases/genética , Hidroximetilbilano Sintase/genética , Porfirias/genética , Splicing de RNA , RNA Mensageiro/genética , Doença Aguda , Sequência de Bases , Clonagem Molecular , DNA/genética , Sondas de DNA , Feminino , Amplificação de Genes , Ligação Genética , Haplótipos , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Linhagem , Fenótipo , Polimorfismo de Fragmento de Restrição , Porfiria Aguda IntermitenteRESUMO
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.
Assuntos
Amônia-Liases/genética , Proteínas de Ligação a DNA/metabolismo , Eritrócitos/enzimologia , Genes , Hidroximetilbilano Sintase/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular , Desoxirribonuclease I , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Globinas/genética , Humanos , Hidroximetilbilano Sintase/sangue , Metilação , Dados de Sequência MolecularRESUMO
We have shown that the promoter of the human beta-globin gene contains three regions in addition to the known CAC, CAAT and TATA box regions that are important for the induction of transcription in erythroid cells. By using DNaseI footprinting and gel mobility shift assays we were able to show that two of these regions bind the erythroid specific nuclear factor NF-E1 (and ubiquitous factors). The third region binds a ubiquitous CAAT-box factor (CP1). Deletion experiments suggest that only the combination of NF-E1 and CP1 binding sites, but not each of the sites alone, are capable of mediating the induction of transcription of a minimal (CAC, CAAT, TATA box) beta-globin promoter in mouse erythroleukaemia (MEL) cells.