RESUMO
In this study, we sought to identify factors responsible for the positive modulation of lactoferrin (LF), a neutrophil-specific, secondary-granule protein gene. Initial reporter gene transfection assays indicated that the first 89 base pairs of the LF promoter are capable of directing myeloid-specific LF gene expression. The presence of a C/EBP site flanked by 2 Sp1 sites within this segment of the LF promoter prompted us to investigate the possible role of these sites in LF expression. Cotransfection studies of LF-89luc plasmid with increasing concentrations of a C/EBPalpha expression vector in myeloid cells resulted in a linear transactivation of luciferase reporter activity. Electrophoretic mobility shift assays found that the C/EBP site is recognized by C/EBPalpha and that both LF Sp1 binding sites bind the Sp1 transcription factor specifically in myeloid cells. Mutation of either Sp1 site markedly reduced activity of the LF-89luc plasmid in myeloid cells, and neither Sp1 mutant plasmid was transactivated by a C/EBPalpha expression plasmid to the same extent as wild-type LF-89luc. We also transfected LF-89luc into Drosophila Schneider cells, which do not express endogenous Sp1, and demonstrated up-regulation of luciferase activity in response to a cotransfected Sp1 expression plasmid, as well as to a C/EBPalpha expression plasmid. Furthermore, cotransfection of LF-89luc plasmid simultaneously with C/EBPalpha and Sp1 expression plasmids resulted in an increase in luciferase activity greater than that induced by either factor alone. Taken together, these observations indicate a functional interaction between C/EBP and Sp1 in mediating LF expression. (Blood. 2000;95:3734-3741)
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Lactoferrina/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Núcleo Celular/metabolismo , Humanos , Lactoferrina/biossíntese , Leucemia Eritroblástica Aguda , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
GABP is a heteromeric transcription factor complex which consists of the ets related protein, GABPalpha, and the Notch-related protein, GABPbeta. We isolated a human genomic DNA fragment which is highly homologous and colinear with human GABPalpha cDNA, but which lacks introns. This processed pseudogene, psihGABPalpha, is expressed as RNA in U937 human myeloid cells, but a mutation at the site that corresponds to the ATG start methionine codon prevents its translation into protein. The pseudogene was localized to chromosome 7 using a somatic cell hybrid mapping panel and it is not syntenic with authentic GABPalpha, which was localized to chromosome 21. We have identified psihGABPalpha, a novel, GABPalpha-related processed pseudogene which is expressed as a RNA transcript in human myeloid cells.
Assuntos
Cromossomos Humanos Par 8 , Proteínas Proto-Oncogênicas/genética , Pseudogenes , Fatores de Transcrição/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA , Humanos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-ets , Transcrição GênicaRESUMO
The adjacent neutrophil elastase, proteinase 3, and azurocidin genes encode serine proteases expressed specifically in immature myeloid cells. Subclones of a 17-kilobase (kb) murine neutrophil elastase genomic clone were assessed for their ability to stimulate the neutrophil elastase promoter in 32D cl3 myeloid cells. Region -9.3 to -7.3 kb stimulated transcription 7-fold, whereas other genomic segments were inactive. This enhancer is located in the second intron of the proteinase-3 gene and so may regulate more than one gene in the myeloid protease cluster. Deletional analysis of the enhancer identified several segments which activated the neutrophil elastase and thymidine kinase promoters 3-6-fold. The most active segment was a 220-base pair region centered at -8.6 kb, which activated transcription 31-fold. This segment contains an Sp1 consensus site, which bound Sp1, flanked by two Ets family consensus sequences, which bound PU.1, GABP, and an Ets factor present in myeloid cell extracts. Mutation of the Sp1-binding site reduced enhancer activity 8-fold in 32D cl3 cells, and mutation of either or both Ets-binding sites reduced activity 3-4-fold. Sp1 activated the distal enhancer 5-fold, GABP 3-fold, and the combination 8-fold in Schneider cells.
Assuntos
Elementos Facilitadores Genéticos , Elastase de Leucócito/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Serina Endopeptidases/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , DNA , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Mieloblastina , Ligação Proteica , Proteínas Proto-Oncogênicas c-etsRESUMO
CD18, the beta chain of the leukocyte integrins, plays a crucial role in immune and inflammatory responses. CD18 is expressed exclusively by leukocytes, and it is transcriptionally regulated during the differentiation of myeloid cells. The ets factors, PU.1 and GABP, bind to three ets sites in the CD18 promoter, which are essential for high level myeloid expression of CD18. We now identify two binding sites for the transcription factor, Sp1, that flank these ets sites. Sp1 is the only factor from myeloid cells that binds to these sites in a sequence-specific manner. Mutagenesis of these sites abrogates Sp1 binding and significantly reduces the activity of the transfected CD18 promoter in myeloid cells. Transfection of Sp1 into Drosophila Schneider cells, which otherwise lack Sp1, activates the CD18 promoter dramatically. GABP also activates the CD18 promoter in Schneider cells. Co-transfection of Sp1 and GABP activates CD18 more than the sum of their individual effects, indicating that these factors cooperate to transcriptionally activate myeloid expression of CD18. These studies support a model of high level, lineage-restricted gene expression mediated by cooperative interactions between widely expressed transcription factors.
Assuntos
Antígenos CD18/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Sondas de DNA , Proteínas de Ligação a DNA/genética , Drosophila melanogaster , Fator de Transcrição de Proteínas de Ligação GA , Mutagênese Sítio-Dirigida , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Neutrophil elastase (NE) is a serine protease that is transcriptionally regulated during early myeloid differentiation. The murine NE (mNE) promoter contains functionally important c-Myb, C/EBP, and ets binding sites. Deletion of the ets site reduced promoter activity by 90%. Although the ets transcription factor, PU.1, bound to this ets site, it only modestly activated the mNE promoter. Here, we show that a second transcription factor from myeloid cells-GABP-binds to the mNE ets site but strongly activates the mNE promoter. GABP is a heteromeric transcription factor complex that consists of GABP alpha, an ets factor, and GABP beta, a Notch-related protein. GABP alpha bound to the mNE ets site and, in turn, recruited GABP beta to form a transcriptionally active complex. GABP alpha and PU.1 competed with each other for binding to the mNE ets site. GABP increased the activity of the mNE promoter sevenfold in U937 myeloid cells. GABP cooperated with c-Myb and C/EBP alpha to activate the mNE promoter more than 85-fold in otherwise nonpermissive, nonhematopoietic NIH 3T3 cells. Thus, GABP binds to the crucial mNE promoter ets site and powerfully activates its expression alone and in cooperation with the transcription factors c-Myb and C/EBP.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Elastase de Leucócito/genética , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Caseína Quinase II , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/genética , Dimerização , Indução Enzimática , Fator de Transcrição de Proteínas de Ligação GA , Humanos , Elastase de Leucócito/biossíntese , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Dados de Sequência Molecular , Família Multigênica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myb , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Células Tumorais CultivadasAssuntos
Antígenos CD18/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Mutagênese , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Antígenos CD18/química , Primers do DNA/química , Desoxirribonuclease HindIII/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese em Gel de Poliacrilamida , Leucócitos/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
CD18 (beta 2 leukocyte integrin) is a leukocyte-specific adhesion molecule that plays a crucial role in immune and inflammatory responses. A 79-nucleotide fragment of the CD18 promoter is sufficient to direct myeloid transcription. The CD18 promoter is bound by the B lymphocyte- and myeloid-restricted ets factor, PU.1, and disruption of the PU.1-binding sites significantly reduces promoter activity. However, PU.1 alone cannot fully account for the leukocyte-specific and myeloid-inducible transcription of CD18. We identified a ubiquitously expressed nuclear protein complex of extremely low electrophoretic mobility that also binds to this region of the CD18 promoter. This binding complex is a heterotetramer composed of GABP alpha, and ets factor, and GABP beta, a subunit with homology to Drosophila Notch. GABP alpha competes with the lineage restricted factor, PU.1, for the same critical CD18 ets sites. The CD18 promoter is activated in myeloid cells by transfection with both GABP alpha and GABP beta together, but not by either factor alone. Transfection of non-hematopoietic cells with the two GABP subunits together with PU.1 is sufficient to activate CD18 transcription in otherwise non-permissive cells. Thus, GABP and PU.1 compete for the same binding sites but cooperate to activate CD18 transcription.
Assuntos
Antígenos CD18/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Núcleo Celular/metabolismo , Pegada de DNA , Sondas de DNA/genética , Fator de Transcrição de Proteínas de Ligação GA , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Proteínas Oncogênicas de Retroviridae , Transcrição Gênica , TransfecçãoRESUMO
Normal cellular differentiation is linked to tightly regulated gene transcription. However, the DNA elements and trans-acting factors that regulate transcription in myeloid cells are poorly defined. CD18, the beta chain of the leukocyte integrins, is transcriptionally regulated during myeloid differentiation. The CD18 promoter is active after transfection into myeloid cells. We demonstrate that a region of the CD18 promoter that contains two binding sites for the PU.1 transcription factor is required for activity in myeloid cells. These sites are bound by in vitro translated PU.1 and by PU.1 from myeloid nuclear extracts. Mutagenesis of these sites abrogates binding by PU.1 and substantially decreases promoter activity in myeloid cells. Thus, the leukocyte-specific transcription factor PU.1 is required for myeloid activity of CD18.
Assuntos
Antígenos CD18/genética , Proteínas de Ligação a DNA/metabolismo , Monócitos/citologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Antígenos CD18/fisiologia , Diferenciação Celular , Linhagem Celular , DNA/metabolismo , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Mutação/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas de Retroviridae , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Pro alpha 1(I) gene in FIC. LTIC expressed abundant transcripts of Pro alpha 1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Pro alpha 1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor beta (TGF beta)-treated LTIC had a preferential increase in the rate of Pro alpha 1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) [Thompson, Simkevich, Holness, Kang and Raghow (1991) J. Biol. Chem. 266, 2549-2556] was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGF beta treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGF beta. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGF beta-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGF beta-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.
Assuntos
Colágeno/genética , Regulação da Expressão Gênica , Fígado/fisiologia , Fator de Transcrição AP-1/fisiologia , Animais , Sequência de Bases , Células Cultivadas , DNA/metabolismo , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Genes jun , Fígado/citologia , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica , Ratos , Ratos Wistar , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The first intron of the human Pro alpha 1(I) collagen gene contains an orientation-dependent enhancer composed of both positive and negative cis-acting elements involved in the transcriptional regulation of this gene. Deletion of a 360 bp Sau 3A intronic fragment spanning nucleotide +494 to +854 (S360) resulted in dramatic down-regulation of pCOL-KT (Thompson et al., J Biol Chem 266: 2549-2556, 1991). Using a DNaseI protection assay, we demonstrate a single footprint located at +590 to +615 in the S360 fragment; nuclear extracts prepared from mesenchymal and nonmesenchymal cells exhibited similar binding characteristics. A double stranded oligonucleotide representing a consensus Ap-1 binding sequence competed with S360 for binding. In contrast to what occurred in response to S360 deletion which was always accompanied by reduced expression, the deletion of the Ap-1 binding site (+598 to +off) caused either increased or decreased expression of the reporter gene depending on the target cell. Site-directed mutations in the Ap-1-like cis-element of Pro alpha 1(I) were also tested in transient expression assays. Consistent with the paradoxical results of Ap-1 deletion, we observed that the functional consequences of mutations in the Ap-1 site also varied in different cells. In A204 cells, one point mutation, which resulted in the loss of protein binding to S360, led to increased CAT activity while another point mutant, which retained binding of the Ap-1 like trans-acting factor(s), showed decreased CAT expression. The effects of these two mutations in the HFL-1 cells were exactly opposite of what was seen for A204 cells. Based on these observations, we postulate that the Ap-1 site plays a critical role in the transcriptional activity of the human Pro alpha 1(I) gene. The implications of an apparently dual mode of regulation through a single cis-regulatory element are discussed.
Assuntos
Colágeno/genética , Proteínas Proto-Oncogênicas c-jun/genética , Sequência de Bases , Análise Mutacional de DNA , Regulação da Expressão Gênica , Genes fos , Genes jun , Humanos , Íntrons , Dados de Sequência Molecular , TransativadoresRESUMO
The transcriptional activity of plasmid pCOL-KT, in which human pro alpha 1 (I) collagen gene upstream sequences up to -804 and most of the first intron (+474 to +1440) drive expression of the chloramphenicol acetyltransferase (CAT) gene [Thompson, Simkevich, Holness, Kang & Raghow (1991) J. Biol. Chem. 266, 2549-2556], was tested in a number of mesenchymal and non-mesenchymal cells. We observed that pCOL-KT was readily expressed in fibroblasts of human (IMR-90 and HFL-1), murine (NIH 3T3) and avian (SL-29) origin and in a human rhabdomyosarcoma cell line (A204), but failed to be expressed in human erythroleukaemia (K562) and rat pheochromocytoma (PC12) cells, indicating that the regulatory elements required for appropriate tissue-specific expression of the human pro alpha 1 (I) collagen gene were present in pCOL-KT. To delineate the nature of cis-acting sequences which determine the tissue specificity of pro alpha 1 (I) collagen gene expression, functional consequences of deletions in the promoter and first intron of pCOL-KT were tested in various cell types by transient expression assays. Cis elements in the promoter-proximal and intronic sequences displayed either a positive or a negative influence depending on the cell type. Thus deletion of fragments using EcoRV (nt -625 to -442 deleted), XbaI (-804 to -331) or SstII (+670 to +1440) resulted in 2-10-fold decreased expression in A204 and HFL-1 cells. The negative influences of deletions in the promoter-proximal sequences was apparently considerably relieved by deleting sequences in the first intron, and the constructs containing the EcoRV/SstII or XbaI/SstII double deletions were expressed to a much greater extent than either of the single deletion constructs. In contrast, the XbaI* deletion (nt -804 to -609), either alone or in combination with the intronic deletion, resulted in very high expression in all cells regardless of their collagen phenotype; the XbaI*/(-SstII) construct, which contained the intronic SstII fragment (+670 to +1440) in the reverse orientation, was not expressed in either mesenchymal or nonmesenchymal cells. Based on these results, we conclude that orientation-dependent interactions between negatively acting 5'-upstream sequences and the first intron determine the mesenchymal cell specificity of human pro alpha 1 (I) collagen gene transcription.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Pró-Colágeno/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Animais , Linhagem Celular , Embrião de Galinha , Deleção Cromossômica , Humanos , Íntrons , Especificidade de Órgãos , Células PC12 , Plasmídeos , Mapeamento por Restrição , TransfecçãoRESUMO
We created pCOL-KT, a plasmid construct in which the promoter/enhancer of human Pro alpha 1(I) gene is linked to the chloramphenicol acetyl transferase reporter gene. The Pro alpha 1(I) promoter/enhancer in pCOL-KT was methylated in vitro and tested for transcriptional activity by transient expression analysis. Methylation of the construct with bacterial methylases reduced transcriptional activity about 25-fold. Site-specific methylation of eight potential canonical sites of eukaryotic methylation within the promoter greatly reduced transcriptional activity. Chromatin conformation of the transfected pCOL-KT DNA was analyzed by nuclease sensitivity. Although both methylated and unmethylated transfected DNA had increased susceptibility to DNase I compared with the endogenous gene, the methylated transfected DNA showed increased resistance to nuclease when compared with unmethylated transfected DNA, indicating that the methylation of the DNA alters the chromatin conformation. We also tested the ability of a human rhabdomyosarcoma cell line that does not express type I collagen to support transcription from an exogenously added Pro alpha 1(I) promoter/enhancer. The transformed cell line is able to support transcription from the Pro alpha 1(I) promoter/enhancer. Treatment of the transformed cell line with 5-azacytidine, a potent inhibitor of DNA methylation, resulted in transcriptional activation of the Pro alpha 1(I) gene. These findings, along with the extreme methylation sensitivity of the Pro alpha 1(I) promoter and enhancer, suggest that DNA methylation may be an important mechanism of transcriptional inactivation of interstitial collagen genes.
Assuntos
Elementos Facilitadores Genéticos , Pró-Colágeno/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Sequência de Bases , Linhagem Celular , Cromatina/metabolismo , Clonagem Molecular , DNA/metabolismo , Regulação da Expressão Gênica , Genes , Humanos , Metilação , Dados de Sequência Molecular , Plasmídeos , Células Tumorais CultivadasRESUMO
Hepatic fatty acid metabolism in the rat is sexually differentiated. Rates of esterification by the liver of fatty acid into triglyceride and other esterification products (phospholipid, diglyceride, cholesteryl esters) are higher in the female than in the male. There is evidence to suggest that GH feminizes other hepatic systems that exhibit sexual dimorphism, including hepatic steroid metabolism, PRL receptors, and estrogen binding. To investigate the role of GH in maintenance of the high rates of fatty acid esterification observed in the female, we assessed rates of [1-14C]oleic acid utilization by hepatocytes prepared from hypophysectomized (hypox) cortisol/T3-replaced female rats with an without continuous in vivo infusion of human (h) GH (5 micrograms/h). In addition, we assessed the effect of in vivo hGH treatment (5 micrograms/h) on [1-14C]oleic acid utilization in the normal male rat. Hypophysectomy was accompanied by a reduction in incorporation of [1-14C]oleic acid into products of esterification (triglyceride, phospholipid, diglyceride) and oxidation (CO2, ketone bodies). Continuous infusion of hGH (5 micrograms/h; 14 days) restored rates of fatty acid esterification in the hypox-cortisol/T3-replaced female rat, with the exception of cholesteryl esters. hGH infusion partially restored rates of fatty acid oxidation in the hypox cortisol/T3-replaced female rat. Treatment of the adult male rat with continuous infusion of hGH (5 micrograms/h; 7 days) resulted in increased rates of incorporation of [1-14C] oleic acid into triglyceride. In contrast, incorporation of oleic acid into phospholipid, diglyceride, and cholesteryl esters was unaltered. These results suggest that GH may be an important regulator of hepatic fatty acid metabolism.