scholarly journals Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer [published erratum appears in Blood 1995 Feb 1;85(3):862]

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1900-1906 ◽  
Author(s):  
JL Miller ◽  
CE Walsh ◽  
PA Ney ◽  
RJ Samulski ◽  
AW Nienhuis
Keyword(s):  
Globin Gene ◽  
K562 Cells ◽  
Single Copy ◽  
Regulatory Elements ◽  
Globin Mrna ◽  
Adeno Associated Virus ◽  
Binding Motifs ◽  
Gamma Globin ◽  
Erythroleukemia Cells ◽  
New Strategy

Abstract The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut- NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single- copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.

scholarly journals Single-copy transduction and expression of human gamma-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: the effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer [published erratum appears in Blood 1995 Feb 1;85(3):862]

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1900-1906 ◽  
Author(s):  
JL Miller ◽  
CE Walsh ◽  
PA Ney ◽  
RJ Samulski ◽  
AW Nienhuis
Keyword(s):  
Globin Gene ◽  
K562 Cells ◽  
Single Copy ◽  
Regulatory Elements ◽  
Globin Mrna ◽  
Adeno Associated Virus ◽  
Binding Motifs ◽  
Gamma Globin ◽  
Erythroleukemia Cells ◽  
New Strategy

The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut- NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single- copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.

Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line

1991 ◽  
Vol 11 (7) ◽  
pp. 3528-3536
Author(s):  
N L Lumelsky ◽  
B G Forget
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Regulation ◽  
Myristate Acetate ◽  
Globin Mrna ◽  
Megakaryocytic Differentiation ◽  
Gamma Globin ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.

Sequences Downstream of the Erythroid Promoter Promote High-Level Expression of the Human α-Spectrin Gene by Providing Boundary Activity and Positive Regulatory Elements.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1572-1572
Author(s):  
Patrick G. Gallagher ◽  
Douglas G. Nilson ◽  
Jolinta Lin ◽  
David M. Bodine
Keyword(s):  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Globin Mrna ◽  
Intron 1 ◽  
Exon 1 ◽  
Globin Promoter ◽  
High Level ◽  
Positive Regulatory Element

Abstract Characterization of the regulatory elements that control α-spectrin (ASp) gene expression is important for understanding the pathogenesis of ASp-linked hemolytic anemia. Our previous studies demonstrated that the ASp promoter directs low levels of expression, and, addition of a downstream region of noncoding exon 1 and intron 1 containing 2 GATA-1 sites conferred a 10-fold increase in activity in transient transfection assays. Transgenic (TG) mouse lines with the ASp promoter, the Asp promoter-exon 1-intron 1, or ASp promoter-exon 1-intron 1 with mutations of both splice sites linked to the human Aγ-globin gene as reporter were created. In reticultocytes, no expression was detected in any of the 8 lines transmitting the ASp promoter-Aγ-globin transgene. TG mice with the ASp promoter-exon1-intron 1 demonstrated significant levels of Aγ-globin gene expression in reticulocytes, with levels of Aγ-globin mRNA of ~0.4% of mouse α-globin mRNA/transgene copy #. This expression was nearly position independent, as 22/24 lines expressed the transgene. Using a FACS-based assay, γ-globin protein was present in 100% of erythrocytes. Expression levels comparable to the Asp promoter-exon 1-intron 1 TG were detected in 9/9 lines with the mutated splice sites, indicating splicing did not contribute to changes in expression. DNaseI hypersensitive site (HS) mapping identified a broad, erythroid-specific HS across exon 1 and intron 1. The presence of a DNaseI HS site suggested the presence of a positive regulatory element or a chromatin modification such as a boundary element. Analysis of a positive regulatory element in vivo was sought by stably transfecting the following luciferase (luc) plasmids into K562 cells: ASp promoter, ASp promoter-exon 1-intron 1, ASp promoter-exon 1, ASp promoter-intron 1, and ASp promoter-exon 1-intron 1 with both GATA-1 sites mutated. Clones with copy # ≤5 were analyzed; ≥9 independent clones/line were analyzed. Normalized luc activity of the ASp promoter-exon 1-intron 1 was significantly higher than the ASp promoter in stably transfected cells, 86±15 v 28±3 (p<0.001). Mutation of both GATA-1 sites in the exon 1-intron 1 plasmid reduced activity to background. Normalized luc activity from the promoter-exon was 46±6; from the promoter-intron 101±31, suggesting the intron functions as a positive regulatory element. A barrier assay was performed by flanking a β-globin promoter-EGFP gene using wild type (WT) exon 1, exon 1 with the GATA site abolished, or WT intron 1, and stably transfecting the plasmids into K562 cells. The WT exon 1 and mutant exon 1 expressed GFP in 10/12 and 7/8 lines, respectively, indicating a barrier function for exon 1 independent of GATA-1 activity. Only 1/9 lines expressed EGFP when the cassette was flanked by the ASp intron and 0/8 expressed EGFP when there were no sequences flanking the β-globin promoter. TG mouse lines with the Asp promoter-exon 1 or the Asp promoter-intron 1 linked to the Aγ-globin gene were created. 1/5 TG lines with ASp promoter-exon 1 expressed at low levels and 3/7 TG lines with ASp promoter-intron 1 expressed at levels comparable to the ASp promoter-exon 1-intron 1. These results demonstrate that 2 elements downstream of the ASp promoter are required for high-level, erythroid-specific expression. Exon 1 has barrier activity and intron 1 functions as a positive regulatory element. This is an excellent candidate region for mutations associated with ASp-linked inherited hemolytic anemia.

scholarly journals Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line.

1991 ◽  
Vol 11 (7) ◽  
pp. 3528-3536 ◽  
Author(s):  
N L Lumelsky ◽  
B G Forget
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Negative Regulation ◽  
Myristate Acetate ◽  
Globin Mrna ◽  
Megakaryocytic Differentiation ◽  
Gamma Globin ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.

Regulation of Globin Gene Expression and Erythroid Differentiation by Sp/KLF Factors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4241-4241
Author(s):  
Jie Hong ◽  
George Stamatoyannopoulos ◽  
Chao-Zhong Song
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Expression System ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Regulatory Elements ◽  
Mrna Levels ◽  
Gene Promoters ◽  
Gamma Globin ◽  
Globin Gene Expression

Abstract Sp/Krüppel-like factor (KLF) family of proteins are characterized by the presence of three highly homologous Cys2His2 type zinc-fingers near the C-terminus that bind GC/CACCC boxes, which are one of the most common regulatory elements found in promoters of many cellular and viral genes. Currently, more than 20 members have been identified in the family. This family of factors plays important roles in cell growth, differentiation, development and homeostasis by regulating the expression of their target genes. The GC and GT/CACCC boxes in the globin gene promoters and the beta globin locus control region play an important role in the tissue- and developmental stage- specific expression of globin genes. We have carried out extensive studies to identify the KLF factors that regulate gamma globin expression and erythroid differentiation. Gene expression analysis revealed that most of the Sp/KLF factors are expressed, albeit at variable levels, in human fetal liver and adult blood cells. To determine the role of the Sp/KLF factors in gamma globin expression and erythroid differentiation, functional studies using systematic RNAi to knockdown selected Sp/KLF factors were performed. We used a lentiviral mediated siRNA expression system for specific silencing selected Sp/KLF factors. Effective knockdown of Sp/KLF factors was achieved as judged by a 70–90% decrease in their mRNA levels in the cells. Analyses of globin gene expression showed that the knockdown of some Sp/KLF factors resulted in changes in globin gene expression in K562 cells. We also observed that knockdown of specific Sp/KLF factors resulted in erythroid differentiation of K562 cells. These results suggest that specific Sp/KLF factors may play a role in regulation of globin gene expression and erythroid differentiation.

scholarly journals AUF-1 and YB-1 are critical determinants of β-globin mRNA expression in erythroid cells

Blood ◽  
2012 ◽  
Vol 119 (4) ◽  
pp. 1045-1053 ◽  
Author(s):  
Sebastiaan van Zalen ◽  
Grace R. Jeschke ◽  
Elizabeth O. Hexner ◽  
J. Eric Russell
Keyword(s):  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Affinity Enrichment

Abstract The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

1987 ◽  
Vol 7 (11) ◽  
pp. 4024-4029
Author(s):  
M Trudel ◽  
J Magram ◽  
L Bruckner ◽  
F Costantini
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Base Pairs ◽  
Beta Globin Gene ◽  
Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

scholarly journals Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2433-2437 ◽  
Author(s):  
SZ Huang ◽  
GP Rodgers ◽  
FY Zeng ◽  
YT Zeng ◽  
AN Schechter
Keyword(s):  
Polymerase Chain Reaction ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Beta Globin ◽  
Chain Reaction ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Polymerase Chain ◽  
Alpha Beta

Abstract We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

scholarly journals Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Eleana F. Stavrou ◽  
Emannuouil Simantirakis ◽  
Meletios Verras ◽  
Carlos Barbas ◽  
George Vassilopoulos ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Bone Marrow Cells ◽  
Globin Gene ◽  
K562 Cells ◽  
Replication Initiation ◽  
Matrix Attachment Region ◽  
Globin Mrna ◽  
Dna Replication Initiation ◽  
Episomal Vectors ◽  
Attachment Region

AbstractWe report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34+ cells, with transfection efficiencies of 46.3 ± 5.2%, 23.0 ± 2.1% and 24.2 ± 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of γ-globin mRNA by 3.3 ± 0.13, of γ-globin protein by 6.75 ± 3.25 and HbF protein by 2 ± 0.2 fold, while the vector remained episomal and non integrated. In murine β-YAC BMCs the vector mediated the activation of the silent human γ-globin gene and in CD34+ cells, increased γ-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of β-globin Replicator, transferred into CD34+ cells, produced CD34+ eGFP+ cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 ± 0.13 fold increased γ-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors.

scholarly journals Augmentation of gamma-globin gene promoter activity by carboxylic acids and components of the human beta-globin locus control region

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3929-3935 ◽  
Author(s):  
S Safaya ◽  
A Ibrahim ◽  
RF Rieder
Keyword(s):  
Gene Expression ◽  
Organic Acids ◽  
Carboxylic Acids ◽  
Transient Expression ◽  
Promoter Activity ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
Hemoglobin Synthesis ◽  
Gamma Globin

Butyric acid increases fetal hemoglobin synthesis in adult animals and in erythroid cells in culture and induces the gamma-globin gene promoter in transient expression experiments in K562 cells (McDonagh KT, Nienhuis AW, Blood 78:255a, 1992 [abstr, suppl 1]). We compared the effect of butyrate and other short-chain carboxylic acids in transient expression studies with K562 cells using an expression plasmid bearing a luciferase reporter gene driven by the normal human A gamma-globin gene promoter. Butyrate (4 carbons) increased the activity of the human A gamma-globin gene promoter up to 123 times. Marked augmentation of the normal gamma-promoter activity was also noted with 5-carbon valeric acid (up to 394 times) and 3-carbon propionic acid (up to 129 times). The branched isobutyric acid as well as phenylacetate showed less ability to increase promoter activity. Addition of the tandemly repeated AP-1/NF-E2 (AP) enhancer sequences from hypersensitive site 2 (HS2) of the locus control region (LCR) increased gamma-promoter activity up to 24 times. Addition of a nearby 16-bp conserved motif (CM) in HS2 (Safaya S, Rieder RF, Blood 78:146a, 1992 [abstr, suppl 1]) to the AP-containing plasmid construct further increased gamma-promoter activity. In the presence of butyrate, the plasmid bearing both the AP and CM sequences showed gene expression up to 477 times greater than that of the basal gamma-promoter-driven luciferase plasmid in the absence of inducer. A plasmid bearing the herpes simplex thymidine kinase promoter was also tested and gene expression was markedly increased by the same organic acids. MEL cells responded to butyrate, valerate, and propionate with induction of hemoglobin synthesis. Responses to isobutyrate and 6-carbon caproate required higher concentrations of the compounds. Thus, other short-chain organic acids as well as butyrate increase gamma-promoter activity in the transient expression system, and this activity can be further augmented by incorporating LCR elements into the expression vector. Nonglobin promoters also respond to the same carboxylic acids.

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