Quantitative PCR analysis of HbF inducers in primary human adult erythroid cells

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 863-869 ◽  
Author(s):  
Reginald D. Smith ◽  
Jin Li ◽  
Constance T. Noguchi ◽  
Alan N. Schechter
Keyword(s):  
Quantitative Pcr ◽  
Butyric Acid ◽  
Trichostatin A ◽  
Quantitative Polymerase Chain Reaction ◽  
Globin Gene ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Human Adult

The development and evaluation of drugs to elevate fetal hemoglobin in the treatment of the genetic diseases of hemoglobin would be facilitated by the availability of reliable cell assays. We have used real-time, quantitative polymerase chain reaction (PCR) analyses of globin messenger RNA (mRNA) levels in a biphasic, erythropoietin-dependent primary culture system for human adult erythroid cells in order to assay compounds for their ability to modulate levels of adult (β) and fetal (γ) globin mRNA. Complementary DNA synthesized from total RNA extracted at timed intervals from aliquots of cells were assayed throughout the period that the culture was studied. γ-globin mRNA levels were found to be much lower (less than 1%) than β-globin mRNA levels. At concentrations of agents chosen for minimal effect on cell division, we find that the 3 drugs studied, 5-azacytidine (5μmol/L), hydroxyurea (40μmol/L), and butyric acid (0.5mmol/L), significantly increase γ-globin mRNA levels. Interestingly, hydroxyurea also had a small stimulatory effect on β-globin mRNA levels, while butyric acid caused a twofold inhibition of β-globin mRNA levels, and 5-azacytidine had little effect on β-globin mRNA levels. The net result of all 3 drugs was to increase the γ/(γ + β) mRNA ratios by threefold to fivefold. These data suggest that the mechanism is distinct for each drug. The profile of butyric-acid–induced changes on globin gene expression is also quite distinct from changes produced by trichostatin A, a known histone deacetylase inhibitor. Quantitative PCR analyses of human erythroid cells should prove useful for studying the mechanism(s) of action of known inducers of γ-globin and identifying new drug candidates.

5-Azacytidine Induction of Human γ-Globin Gene Expression: Experimental Evaluation of Current Models.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1581-1581
Author(s):  
Rodwell Mabaera ◽  
Christine Richardson ◽  
Sarah Conine ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Maximal Response ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Potent Inducer ◽  
Cellular Dna ◽  
Globin Gene Expression

Abstract 5-Azacytidine (5-Aza) was demonstrated to be a potent inducer of human fetal globin gene expression more than 20 years ago. More recently, 5-Aza-2-deoxycytidine has been shown to have similar properties. Since the 1980’s there have been two predominant hypotheses to explain the action of these agents. The first is based on the observation that these, and several other active inducing agents, are cytotoxic to differentiating erythroid cells and that drug treatment alters the kinetics of erythroid differentiation. This has been proposed to result in prolonged expression of the γ-globin genes which are normally expressed only early in differentiation. The second is based on the observation that both agents are DNA methyltransferase inhibitors and are presumed to cause demethylation of cellular DNA including the γ-globin gene promoters leading to activation of the genes. These two models lead to specific predictions that we have evaluated using an in vitro erythroid differentiation system. In this system, human adult CD34+ cells are cultured in SCF, Flt3 ligand and IL-3 for 7 days and then switched to Epo for 14 days. This results in an exponential expansion of erythroid cells. As has been described for normal human differentiation, these cells express small amounts of γ-globin mRNA early in differentiation followed by a much larger amount of β-globin mRNA. HPLC at the end of the culture period shows 99% HbA and 1% HbF. Treatment of cultures on a daily basis with 5-Aza starting on day 10 results in dose dependent increases in γ-globin mRNA, Gγ- and Aγ-chain production and HbF. The cytotoxicity model predicts that γ-globin expression will be prolonged to later in differentiation - and this is seen. However, a daily 5-Aza dose of 300 nM, which produces ~80% of the maximal response in γ-globin mRNA and HbF, has no effect on cell growth or differentiation kinetics. This argues against the toxicity model. We next examined the effect of 5-Aza on γ-globin promoter methylation using the bisulfite method. We studied CpGs at −344, −252, −162, −53, −50, +6, +19 and +50 relative to the start site. For untreated controls, all of the sites are nearly 100% methylated at day 1. By day 3, the upstream sites become ~50% methylated except the −53 CpG which was <20%. This pattern persisted at day 10. By day 14 the promoters had become largely remethylated. For cells treated with 5-Aza starting on day 10, there was no change in the levels of methylation seen on days 1,3 and 10, but at day 14 the low levels of upstream methylation persisted - just as γ-globin expression does. However, in both treated and untreated cells, down-stream CpG sites were highly methylated at all time points. This suggests that γ promoter demethylation may be due to a local and not a generalized effect of 5-Aza on cellular DNA methylation. We also made two unexpected observations. At a 300nM dose of 5-Aza, γ-globin mRNA is ~doubled while β-globin mRNA levels are ~halved - indicating that 5-Aza not only induces γ-globin expression also suppresses β-globin. Also despite only a doubling in γ-globin mRNA, there was an ~50-fold increase in HbF, from ~1% to more than 50%, while total per cell Hb levels were unchanged. Neither of these results are easily explained by current models of γ-globin gene induction. Our results raise the possibility that mechanisms beyond cytotoxicity and generalized DNA demethylation may be responsible for pharmacologic induction of γ-globin mRNA and HbF.

scholarly journals Regulation of histone and beta A-globin gene expression during differentiation of chicken erythroid cells.

1987 ◽  
Vol 7 (10) ◽  
pp. 3663-3672 ◽  
Author(s):  
M Affolter ◽  
J Côté ◽  
J Renaud ◽  
A Ruiz-Carrillo
Keyword(s):  
Transcriptional Activity ◽  
Globin Gene ◽  
The Other ◽  
Mrna Levels ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Mature Erythrocyte ◽  
Tightly Coupled ◽  
Histone H5

The expression of the genes for several histones and beta A-globin was examined in the chicken erythroid cells lineage. During the transition from CFU-(E) to the mature erythrocyte, histone H5 gradually increased fourfold in nuclei with little concomitant displacement of the H1 histones. This resulted in a 70% net increase in linker histone (H1 plus H5) content. The differential accumulation of H5 reflected (i) an increase in the transcriptional activity of the H5 gene occurring at the erythroblast stage, (ii) an apparent longer half-life of H5 mRNA, and (iii) a higher stability of the protein. Although the transcriptional activity of the histone genes (except H5) decreased with cell age, it was not tightly coupled to the S phase. On the other hand, the mRNA levels for these histones were tightly regulated during the cell cycle. Use of protein and DNA synthesis inhibitors indicated that the content of H5 mRNA was regulated at the posttranscriptional level by a control mechanism(s) differing from those for the other histones. Although the transcription rates of the H5 and beta A-globin genes were comparable, differential accumulation of beta A-globin mRNA led to a 30- to 170-fold-higher copy number of the beta A-globin mRNA as the cell matured.

Regulation of histone and beta A-globin gene expression during differentiation of chicken erythroid cells

1987 ◽  
Vol 7 (10) ◽  
pp. 3663-3672
Author(s):  
M Affolter ◽  
J Côté ◽  
J Renaud ◽  
A Ruiz-Carrillo
Keyword(s):  
Transcriptional Activity ◽  
Globin Gene ◽  
The Other ◽  
Mrna Levels ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Mature Erythrocyte ◽  
Tightly Coupled ◽  
Histone H5

The expression of the genes for several histones and beta A-globin was examined in the chicken erythroid cells lineage. During the transition from CFU-(E) to the mature erythrocyte, histone H5 gradually increased fourfold in nuclei with little concomitant displacement of the H1 histones. This resulted in a 70% net increase in linker histone (H1 plus H5) content. The differential accumulation of H5 reflected (i) an increase in the transcriptional activity of the H5 gene occurring at the erythroblast stage, (ii) an apparent longer half-life of H5 mRNA, and (iii) a higher stability of the protein. Although the transcriptional activity of the histone genes (except H5) decreased with cell age, it was not tightly coupled to the S phase. On the other hand, the mRNA levels for these histones were tightly regulated during the cell cycle. Use of protein and DNA synthesis inhibitors indicated that the content of H5 mRNA was regulated at the posttranscriptional level by a control mechanism(s) differing from those for the other histones. Although the transcription rates of the H5 and beta A-globin genes were comparable, differential accumulation of beta A-globin mRNA led to a 30- to 170-fold-higher copy number of the beta A-globin mRNA as the cell matured.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression

1985 ◽  
Vol 5 (4) ◽  
pp. 649-658
Author(s):  
G M Veldman ◽  
S Lupton ◽  
R Kamen
Keyword(s):  
Dna Replication ◽  
Globin Gene ◽  
Mrna Levels ◽  
Beta Globin ◽  
Transcriptional Enhancer ◽  
Globin Mrna ◽  
Enhancer Region ◽  
Sequence Elements ◽  
Multiple Copies ◽  
A Site

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

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.

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 The stability of human beta-globin mRNA is dependent on structural determinants positioned within its 3' untranslated region

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5314-5323 ◽  
Author(s):  
JE Russell ◽  
SA Liebhaber
Keyword(s):  
Mrna Stability ◽  
Globin Gene ◽  
Structural Features ◽  
Structural Basis ◽  
Dependent Manner ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Coding Region ◽  
Globin Mrna ◽  
Alpha Globin

Controls that act at both transcriptional and posttranscriptional levels assure that globin genes are highly expressed in developing erythroid cells. The extraordinary stabilities of alpha- and beta- globin mRNAs permit globin proteins to accumulate to substantial levels in these cells, even in the face of physiologic transcriptional silencing. Structural features that determine alpha-globin mRNA stability have recently been identified within its 3′UTR; in contrast, the structural features that determine beta-globin mRNA stability remain obscure. The current study begins to define the structural basis for beta-globin mRNA stability. Two tandem antitermination mutations are introduced into the wild-type human beta-globin gene that permit ribosomes to read into the 3′UTR of the encoded beta-globin mRNA. The readthrough beta-globin mRNA is destabilized in cultured erythroid cells, indicating that, as in human alpha-globin mRNA, an unperturbed 3′UTR is crucial to maintaining mRNA stability. Additional experiments show that the beta-globin and alpha-globin mRNA 3′UTRs provide equivalent levels of stability to a linked beta-globin mRNA coding region, suggesting a parallel in their functions. However, destabilization of the antiterminated beta-globin mRNA is independent of active translation into the 3′UTR, whereas translation into the alpha-globin mRNA 3′UTR destabilizes a linked beta-globin coding region in a translationally dependent manner. This indicates that the alpha- and beta-globin 3′UTRs may stabilize linked mRNAs through distinct mechanisms. Finally, it is shown that neither of the two mutations that, in combination, destabilize the beta-globin mRNA have any effect on beta-globin mRNA stability when present singly, suggesting potential redundancy of stabilizing elements. In sum, the current study shows that a functionally intact beta-globin mRNA 3′UTR is crucial to maintaining beta-globin mRNA stability and provides a level of stability that is functionally equivalent to, although potentially mechanistically distinct from, the previously characterized alpha- globin mRNA 3′UTR stability element.

scholarly journals Pattern of chick gene activation in chick erythrocyte heterokaryons.

1982 ◽  
Vol 95 (3) ◽  
pp. 885-892 ◽  
Author(s):  
S Linder ◽  
S H Zuckerman ◽  
N R Ringertz
Keyword(s):  
Hybrid Cell ◽  
Globin Gene ◽  
Gene Activation ◽  
Parental Cell ◽  
Mrna Levels ◽  
Cell Type ◽  
Globin Mrna ◽  
Two Dimensional Gel Electrophoresis ◽  
L6 Myoblast ◽  
Globin Synthesis

The reactivation of chicken erythrocyte nuclei in chick-mammalian heterokaryons resulted in the activation of chick globin gene expression. However, the level of chick globin synthesis was dependent on the mammalian parental cell type. The level of globin synthesis was high in chick erythrocyte-rat L6 myoblast heterokaryons but was 10-fold lower in chick erythrocyte-mouse A9 cell heterokaryons. Heterokaryons between chick erythrocytes and a hybrid cell line between L6 and A9 expressed chick globin at a level similar to that of A9 heterokaryons. Erythrocyte nuclei reactivated in murine NA neuroblastoma, 3T3, BHK and NRK cells, or in chicken fibroblasts expressed less than 5% chick globin compared with the chick erythrocyte-L6 myoblast heterokaryons. The amount of globin expressed in heterokaryons correlated with globin mRNA levels. Hemin increased beta globin synthesis two- to threefold in chick erythrocyte-NA neuroblastoma heterokaryons; however, total globin synthesis was still less than 10% that of L6 heterokaryons. Distinct from the variability in globin expression, chick erythrocyte heterokaryons synthesized chick constitutive polypeptides in similar amounts independent of the mammalian parental cell type. Approximately 40 constitutive chick polypeptides were detected in heterokaryons after immunopurification and two-dimensional gel electrophoresis. The pattern of synthesis of these polypeptides was similar in heterokaryons formed by fusing chicken erythrocytes with rat L6 myoblasts, hamster BHK cells, or mouse neuroblastoma cells. Three polypeptides synthesized by non-erythroid chicken cells but less so by embryonic erythrocytes were conspicuous in heterokaryons. Two abundant erythrocyte polypeptides were insignificant in non-erythroid chicken cells and in heterokaryons.

scholarly journals Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

1998 ◽  
Vol 18 (11) ◽  
pp. 6634-6640 ◽  
Author(s):  
Denise E. Sabatino ◽  
Amanda P. Cline ◽  
Patrick G. Gallagher ◽  
Lisa J. Garrett ◽  
George Stamatoyannopoulos ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fetal Liver ◽  
Globin Gene ◽  
Gene Promoter ◽  
Yolk Sac ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Globin Promoter ◽  
In Erythroid Cells

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.

Human Beta-Globin Expression during Erythroid Cells Development in Transgenic Mice Bearing Human Beta-Globin Gene with Mutated BP1 Binding Site.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3651-3651
Author(s):  
Olga P. Zoueva ◽  
David Bodine ◽  
Griffin P. Rodgers
Keyword(s):  
Transgenic Mice ◽  
Binding Site ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fold Increase ◽  
Mouse Embryos ◽  
Mrna Levels ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Beta Globin Gene

Abstract Binding of beta protein 1 (BP1) to its site on the promoter of adult beta-globin gene has silencing effect on beta-globin transcription in vitro. To better understand the mechanism of the negative regulation of beta-globin expression by BP1 we have developed transgenic mice. Specifically, we introduced a mutated BP1 binding site into the promoter of beta-globin gene sequence of 35 kb cosmid construct. This construct containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into the single cell mouse embryos. To detect the differences in developmental regulation of the human beta-globin gene expression in the transgenic mice, we studied the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5. In addition, we analyzed adult erythroid cells. To minimize experimental error, samples from individual animals of three transgenic lines were analyzed independently using real-time PCR assays. Levels of expression of murine alpha-globin mRNA were used as internal controls. The BP1 gene and its mouse analog Dlx4 belongs to the Distal-less family of homeobox genes, which are expressed during early development. We found that the mRNA levels of human beta-globin in transgenic mice containing mutated BP1 binding site were higher at all stages of erythroid cells development as compared with control transgenic mice bearing cosmid construct with wild type sequence of BP1 site. Particularly, we detected up to 20-fold increase in human beta-globin expression in embryonic blood at E10.5, 3-fold increase in fetal livers of transgenic mice at E13.5, and up to 1.4-fold increase in adult reticulocytes. We also found that increase in human beta-globin expression was correlated with expression pattern of murine Dlx4 which mRNA was predominantly expressed in embryonic blood at E10.5. Thus, our data indicate that transgenic mice bearing human beta-globin gene with mutated BP1 site have significantly higher human beta-globin transcripts levels in blood cells from primitive erythropoesis than control mice. These results may help develop the novel clinic approaches for the inhibition of the expression of abnormal beta-globin genes, such as sickle (hbs) and hbc.

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