A Novel C to A Substitution in the CCAAT Box of beta Globin Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3810-3810
Author(s):  
Chiara Refaldi ◽  
Elena Di Pierro ◽  
Maria C. Mocellini ◽  
Maria D. Cappellini
Keyword(s):  
Transcription Factor ◽  
Globin Gene ◽  
Tata Box ◽  
Sequencing Analysis ◽  
Beta Globin ◽  
Specific Transcription Factor ◽  
Beta Globin Gene ◽  
Factor C ◽  
Ccaat Box

Abstract The promoter of the human beta-globin gene contains three positive cis-acting elements required for maximal transcription: the CACCC box located between −86 and −90, the CCAAT box located between −72 and − 76 and the TATA box located between −28 and −31 relative to the start site of transcription. Naturally occurring mutations within the TATA and the CACCC box regions have been recorded in patients with beta+ thalassemia. Mutations within the TATA box disrupt assembly of the basal transcription complex, while mutations at the CACCC box prevent binding of an erythroid-specific transcription factor EKLF. Surprisingly, no mutations have so far been identified in the highly conserved element CCAAT box and the transcription factors responsible for the regulatory activity of the CCAAT site in vivo have been less intensively studied. We report a novel mutation −76 C>A (HBB c. −126) detected by sequencing analysis of beta globin gene in a Italian beta+ thalassemic patient. The transversion C>A hits the first nucleotide in the CCAAT box of the beta globin gene. The carrier, a male 44 years old, shows a mild hypochromic and microcytic anaemia with reduced mean corpuscular volume and mean corpuscular haemoglobin (MCV 75 fl, MCH 25 pg) and Hb A2 level slightly increased (3.9%). Recently, studies in vitro in gel-shift and reporter assays, investigating the transcriptional activity of human beta globin CCAAT box, have identified five factors: NF-Y (CP1) a ubiquitous CCAAT box binding complex, GATA-1 an erythroid-specific transcription factor, C/EBPbeta, C/EBPgamma and C/EBPdelta members of CCAAT/enhancer-binding protein family involved in hemapoietic regulation. This represents the first report of a natural mutation of the human beta-globin CCAAT box and confirms its functional significance for in vivo transcription.

scholarly journals A novel mutation in the TATA box in a Japanese patient with beta +- thalassemia

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 547-550 ◽  
Author(s):  
Y Takihara ◽  
T Nakamura ◽  
H Yamada ◽  
Y Takagi ◽  
Y Fukumaki
Keyword(s):  
Novel Mutation ◽  
Globin Gene ◽  
Japanese Woman ◽  
Tata Box ◽  
Proximal Promoter ◽  
Base Substitution ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Beta Globin Gene

Abstract A single base substitution (A-G) at position -31 within the highly conserved proximal promoter element, the TATA box, was identified in the beta-globin gene cloned from a Japanese woman with beta +- thalassemia. It appears that she is homozygous for this specific allele, as determined by haplotype analysis using seven different polymorphic sites in the beta-globin gene cluster. Transient expression of the mutant gene in COS cells revealed a 45% reduction in beta-globin RNA production, relative to normal. These results establish the functional significance of the second base of the TATA box for in vivo transcription of the human beta-globin gene.

scholarly journals A novel mutation in the TATA box in a Japanese patient with beta +- thalassemia

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 547-550
Author(s):  
Y Takihara ◽  
T Nakamura ◽  
H Yamada ◽  
Y Takagi ◽  
Y Fukumaki
Keyword(s):  
Novel Mutation ◽  
Globin Gene ◽  
Japanese Woman ◽  
Tata Box ◽  
Proximal Promoter ◽  
Base Substitution ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Beta Globin Gene

A single base substitution (A-G) at position -31 within the highly conserved proximal promoter element, the TATA box, was identified in the beta-globin gene cloned from a Japanese woman with beta +- thalassemia. It appears that she is homozygous for this specific allele, as determined by haplotype analysis using seven different polymorphic sites in the beta-globin gene cluster. Transient expression of the mutant gene in COS cells revealed a 45% reduction in beta-globin RNA production, relative to normal. These results establish the functional significance of the second base of the TATA box for in vivo transcription of the human beta-globin gene.

scholarly journals The inactive beta globin gene on a gamma delta beta thalassemia chromosome has a normal structure and functions normally in vitro

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 766-770
Author(s):  
PT Curtin ◽  
YW Kan
Keyword(s):  
Globin Gene ◽  
Large Deletion ◽  
Beta Thalassemia ◽  
Ribonuclease Protection Assay ◽  
Beta Globin ◽  
Gamma Delta ◽  
Beta Globin Gene ◽  
Thalassemia Minor

We have previously described an English family with gamma delta beta- thalassemia in which a large deletion stops 25 kilobases (kb) upstream from the beta-globin gene locus, and yet the beta-globin gene is inactive in vivo. Affected family members had a beta-thalassemia minor phenotype with a normal hemoglobin A2 level. Gene mapping showed that these subjects were heterozygous for a chromosome bearing a large deletion that began in the G gamma-globin gene, extended through the epsilon-globin gene, and continued upstream for at least 75 kb. The A gamma-, delta-, and beta-globin gene loci on this chromosome were intact. To examine the possibility that an additional defect was present in the beta-globin gene, we cloned, sequenced, and examined the expression of the beta-globin gene from the affected chromosome. No mutation was found in the beta-globin gene sequence from 990 base-pairs 5′ to the cap site to 350 basepairs 3′ to the polyadenylation signal. The gene was subcloned into an expression vector and introduced into HeLa cells. Analysis of RNA derived from these cells, using a ribonuclease protection assay, revealed qualitatively and quantitatively normal transcription. Thus a structurally and functionally normal beta-globin gene is inactive in the presence of a large deletion more than 25 kb upstream. The loss of beta-globin gene function may be due to disturbance of chromatin conformation caused by the deletion or may be the result of loss of upstream sequences that are necessary for beta-globin gene expression in vivo.

scholarly journals A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1384-1392 ◽  
Author(s):  
I Plavec ◽  
T Papayannopoulou ◽  
C Maury ◽  
F Meyer
Keyword(s):  
Bone Marrow Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

scholarly journals Homology requirements for unequal crossing over in humans.

Genetics ◽  
1991 ◽  
Vol 128 (1) ◽  
pp. 143-161 ◽  
Author(s):  
A B Metzenberg ◽  
G Wurzer ◽  
T H Huisman ◽  
O Smithies
Keyword(s):  
Homologous Recombination ◽  
Gene Cluster ◽  
Fusion Gene ◽  
Globin Gene ◽  
Crossing Over ◽  
Beta Globin ◽  
Unequal Crossover ◽  
Unequal Crossing Over ◽  
Beta Globin Gene

Abstract To gain insight into mechanisms of unequal homologous recombination in vivo, genes generated by homologous unequal crossovers in the human beta-globin gene cluster were examined by nucleotide sequencing and hybridization experiments. The naturally occurring genes studied included one delta-beta Lepore-Baltimore fusion gene, one delta-beta Lepore-Hollandia fusion gene, 12 delta-beta Lepore-Boston genes, one A gamma-beta fusion Kenya gene, one A gamma-G gamma fusion (the central gene of a triplication) and one G gamma-A gamma fusion. A comparison of the nucleotide sequences of three Lepore-Boston genes indicates that they were derived from at least two independent homologous but unequal crossover events, although the crossovers occurred within the same 58-bp region. Nine additional Lepore-Boston genes from individuals of various ethnic origins were shown, by hybridization to specific oligonucleotide probes, to have been generated by a crossover in the same region as the sequenced genes. Evidence for gene conversion accompanying a homologous unequal crossover event was found in only one case (although some of the single nucleotide differences observed in other genes in this study may be related to the crossover events in ways that we do not presently understand). Thus, as judged by this limited sample, concurrent gene conversions are not commonly associated with homologous but unequal exchange in humans in vivo. Classification of the recombinant chromosomes by their polymorphic restriction sites in the beta-globin gene cluster indicated that the Lepore-Boston genes are found in at least six different haplotype backgrounds. Therefore the total number of independent examples in this study is at least 6, and at most 12. We have shown that in at least six cases of genes that have arisen by homologous but unequal crossing over in vivo, each event occurred in a relatively extensive region of uninterrupted identity between the parental genes. This preference cannot be explained by a mechanism whereby crossovers occur at random within misaligned related but not identical genes. In general, crossovers occur in regions that are among the largest available stretches of identity for a particular pair of mismatched genes. Our data are in agreement with those of other types of studies of homologous recombination, and support the idea that sequence identity, rather than general homology, is a critical factor in homologous recombination.

Altered Regulation of β like Globin Genes by a Redesigned Erythroid Transcription Factor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1212-1212
Author(s):  
Deepa Manwani ◽  
Mariann Galdass ◽  
James J. Bieker
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Zinc Finger ◽  
Embryoid Body ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Beta Globin Gene ◽  
Body Development ◽  
Globin Promoter

Abstract The well characterized switch during ontogeny of globin gene expression from embryonic/ fetal to adult type is a result of a complex interplay between cis and trans acting regulatory elements at the beta globin locus. Trans acting elements include tissue specific transcription factors that bind specific motifs within the beta globin gene cluster with high specificity. Erythroid Kruppel like factor (EKLF) is one such erythroid specific, zinc finger transcription factor that is critical for the activation of the beta globin promoter and for consolidating the switch from gamma to beta globin during development. The ability to willfully regulate the expression of endogenous genes using redesigned zinc finger transcription factors is an emerging field. There is tremendous appeal in utilizing the understanding of transcriptional control pathways to design tools that will elucidate molecular mechanisms and provide potential therapeutic tools. To this end we redesigned Erythroid Kruppel Like Factor (EKLF) as a transcriptional repressor. The zinc finger DNA binding domain was linked to the repressor domain from the Drosophila Engrailed protein with the prediction that this construct (ENG/ZNF) would bind the beta globin promoter and repress it. It was hypothesized that embryonic/fetal globin activation would result by a competitive mechanism. When introduced transiently into cells these transcription factors are effective in repressing the adult beta globin promoter CACCC element, the natural target for EKLF. In stable MEL clones, repression of the adult beta globin gene is accompanied by a reactivation of the endogenous embryonic globin gene. In order to study this effect in the context of a whole animal we generated transgenic mice expressing ENG/ZNF. A 271 bp region 5′of the ANK-1 gene was chosen to drive expression in transgenic mice as it provides erythroid specific expression with copy number dependence and minimal position dependence. D13.5 fetal livers were subject to RT-PCR analysis in the linear range to quantitate the ratios of BH1 to alpha globin transcripts. The 9 ENG/ZNF transgenic embryos express BH1 mRNa in a range of values that is statistically higher than in 9 control littermates (Mann Whitney U test, p value 0.02) and beta major globin mRNA at lower levels. We further studied ENG/ZNF in the developmentally plastic environment of differentiating murine embryonic stem cells. The construct was stably integrated into a targeting site upstream of the HPRT locus under the control of a tetracycline inducible promoter. The Doxycycline induction of ENG/ZNF transgene expression results in a 4 fold activation of embryonic globin at day 6 of embryoid body development; however there is no evidence of beta globin repression. Since at this stage of embryoid body development, primitive erythroid cells are 100–500 fold more abundant than definitive erythroid cells, this may reflect a differential effect of EKLF in primitive erythroid cells. To evaluate this further, we are currently performing analyses in primitive versus definitive erythroid colonies. In conclusion, our studies support the competitive model of globin switching and may contribute to the delineation of a stage specific role of EKLF. In addition, transcriptional reagents that augment gamma globin expression hold promise as novel therapeutic agents for sickle cell disease and other hemoglobinopathies.

scholarly journals Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2031-2037 ◽  
Author(s):  
GW Hall ◽  
S Thein
Keyword(s):  
Globin Gene ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Terminal Exon ◽  
Mrna Accumulation ◽  
Beta Globin Gene ◽  
Globin Chains ◽  
Nonsense Codon ◽  
Nonsense Codons

We present in vivo evidence that there is no reduction in beta-mRNA accumulation in patients with nonsense codons in the terminal exon of the beta-globin gene. Using reverse transcriptase/polymerase chain reaction (RT-PCR), beta-globin cDNA was isolated from the reticulocytes of individuals heterozygous for nonsense codon mutations in exons II and III of the beta-globin gene. Clinically asymptomatic individuals heterozygous for mutations causing premature termination of translation in exon II [beta(0)39(C-T) and F/S71/72(+A)] were found to have almost no mutant beta-cDNA, whereas patients with nonsense codon mutations in exon III [beta 121(G-T) and beta 127(C-T)] with the clinical phenotype of thalassemia intermedia had comparable levels of mutant and normal beta-cDNA. Translation of the mutant beta-mRNA from patients with nonsense codon mutations in exon III would give rise to truncated beta- globin chains, which could explain the more severe phenotype seen in these individuals.

scholarly journals The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1470-1474 ◽  
Author(s):  
GF Atweh ◽  
XX Zhu ◽  
HE Brickner ◽  
CH Dowling ◽  
HH Jr Kazazian ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Promoter Mutation ◽  
Beta Globin Gene ◽  
Gamma Globin ◽  
New Type

A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta- thalassemia chromosome. An A to G transversion is identified in the “ATA” sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

Highly efficient beta globin transcription in the absence of both a viral enhancer and erythroid factors

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 815-827 ◽  
Author(s):  
M.E. Walmsley ◽  
R.K. Patient
Keyword(s):  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Globin Gene ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Highly Efficient ◽  
Progesterone Treatment ◽  
Sv40 Enhancer ◽  
Site Versus

We have studied the transcription of the Xenopus major adult beta globin gene in microinjected Xenopus oocytes at various levels of injected template, with or without the SV40 enhancer. We find that enhancer-independent transcription is highly efficient, being only two orders of magnitude below the calculated in vivo rate. Linkage to the SV40 enhancer has very little stimulatory effect. We have also tested the effect of replication on transcription in the oocyte system where replication was induced by progesterone treatment followed by prick activation. We found that the presence of replicated templates did not stimulate expression of the Xenopus beta globin gene either in the presence or absence of the SV40 enhancer. In addition, we found that specificity of transcription, in cryptic of initiation at the cap site versus initiation at cryptic promoters upstream of the cap site, was dramatically improved by the injection of higher numbers of beta globin templates, by oocyte maturation and activation or by the presence of the SV40 enhancer.

Specific Activation of Human beta-Globin Gene Expression by the Transcription Factor Ikaros.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3641-3641
Author(s):  
Andrew C. Perkins ◽  
Peter Papathanasiou ◽  
Christopher C. Goodnow ◽  
Janelle R. Keys
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Fetal Liver ◽  
Globin Gene ◽  
Regulatory Control ◽  
Haematopoietic Stem Cell ◽  
Beta Globin ◽  
Relative Expression ◽  
Beta Globin Gene ◽  
Globin Gene Expression

Abstract The zinc finger transcription factor Ikaros is recognized as a key regulator of lymphocyte differentiation. Recently generated dominant negative mutants have hinted at a broader role in haematopoietic stem cell generation. Most recently, a mouse strain, IkarosPlastic, with a point mutation in Ikaros that disrupts DNA binding but preserves efficient assembly of Ikaros protein complexes, is embryonically lethal due to severe defects in erythrocyte differentiation (Papathanasiou P, et al,. Immunity, 2003). (1). These mice display normal murine globin gene expression in the fetal liver. However in humans the globin locus is under alternative regulatory control, particularly with respect to the fetal-to-adult globin switch. Thus, to determine if Ikaros plays a role in human globin switching we crossed the IkarosPlastic mice with mice transgenic for a YAC containing the entire human b-globin locus, which show human fetal to adult globin gene switching from E12 to E17. Embryos were harvested from E12.5 to E15.5 and globin expression was determined in the fetal liver by real-time PCR (relative to actin). At all time points human gamma-globin gene expression was not significantly altered by the presence of the IkarosPlastic mutatation (relative expression Ikaroswt/wt 1±0.11, IkarosPlastic/Plastic 0.82±0.12). In contrast, human beta-globin gene expression was significantly down-regulated in IkarosPlastic fetal livers (relative expression Ikaroswt/wt 1±0.14, IkarosPlastic/Plastic 0.18±0.07). Interestingly, neither murine a- or b-globin gene expression was significantly different to wild type mice, which suggests that the transcription factor Ikaros plays a specific role in the transcriptional activation of the human b-globin gene during development. The mechanism by which this occurs remains to be elucidated, however it is intriguing to consider that Ikaros may act as a potentiator of transcription for erythroid specific transcription factors such as EKLF. Experiments to address this will be presented.

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