Developmental regulation of β-globin gene switching

Abstract We have examined the pattern of human globin gene switching in transgenic mice containing three different γ and β gene constructs (HS2GγAγδβ, HS2Aγβneo, and HS2Aγenβ) and compared the results with previously described transgenics (HS2Aγβ, HS2GγAγ-117δβ, and LCRεGγAγδβ). Developmental regulation was observed in all cases with identical patterns in lines bearing the same construct. Three different patterns of switching were observed: LCRεGγAγδβ and HS2Aγβneo mice switched rapidly, HS2GγAγδβ and HS2GγAγ-117δβ at an intermediate rate, and HS2Aγβ and HS2Aγenβ mice showed delayed switching, with a plateau in late fetal-early neonatal life and readily detectable levels of γ mRNA in adults. No difference was observed in the time of switching of the HS2GγAγδβ mice compared with those with the Aγ-117 hereditary persistence of fetal hemoglobin mutation, but adult levels of γ mRNA were significantly higher (≈5%) in lines carrying the mutation than in those without (≈1%). Reversion to the rapid switch of the LCRεGγAγδβ mice was observed in three lines with the HS2Aγβ neo construct in which expression of the tk-neo gene was approximately equal to that of the globin genes. The inclusion of the Aγ enhancer in HS2Aγβ mice did not alter the pattern of switching, or reduce the relatively high levels of γ mRNA in these lines. However, unlike other HS2 mice, the combination of HS2 and the Aγ enhancer resulted in copy number-dependent expression in HS2Aγenβ lines, with intrauterine death at ≈12.5 days gestation at high copy numbers. These results demonstrate that numerous elements throughout the β globin gene cluster interact to produce the correct pattern of developmental regulation of these genes. Furthermore, extinction of γ gene expression in adult life is not completely autonomous and is incomplete when HS2 is the only LCR element present.

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Hb F Malta I in Association with Hb F Sardinia (AyT) and Hb Valletta in Heterozygotes: Quantification of the Six Globins Suggests Developmental Control of the XMN-I Site and Interplay with the (AT)xTy Sequence in Connection with Globin Gene Switching.

Blood ◽

10.1182/blood.v108.11.3830.3830 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3830-3830

Author(s):

Alexander Felice ◽

Joseph Borg ◽

Wilma Cassar ◽

Ruth Galdies ◽

Monica Pizzuto ◽

...

Keyword(s):

Developmental Regulation ◽

Globin Gene ◽

Genotypic Variation ◽

Beta Thalassemia ◽

Globin Genes ◽

Haplotype Data ◽

Gene Switching ◽

Regulatory Dna ◽

Compound Heterozygotes

Abstract Although the precise biochemical mechanisms of globin gene switching remain elusive, considerable insight is gained by in vivo expression profiling through quantification of the hemoglobin / globin phenotype of informative heterozygosities and homozygosities / compound heterozygosities in the context of specific regulatory DNA sequence diversity such as the XMN-I or the [(AT)xTy] sequence polymorphisms. The quantification of normal and abnormal globins of Hb F Malta-I (or a2b2, 117(G19)His>Arg) heterozygotes which are in tight linkage disequilibrium with Hb Valletta (or a2b2 287(f3)Thr>Pro) i.e. Gyo, GyFMalta-I, AyI, bV and bA together with extensive haplotyping of homozygotes and heterozygotes including the XMN-I dimorphism in the Gy promoter and the (AT)xTy polymorphism (BP1 binding site) 5′ to the b globin genes had suggested that the XMN-I dimorphism was largely inactive in the normal newborn. In contrast the Hb F levels and the proportion of Gy globin in anemic adult beta-thalassemia homozygotes and compound heterozygotes differed significantly, depending on the XMN-I genotype (TT, TC or CC) Here, we document the occurrence of seven newborn who were heterozygous at three globin loci permitting quantification by reverse phase liquid chromatography of the six globin products; Gyo, GyFMalta-I, AyI, AyT, bV and bA in the context of genotypic variation at the XMN-I and (AT)xTy sequences. The data were compared with those of newborn HbF-Malta-I-Hb-Valletta heterozygotes and anemic adult beta thalassemia homozygotes / compound heterozygotes. The globin quantification together with haplotype data were analysed using the general linear model (two-way ANOVA) by SPSS version 12. The data excluded significant effect of the XMN-I dimorphism alone on relative y/b globin gene expression in the newborn. On the other hand, the (AT)xTy polymorphism with BP1 binding sites of 21 [(AT)7T7], 23 [(AT)9T5], or 25 [(AT)11T3], nucleotides in trans over-ride XMN-I. In contrast, it is the XMN-I dimorphism that over-rides the (AT)xTy diversity in the anemic adult beta thalassemia homozygotes or compound heterozygotes. The GyFMalta-I/Gyo ratio of the newborn heterozygotes with Hb F Malta-I and the AyT/AyI ratio of the newborn heterozygotes with HbF-Malta-I and HbF-Sardinia suggested that the developmental regulation of the XMN-I site may be subject to cis/trans interplay with the (AT)xTy sequences.

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Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1561 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1561-1567 ◽

Cited By ~ 39

Author(s):

J A Lloyd ◽

J M Krakowsky ◽

S C Crable ◽

J B Lingrel

Keyword(s):

Transgenic Mice ◽

Developmental Regulation ◽

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

High Level ◽

Gene Switching ◽

Globin Gene Expression

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.

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Developmental regulation of fetal to adult globin gene switching in human fetal erythroid × mouse erythroleukemia cell hybrids

Developmental Biology ◽

10.1016/0012-1606(91)90323-u ◽

1991 ◽

Vol 148 (1) ◽

pp. 129-137 ◽

Cited By ~ 15

Author(s):

Tariq Enver ◽

Martha Brice ◽

Joyce Karlinsey ◽

George Stamatoyannopoulos ◽

Thalia Papayannopoulou

Keyword(s):

Developmental Regulation ◽

Globin Gene ◽

Cell Hybrids ◽

Erythroleukemia Cell ◽

Gene Switching ◽

Mouse Erythroleukemia

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Human gamma- to beta-globin gene switching using a mini construct in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1561-1567.1992 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1561-1567

Author(s):

J A Lloyd ◽

J M Krakowsky ◽

S C Crable ◽

J B Lingrel

Keyword(s):

Transgenic Mice ◽

Developmental Regulation ◽

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Gamma Globin ◽

High Level ◽

Gene Switching ◽

Globin Gene Expression

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.

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Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice

Nature ◽

10.1038/344309a0 ◽

1990 ◽

Vol 344 (6264) ◽

pp. 309-313 ◽

Cited By ~ 185

Author(s):

Tariq Enver ◽

Natacha Raich ◽

Allen J. Ebens ◽

Thalia Papayannopoulou ◽

Frank Costantini ◽

...

Keyword(s):

Transgenic Mice ◽

Developmental Regulation ◽

Globin Gene ◽

Gene Switching

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Serum factors can modulate the developmental clock of gamma- to beta-globin gene switching in somatic cell hybrids.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4844 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4844-4851 ◽

Cited By ~ 4

Author(s):

G Zitnik ◽

Q Li ◽

G Stamatoyannopoulos ◽

T Papayannopoulou

Keyword(s):

Globin Gene ◽

Cumulative Number ◽

Beta Globin ◽

Serum Factors ◽

Beta Globin Gene ◽

Cell Divisions ◽

Growth Promoting ◽

Gene Switching ◽

Mouse Erythroleukemia

The fusion of human fetal erythroid (HFE) cells with mouse erythroleukemia (MEL) cells produces stable synkaryons (HFE x MEL) which can be monitored for extended periods of time in culture. Initially these hybrids express a human fetal globin program (gamma >> beta), but after weeks or months in culture, they switch to an adult pattern of globin expression (beta >> gamma). The rate at which hybrids switch to the adult phenotype is roughly dependent on the gestational age of the fetal erythroid cells used in the fusion, suggesting that the rate of switching in vitro may be determined by a developmental clock type of mechanism, possibly involving the cumulative number of divisions experienced by the human fetal cells. To investigate whether the number or rate of cell divisions postfusion can influence the rate of switching, we monitored the rate of switching in hybrids from independent fusions under growth-promoting (serum-replete) and growth-suppressing (serum-deprived) conditions. We found that hybrids grown under serum-deprived or serumless conditions switched more rapidly to adult globin expression than did their counterparts in serum-replete conditions. Neither the number of cumulative cell divisions nor time in culture per se predicted the rate of switching in vitro. Our data suggest that factors present in serum either retard switching of hybrids by their presence or promote switching by their absence, indicating that globin switching in vitro can be modulated by the environment; however, once switching in HFE x MEL hybrids is complete, serum factors cannot reverse this process.

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