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.

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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Globin Gene Switching in Transgenic Mice Carrying HS2-Globin Gene Constructs

Blood ◽

10.1182/blood.v89.2.713 ◽

1997 ◽

Vol 89 (2) ◽

pp. 713-723 ◽

Cited By ~ 13

Author(s):

N.A. Roberts ◽

J.A. Sloane-Stanley ◽

J.A. Sharpe ◽

S.J. Stanworth ◽

W.G. Wood

Keyword(s):

Transgenic Mice ◽

Developmental Regulation ◽

Globin Gene ◽

Adult Life ◽

Fetal Hemoglobin ◽

Intrauterine Death ◽

Globin Genes ◽

Copy Numbers ◽

Correct Pattern ◽

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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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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Mild dyserythropoiesis and β-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

Human Genomics ◽

10.1186/s40246-020-00283-3 ◽

2020 ◽

Vol 14 (1) ◽

Author(s):

Petros Papadopoulos ◽

Athanassia Kafasi ◽

Iris M. De Cuyper ◽

Vilma Barroca ◽

Daniel Lewandowski ◽

...

Keyword(s):

Globin Gene ◽

Family Members ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Perinatal Period ◽

Globin Genes ◽

Hemoglobin Switching ◽

Suppression Mechanism ◽

Gene Switching

Abstract The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

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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

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Mild dyserythropoiesis and b-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

10.21203/rs.3.rs-44936/v3 ◽

2020 ◽

Author(s):

Petros Papadopoulos ◽

Athanassia Kafasi ◽

Iris de Cuyper ◽

Vilma Barroca ◽

Daniel Lewandowski ◽

...

Keyword(s):

Globin Gene ◽

Family Members ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Perinatal Period ◽

Globin Genes ◽

Hemoglobin Switching ◽

Suppression Mechanism ◽

Gene Switching

Abstract The expression of the human b-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (e to g), and the second one during the perinatal period (g to b). The g to b globin gene switching mechanism includes suppression of fetal (g-globin, HbF) and activation of adult (b-globin, HbA) globin gene transcription. In Hereditary Persistence of Fetal Hemoglobin (HPFH), the g-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the g to b globin switch. Previously a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF amongst family members, and those from other reported families carrying genetic variants in KLF1, suggest additional contributors to globin switching. ASF1B was downregulated in family members with HPFH. Here, we investigate the role of ASF1B in g to b globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

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Mild dyserythropoiesis and b-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

10.21203/rs.3.rs-44936/v1 ◽

2020 ◽

Author(s):

Petros Papadopoulos ◽

Athanassia Kafasi ◽

Iris de Cuyper ◽

Vilma Barroca ◽

Daniel Lewandowski ◽

...

Keyword(s):

Globin Gene ◽

Family Members ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Perinatal Period ◽

Globin Genes ◽

Hemoglobin Switching ◽

Suppression Mechanism ◽

Gene Switching

Abstract The expression of the human b-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (e to g), and the second one during the perinatal period (g to b). The g to b globin gene switching mechanism includes suppression of fetal (g-globin, HbF) and activation of adult (b-globin, HbA) globin gene transcription. In Hereditary Persistence of Fetal Hemoglobin (HPFH), the g-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the g to b globin switch. Previously a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF amongst family members, and those from other reported families carrying genetic variants in KLF1, suggest additional contributors to globin switching. ASF1B was downregulated in family members with HPFH. Here, we investigate the role of ASF1B in g to b globin switching and erythropoiesis in vivo . Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

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Mild dyserythropoiesis and b-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

10.21203/rs.3.rs-44936/v2 ◽

2020 ◽

Author(s):

Petros Papadopoulos ◽

Athanassia Kafasi ◽

Iris de Cuyper ◽

Vilma Barroca ◽

Daniel Lewandowski ◽

...

Keyword(s):

Globin Gene ◽

Family Members ◽

Fetal Hemoglobin ◽

Erythroid Differentiation ◽

Perinatal Period ◽

Globin Genes ◽

Hemoglobin Switching ◽

Suppression Mechanism ◽

Gene Switching

Abstract The expression of the human b-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (e to g), and the second one during the perinatal period (g to b). The g to b globin gene switching mechanism includes suppression of fetal (g-globin, HbF) and activation of adult (b-globin, HbA) globin gene transcription. In Hereditary Persistence of Fetal Hemoglobin (HPFH), the g-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the g to b globin switch. Previously a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF amongst family members, and those from other reported families carrying genetic variants in KLF1, suggest additional contributors to globin switching. ASF1B was downregulated in family members with HPFH. Here, we investigate the role of ASF1B in g to b globin switching and erythropoiesis in vivo . Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

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Developmental regulation of β-globin gene switching

Cell ◽

10.1016/0092-8674(88)90005-0 ◽

1988 ◽

Vol 55 (1) ◽

pp. 17-26 ◽

Cited By ~ 157

Author(s):

Ok-Ryun Baik Choi ◽

James Douglas Engel

Keyword(s):

Developmental Regulation ◽

Globin Gene ◽

Gene Switching

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

Blood ◽

10.1182/blood.v71.3.766.bloodjournal713766 ◽

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.

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Beta zero-thalassemia in association with a gamma-globin gene quadruplication

Blood ◽

10.1182/blood.v68.6.1394.bloodjournal6861394 ◽

1986 ◽

Vol 68 (6) ◽

pp. 1394-1397

Author(s):

KG Yang ◽

JZ Liu ◽

F Kutlar ◽

A Kutlar ◽

C Altay ◽

...

Keyword(s):

Clinical Condition ◽

Globin Gene ◽

Normal Chromosome ◽

Gene Arrangement ◽

Beta Thalassemia ◽

Globin Genes ◽

Thalassemia Intermedia ◽

Gamma Globin ◽

Alpha Globin

We have studied the hematology, hemoglobin composition, and globin gene arrangements in one young Turkish boy with a beta zero-thalassemia homozygosity and in 11 of his relatives. Evidence is presented that the chromosome with the beta zero-thalassemia determinant carries a gamma- globin gene quadruplication, perhaps in a -G gamma-G gamma-G gamma-A gamma-gene arrangement. The eight gamma-globin genes in this patient produced G gamma and A gamma chains in a 95 to 5 ratio, and nearly 99% of the patient's hemoglobin was of the fetal type. The clinical condition resembled that of a thalassemia intermedia. HbF levels in eight beta-thalassemia heterozygotes varied between 0.5 and 4.2% and the percentages of G gamma in this HbF averaged at 87% or 95%; this level is to some extent related to the haplotype of the normal chromosome. All subjects carried four alpha-globin genes; a new BglII polymorphism was observed within the psi alpha-globin gene.

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