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

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.

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

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.

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

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.

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

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.

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

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 713-723 ◽  
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.

Recapitulation of the Fetal Pattern of Expression of the Iγ and Vγ-Globin Genes in Cultured Baboon CD34+ Bone Marrow Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1590-1590
Author(s):  
Donald Lavelle ◽  
Kestis Vaitkus ◽  
Mahipal Singh ◽  
Maria Hankewych ◽  
Joseph DeSimone
Keyword(s):  
Bone Marrow ◽  
Amino Acid ◽  
Bone Marrow Cells ◽  
Globin Gene ◽  
Single Amino Acid ◽  
Globin Genes ◽  
Globin Chain ◽  
Hemoglobin Switching ◽  
Globin Chains

Abstract The human Gγ-globin and Aγ-globin genes differ by the presence of a single amino acid, either glycine or alanine, at position 136. The ratio of Gγ/Aγ-globin expression is approximately 7/3 at birth and changes to 2/3 in the adult. The mechanism responsible for this developmental switch is unknown. In the baboon, the duplicated γ-globin genes differ by the presence of a single amino acid at position 75. The Iγ-globin gene contains isoleucine at position 75, while the Vγ-globin gene contains valine at this position. The ratio of expression of the Iγ and Vγ-globin chains also differs in the fetal and adult stages. The Iγ/Vγ ratio is 3/2 in the fetus and 2/3 in the adult. Thus the pattern of expression of the baboon Iγ-globin gene is analogous to the human Gγ-globin gene, and that of the Vγ-globin gene is analogous to the human Aγ-globin gene. During stress erythropoiesis, moderately increased HbF levels are observed (5–10% HbF) and the Iγ/Vγ-globin chains are expressed in the characteristic adult ratio. Decitabine treatment reactivates HbF expression to high levels (50–70% HbF) and Iγ/Vγ ratios of approximately 1:1 have been observed following decitabine treatment. Thus decitabine treatment alters the Iγ/Vγ ratio but does not cause a complete reversion to the fetal pattern of expression. HbF is also reactivated to high levels in cultured baboon BFUe. In this investigation the pattern of expression of the Iγ- and Vγ-globin genes in cultured baboon CD34+ bone marrow (BM) cells was analyzed to determine whether reactivation of HbF in culture was associated with a change in the pattern of expression of the Iγ-and Vγ-globin genes. CD34+ cells were enriched from baboon BM using the 12.8 monoclonal antibody in combination with immunomagnetic microbead columns (Miltenyi) and cultured in Iscove’s media supplemented with 30% fetal bovine serum, stem cell factor (SCF; 200ng/ml), erythropoietin (EPO; 2U/ml), and dexamethasone (Dex; 1μM). The pattern of globin chain expression in d12 cultures, cord blood (CB) of a 58d fetus, and peripheral blood (PB) of adult baboons following phlebotomy and decitabine treatment was compared by HPLC analysis of hemolysates. The baboon 58d CB contained >90% HbF and the ratio of Iγ/Vγ was 1.85. In the adult (phlebotomized) PB the level of HbF was 8.1% and the Iγ/Vγ ratio was 0.75 thus confirming that the ratio of the baboon Iγ and Vγ-globin chains differs in the fetal and adult stages of development in a manner similar to that of the human Gγ and Aγ-globin chains. Following decitabine treatment (PA 7002) an HbF level of 55% was attained with an Iγ/Vγ ratio of 1.1. Hemolysates prepared from d12 cultures of CD34+ baboon (PA 7002) BM cells grown in the presence of SCF, EPO, and Dex contained 57.6% HbF, nearly the same level observed following decitabine treatment in vivo. The Iγ/Vγ ratio was 1.94, markedly different from that observed in this same baboon following decitabine in vivo and, moreover, nearly identical to the fetal ratio. Thus HbF reactivation in cultured adult baboon CD34+ BM cells was associated with a change in the ratio of expression of the two baboon γ-globin genes to that characteristic of the fetal stage. Recapitulation of the fetal pattern of γ-globin chain expression in cultured baboon CD34+ progenitors demonstrates yet another advantage of the baboon model for investigations of hemoglobin switching.

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 ◽  
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.

In Vivo Efficacy of Bcl11a Erythroid-Enhancer Deletion in Humanized Mouse Models of Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2465-2465 ◽  
Author(s):  
Thomas M Ryan ◽  
Suean Daimia Fontenard ◽  
Shanrun Liu ◽  
Jonathan Lockhart ◽  
Michael Berlett
Keyword(s):  
Mouse Models ◽  
Fetal Hemoglobin ◽  
Thalassemia Major ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Humanized Mouse ◽  
Gamma Globin ◽  
Hemoglobin Switching ◽  
Humanized Mouse Models

Abstract Autologous cell therapy holds great promise for the treatment of beta thalassemia major and hemoglobinopathies like sickle cell anemia. Gene editing of a patient's own stem cells to reactivate the silenced gamma globin gene is one approach under active development. Prior to directly testing these new therapies in patients, we can answer some basic questions about their in vivo efficiency and efficacy in humanized mouse models of anemia. These models have their endogenous adult alpha and beta globin genes replaced with human alpha, gamma, and beta globin genes. These mice synthesize high level of human fetal hemoglobin during fetal life and complete their fetal-to-adult hemoglobin switch after birth. Experimental strategies designed to reactivate the silenced fetal gamma globin genes in adult erythroid cells are easily tested in vivo in these humanized hemoglobin switching mouse models. The silenced human fetal gamma globin genes can be activated by mutating the erythroid-specific enhancer of Bcl11a by gene editing. CRISPR sgRNAs, designed to target the +62 kb DNase I hypersensitive site in the second intron of Bcl11a, were microinjected along with Cas9 mRNA, into fertilized mouse embryos collected from humanized hemoglobin (Hb A) mice. The indel mutations that were generated in the founder animals were characterized and bred to homozygosity. The data demonstrates that the sgRNAs tested were successful in creating multiple unique mutations at the erythroid enhancer target sites. These mutations were transmitted through the germline allowing the effect of individual edited alleles to be analyzed. The majority of the mutations showed marginal increases in the number of F-cells over control animals. Significantly, despite having homozygous mutation of the erythroid-enhancer in all cells, fetal hemoglobin expression remains heterocellular. Importantly, the therapeutic efficacy of reactivating fetal hemoglobin with specific Bcl11a erythroid-enhancer mutations for the treatment of beta thalassemia major and sickle cell anemia was directly measured in vivo in these humanized models of disease. The reactivation of gamma globin in these humanized mouse models provides us with an opportunity to further interrogate the Bcl11a enhancer element, identify additional factors involved in hemoglobin switching and elucidate the mechanism driving pancellular vs heterocellular fetal hemoglobin expression. Disclosures No relevant conflicts of interest to declare.

Persistent Human γ-Globin Expression in Adult Transgenic Mice Following Selective Deletion of Two Repressor Elements Located 3′ to the Aγ-Globin Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1585-1585
Author(s):  
Maria Gazouli ◽  
Elena Katsantoni ◽  
Theodore Kosteas ◽  
Nicholas P. Anagnou
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Globin Gene ◽  
Adult Life ◽  
Single Copy ◽  
Perinatal Period ◽  
Globin Genes ◽  
Cis Acting ◽  
Transgenic Lines

Abstract Adult β-globin gene expression is tightly regulated during development and hematopoiesis. The human globin genes undergoing two developmental switches are regulated by a complex interplay between cis-acting elements and stage-specific trans-acting factors. Understanding the molecular basis of globin gene switching is of particular interest as persistent expression of the fetal γ-globin genes in the adult ameliorates the effects of hemoglobinopathies. Natural occurring deletions within the human β-globin gene cluster lead to specific clinical syndromes characterized by increased production of fetal hemoglobin (HbF) in adult life. These clinical syndromes provide an excellent model to reveal and delineate novel cis-acting elements involved in the developmental control of hemoglobin switching. One major hypothesis, which accounts for these distinct phenotypic features, assumes that silencers located within the Aγ to δ gene region, are deleted in both HPFH and δβ-thalassemias leading to the failure of switching. Previous studies of our laboratory suggested that four elements (Enh, F, O and P) located within the Aγ toδ globin intergenic region, exhibited silencer activity in transient assays (Clin Res 41:308, 1993 and Blood 84:506, 1994) and that the Enh and F elements were capable of down-regulating transcription of the human β-globin locus in an embryonic-specific manner in transgenic mice (Exp Hematol 32:224, 2004). In the present study, we sought to further clarify the in vivo role of the Enh and F elements in the silencing of the fetal Aγ-gene. To this end, we have generated transgenic mice by using cosmid constructs containing the full length human globin LCR linked to the 3.3 kb Aγ gene, lacking both the Enh and F elements. As controls, we used transgenic lines containing the full length LCR linked to the 5.6 kb Aγ-gene construct, which includes both the Enh and F elements, previously shown by us (Blood102:3412, 2003) and others (Nature350:252, 1991) to be autonomously regulated during the perinatal period. Three transgenic lines for the LCR 3.3 kb Aγ-gene construct have been generated. Cosmid integrity and copy numbers (2, 3 and 4 copies respectively) were determined by Southern blot analysis. Expression analysis in adult blood RNA performed by S1 nuclease protection and real-time reverse transcriptase PCR, documented persistence of expression of Aγ-gene in adult life. To further investigate whether the persistence of Aγ-gene expression was not a non-specific effect of the multicopy integrants, we generated a new series of single copy mice by cross-breeding the three transgenic lines with a line expressing the Cre recombinase gene (CAG-Cre). As expected, in the control LCR-5.6 kb Aγ lines, containing the Enh and F elements, the Aγ-globin gene was silenced in all lines tested in the adult stage. In contrast, high levels of Aγ-globin gene expression, similar to those of multicopy integrants were documented in all three generated single copy LCR-3.3 kb Aγ lines, lacking the Enh and F elements. Thus, this study documents directly for the first time the in vivo role of of these two gene-proximal negative regulatory elements on the silencing of the Aγ-gene in the perinatal period and may permit the design of future therapeutic strategies for their exploitation in therapeutic approaches for thalassemias.

scholarly journals Induction of erythroid differentiation and fetal hemoglobin production in human leukemic cells treated with phenylacetate

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1576-1581 ◽  
Author(s):  
D Samid ◽  
A Yeh ◽  
P Prasanna
Keyword(s):  
Antitumor Agents ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Leukemic Cells ◽  
Amino Acid Derivative ◽  
Human Leukemic Cells ◽  
Differentiation Inducers ◽  
Hemoglobin Production

Abstract There is considerable interest in identifying nontoxic differentiation inducers for the treatment of various malignant and nonmalignant blood disorders, including inborn beta-chain hemoglobinopathies. Using the human leukemic K562 cell line as a model, we explored the efficacy of phenylacetate, an amino acid derivative with a low toxicity index when administered to humans. Treatment of K562 cultures with pharmacologically attainable concentrations of phenylacetate resulted in erythroid differentiation, evident by the reduced growth rate and increased hemoglobin production. The effect was time- and dose- dependent, further augmented by glutamine starvation (phenylacetate is known to deplete circulating glutamine in vivo), and reversible upon cessation of treatment. Molecular analysis showed that phenylacetate induced gamma globin gene expression with subsequent accumulation of the fetal form of hemoglobin (HbF). Interestingly, the addition of phenylacetate to antitumor agents of clinical interest, eg, hydroxyurea and 5-azacytidine, caused superinduction of HbF biosynthesis. The results suggest that phenylacetate, used alone or in combination with other drugs, might offer a safe and effective new approach to treatment of some hematopoietic neoplasms and severe hemoglobinopathies.

Human Gamma Globin Gene Regulation in Knock-In Mouse Models of Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1779-1779
Author(s):  
Sean C. McConnell ◽  
Yongliang Huo ◽  
Shan-Run Liu ◽  
Ting-Ting Zhang ◽  
Clayton L. Ulrey ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Mouse Models ◽  
Adult Mouse ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Model Systems ◽  
Globin Genes ◽  
Human Phenotype ◽  
Globin Gene Regulation ◽  
Gene Switching

Abstract The generation of transgenic and gene targeted mouse models of human hemoglobinopathies provides valuable opportunities to test mechanisms of human globin gene regulation and experimental therapies. Yet mice do not naturally have a fetal hemoglobin, challenging our ability to adequately model the developmental onset of disease. Transgenic model systems that contain the entire human β-globin locus present obstacles to the study of human globin gene switching, including a fetal to adult globin gene switch that occurs too early in development. The generation of genetically engineered mice with a delayed human γ to β hemoglobin switch has been a major topic of interest for our laboratory. Delayed γ globin gene expression improves the clinical progression in patients as well as animal models with hemoglobinopathies. However, molecular mechanisms involved in globin gene switching are not well understood. In this study the transcriptional and epigenetic regulation of human γ to β hemoglobin switching are analyzed in novel human knock-in (KI) mouse models that complete the switch from fetal to adult hemoglobin after birth. These KI mice were generated by replacement of the adult mouse β-globin genes by homologous recombination in embryonic stem cells with a delayed switching human γ to β globin gene construct. Quantitative real-time PCR and HPLC were used to measure mouse and human embryonic, fetal, and adult globin genes through development and show that we have given the mouse a true fetal hemoglobin. Heterozygous mice express human β-like globin genes at a high level comparable to the adult mouse β globin genes. Mutations responsible for hereditary persistence of fetal hemoglobin (HPFH) in the γ globin promoter recapitulate the human phenotype in KI mice, with over 50 fold γ globin gene upregulation in adults. These HPFH KI mice also display higher γ globin levels at birth and markedly delayed γ globin gene downregulation in the weeks following birth. These studies in KI mice demonstrate that human β-like globin genes interacting with the mouse LCR are regulated in a manner similar to what is seen in humans and may be used to study the mechanisms of globin gene switching. Greater understanding of γ-globin gene regulation will be required for achieving the therapeutic goal of reactivating silenced γ-globin genes to ameliorate severe human hemoglobinopathies.

Sign in / Sign up

Export Citation Format

Share Document