Developmental switch in the relative expression of the alpha 1- and alpha 2-globin genes in humans and in transgenic mice

Human alpha-globin is encoded by two adjacent genes, alpha 2 and alpha 1. Despite their remarkable level of structural identity, the more 5′ (alpha 2) gene is the major alpha-globin locus in the normal adult, expressed at 2.6-fold higher levels than the adjacent and more 3′ (alpha 1) globin gene. In light of the well-characterized pattern of gene activation in the human alpha- and beta-globin gene clusters during development, we considered the possibility that the relative expression of these two alpha-globin loci might be developmentally controlled. Analysis of human embryonic and early fetal erythroid RNA samples confirmed this possibility; levels of mRNA encoded by the two alpha-globin loci are equal in the embryo and subsequently shift to dominant expression of the alpha 2-globin locus at week 8 in utero. In transgenic mice carrying the entire human alpha-globin cluster (except for the theta gene) we show the same shift from equal expression of the alpha 1- and alpha 2-globin loci at the embryonic stage to predominance of the alpha 2-globin locus in the adult. These data demonstrate a switch in the expression of the two adjacent alpha-globin genes during the embryonic-to-fetal switch in erythroid development and provide an experimental system for its further characterization.

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Developmental switch in the relative expression of the alpha 1- and alpha 2-globin genes in humans and in transgenic mice

Blood ◽

10.1182/blood.v79.9.2471.2471 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2471-2474 ◽

Cited By ~ 13

Author(s):

M Albitar ◽

FE Cash ◽

C Peschle ◽

SA Liebhaber

Keyword(s):

Transgenic Mice ◽

Globin Gene ◽

Gene Activation ◽

Experimental System ◽

Gene Clusters ◽

Globin Genes ◽

Beta Globin ◽

Relative Expression ◽

Structural Identity ◽

Alpha Globin

Abstract Human alpha-globin is encoded by two adjacent genes, alpha 2 and alpha 1. Despite their remarkable level of structural identity, the more 5′ (alpha 2) gene is the major alpha-globin locus in the normal adult, expressed at 2.6-fold higher levels than the adjacent and more 3′ (alpha 1) globin gene. In light of the well-characterized pattern of gene activation in the human alpha- and beta-globin gene clusters during development, we considered the possibility that the relative expression of these two alpha-globin loci might be developmentally controlled. Analysis of human embryonic and early fetal erythroid RNA samples confirmed this possibility; levels of mRNA encoded by the two alpha-globin loci are equal in the embryo and subsequently shift to dominant expression of the alpha 2-globin locus at week 8 in utero. In transgenic mice carrying the entire human alpha-globin cluster (except for the theta gene) we show the same shift from equal expression of the alpha 1- and alpha 2-globin loci at the embryonic stage to predominance of the alpha 2-globin locus in the adult. These data demonstrate a switch in the expression of the two adjacent alpha-globin genes during the embryonic-to-fetal switch in erythroid development and provide an experimental system for its further characterization.

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Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3786 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3786-3794 ◽

Cited By ~ 14

Author(s):

M Albitar ◽

M Katsumata ◽

S A Liebhaber

Keyword(s):

Transgenic Mice ◽

Control Region ◽

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Developmental Control ◽

Beta Globin Gene ◽

Human Adult ◽

Alpha Globin ◽

In Cis

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

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Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3786-3794.1991 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3786-3794

Author(s):

M Albitar ◽

M Katsumata ◽

S A Liebhaber

Keyword(s):

Transgenic Mice ◽

Control Region ◽

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Developmental Control ◽

Beta Globin Gene ◽

Human Adult ◽

Alpha Globin ◽

In Cis

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Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.7.11.4024-4029.1987 ◽

1987 ◽

Vol 7 (11) ◽

pp. 4024-4029

Author(s):

M Trudel ◽

J Magram ◽

L Bruckner ◽

F Costantini

Keyword(s):

Transgenic Mice ◽

Fusion Gene ◽

Globin Gene ◽

Regulatory Elements ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Base Pairs ◽

Beta Globin Gene ◽

Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

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A silencer element from the alpha-globin gene inhibits expression of beta-like genes

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.5047-5051.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 5047-5051

Author(s):

G F Atweh ◽

J M Liu ◽

H E Brickner ◽

X X Zhu

Keyword(s):

Globin Gene ◽

Expression System ◽

Globin Genes ◽

Beta Globin ◽

Base Pair Fragment ◽

Alpha Globin ◽

In Trans ◽

Cis And Trans ◽

In Cis ◽

Silencer Element

We have studied the cis and trans interactions of the alpha- and beta-globin genes in a transient expression system. We found that the alpha-globin gene inhibited beta-globin expression in cis but not in trans. The silencer element responsible for this inhibition was localized to a 259-base-pair fragment at the 5' end of the alpha-globin gene.

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An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age

Blood ◽

10.1182/blood.v85.4.1111.bloodjournal8541111 ◽

1995 ◽

Vol 85 (4) ◽

pp. 1111-1117 ◽

Cited By ~ 79

Author(s):

YC Chang ◽

KD Smith ◽

RD Moore ◽

GR Serjeant ◽

GJ Dover

Keyword(s):

Sickle Cell ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Beta Globin ◽

Gene Number ◽

Cohort Group ◽

Five Factors ◽

F Cells ◽

Alpha Globin

Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < .05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40% of Hb F variation. beta-Globin haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).

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Expression of the chicken beta-globin gene cluster in mice: correct developmental expression and distributed control.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.407 ◽

1995 ◽

Vol 15 (1) ◽

pp. 407-414 ◽

Cited By ~ 29

Author(s):

M M Mason ◽

E Lee ◽

H Westphal ◽

M Reitman

Keyword(s):

Developmental Stage ◽

Globin Gene ◽

Stage Iv ◽

Gene Clusters ◽

Developmental Expression ◽

Single Element ◽

Globin Genes ◽

Beta Globin ◽

Variable Expression ◽

Hypersensitive Sites

To investigate the regulation of gene clusters, we introduced the entire chicken beta-globin cluster into mice. This 35-kb region includes the four globin genes (rho-beta H-beta A-epsilon), the four upstream hypersensitive sites, and the intergenic beta A/epsilon enhancer. The chicken globins are not arranged in order of developmental expression, which is unlike the case for the human beta-globin cluster, in which gene order plays a role in the regulation of globin expression. Mice carrying the chicken cluster expressed the transgenes with the same developmental patterns as seen in the chicken. Therefore, stage-specific erythroid transcriptional milieus existed before the divergence of birds and mammals and have been conserved since then. Mice bearing the complete cluster except for a deletion removing the beta A/epsilon enhancer displayed markedly reduced expression of the beta H, beta A, and epsilon genes with efficient (but variable) rho expression. Mice carrying the four genes and beta A/epsilon enhancer but without the upstream hypersensitive sites showed reduced expression of rho, beta H, and beta A, with variable expression of epsilon. We conclude that (i) all of the genes (except possibly rho) are under the control of both the upstream hypersensitive sites and the enhancer, (ii) the influence of the control elements can extend beyond the nearest active gene, (iii) a single element (the enhancer) can influence more than one gene in a single developmental stage, (iv) the enhancer can work bidirectionally, and (v) neither the upstream sites (as a group) nor the enhancer showed developmental stage specificity. Thus, the regulation of this cluster is achieved by interaction of two distinct control regions with each of the globin genes.

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The detection and use of hemoglobin mutants in the direct analysis of human globin genes

Blood ◽

10.1182/blood.v55.6.1060.1060 ◽

1980 ◽

Vol 55 (6) ◽

pp. 1060-1062 ◽

Cited By ~ 6

Author(s):

PF Little ◽

E Whitelaw ◽

G Annison ◽

R Williamson ◽

JM Kooter ◽

...

Keyword(s):

Restriction Site ◽

Globin Gene ◽

Direct Analysis ◽

Globin Genes ◽

Beta Globin ◽

Gene Sequences ◽

Gamma Globin ◽

Alpha Globin ◽

Specific Restriction ◽

Alpha Beta

Abstract Many human globin-chain mutants contain amino acid replacements that result from single base changes in the corresponding globin gene. Using recombinants, the coding sequences of each of the alpha-, beta-, Ggamma- , and Agamma-globin genes have now been determined. Those sequences of DNA that are cleaved by a number of specific restriction endonucleases have been identified and accurately positioned. Mutations at these sequences abolish the restriction site, and therefore, the pattern of DNA fragments containing hybridizing globin-gene sequences is altered compared to DNA from normal persons. This allows the identification of one of a pair of cross-hybridizing human globin-gene sequences, as is shown here for the two alpha-globin, the two gamma-globin, and the delta- and beta-globin genes.

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Alpha-thalassemia caused by a large (62 kb) deletion upstream of the human alpha globin gene cluster

Blood ◽

10.1182/blood.v76.1.221.bloodjournal761221 ◽

1990 ◽

Vol 76 (1) ◽

pp. 221-227

Author(s):

CS Hatton ◽

AO Wilkie ◽

HC Drysdale ◽

WG Wood ◽

MA Vickers ◽

...

Keyword(s):

Sequence Analysis ◽

Dna Sequence ◽

Interspecific Hybrid ◽

Globin Gene ◽

Regulatory Sequences ◽

Globin Genes ◽

Beta Globin ◽

Alpha Thalassemia ◽

Alpha Globin ◽

Globin Synthesis

We describe a family in which alpha-thalassemia occurs in association with a deletion of 62 kilobases from a region upstream of the alpha globin genes. DNA sequence analysis has shown that the transcription units of both alpha genes downstream of this deletion are normal. Nevertheless, they fail to direct alpha globin synthesis in an interspecific hybrid containing the abnormal (alpha alpha)RA chromosome. It seems probable that previously unidentified positive regulatory sequences analogous to those detected in a corresponding position of the human beta globin cluster are removed by this deletion.

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Influence of Globin Gene Modifiers on Baseline and Hydroxyurea-Induced Fetal Hemoglobin Levels in Children with Sickle Cell Anemia.

Blood ◽

10.1182/blood.v106.11.3171.3171 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3171-3171

Author(s):

Russell E. Ware ◽

Barry Eggleston ◽

Tatiana Abramova ◽

Sherri A. Zimmerman ◽

Alice Lail ◽

...

Keyword(s):

Sickle Cell ◽

Sickle Cell Anemia ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Globin Genes ◽

Beta Globin ◽

Gene Number ◽

Gamma Globin ◽

Alpha Globin ◽

Hydroxyurea Therapy

Abstract Fetal hemoglobin (HbF) is recognized as a major determinant of clinical disease severity in children and adults with sickle cell anemia (SCA). Patients with elevated HbF levels have a milder disease course, and many current therapeutic protocols for SCA include pharmacological induction of HbF. However, baseline and treatment HbF levels vary widely due to presumed genetic and environmental factors. Recognized globin gene modifiers of HbF include the beta globin haplotype and a potential contribution from concomitant alpha thalassemia. To characterize more fully the influence of globin gene modifiers on both baseline and treatment HbF levels, we retrospectively determined the beta globin haplotype (Benin, CAR, Senegal, Cameroon, or Arab-Indian) by selective gamma globin gene nucleotide sequencing and the alpha globin gene number (2, 3, or 4) by PCR for 67 African-American children with SCA receiving hydroxyurea therapy at stable maximal tolerated dose (MTD). The four beta globin haplotypes and frequencies identified in our cohort of children include Benin (0.61), CAR (0.17), Senegal (0.12), and Cameroon (0.10). The number of alpha globin genes and frequencies identified were 4 genes (0.72), 3 genes (0.25) and 2 genes (0.03). Baseline and MTD HbF levels were analyzed according to each variable. The average baseline HbF value for the entire cohort of children was 7.7 ± 4.4% (median 7.6%, range 1.3 – 19.3%), while the average treatment HbF value was 23.9 ± 7.2 % (median 22.9%, range 10.2 – 40.7%). All 67 children increased their HbF in response to hydroxyurea therapy (median 16.7%, range 5.0 – 28.8%). There was a modest but statistically significant correlation between the baseline and treatment HbF (r=0.66, p<.0001). The estimated effect of one unit change in baseline HbF on treatment HbF was 1.11 (95% CI of 0.78, 1.43). When baseline %HbF was analyzed according to the beta globin haplotype, the overall ANOVA had a p-value of 0.02, indicating a statistically significant influence. Further analysis confirmed associations previously identified in adults with SCA, i.e. children with at least one copy of the CAR haplotype had a lower baseline HbF (5.9% vs 8.4%, p=.05), while those with at least one copy of the Senegal haplotype had a higher baseline HbF (11.1% vs 6.7%, p<.001). When hydroxyurea MTD (treatment) HbF values were analyzed according to beta globin haplotype while adjusting for baseline HbF, however, the effect of beta globin haplotype was not statistically significant (p=.13). Analyses of HbF according to alpha globin gene number revealed no statistically significant effects on either baseline or treatment HbF values. Taken together, these data support the hypothesis that beta globin haplotype significant influences baseline HbF values for children with SCA, but has no significant effects on hydroxyurea MTD HbF values. Accordingly, children with SCA should be offered hydroxyurea based solely on clinical indications, without consideration of baseline HbF or beta globin haplotype. Even children with low baseline HbF values or the CAR beta globin haplotype can respond to hydroxyurea therapy with an elevated %HbF. Future studies designed to identify genetic modifiers of treatment HbF values should focus on sequence polymorphisms in non-globin genes that have trans-acting effects on gamma globin gene expression.

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