Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.

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In situ hybridization reveals co-expression of embryonic and adult alpha globin genes in the earliest murine erythrocyte progenitors

Development ◽

10.1242/dev.116.4.1041 ◽

1992 ◽

Vol 116 (4) ◽

pp. 1041-1049 ◽

Cited By ~ 2

Author(s):

A. Leder ◽

A. Kuo ◽

M.M. Shen ◽

P. Leder

Keyword(s):

In Situ Hybridization ◽

Embryonic Development ◽

Fetal Liver ◽

Globin Gene ◽

Yolk Sac ◽

Globin Genes ◽

Globin Mrna ◽

Mature Adult ◽

Alpha Globin

Murine erythropoiesis begins with the formation of primitive red blood cells in the blood islands of the embryonic yolk sac on day 7.5 of gestation. By analogy to human erythropoiesis, it has been thought that there is a gradual switch from the exclusive expression of the embryonic alpha-like globin (zeta) to the mature adult form (alpha) in these early mouse cells. We have used in situ hybridization to assess expression of these two globin genes during embryonic development. In contrast to what might have been expected, we find that there is simultaneous expression of both zeta and alpha genes from the very onset of erythropoiesis in the yolk sac. At no time could we detect expression of embryonic zeta globin mRNA without concomitant expression of adult alpha globin mRNA. Indeed, adult alpha transcripts exceed those of embryonic zeta in the earliest red cell precursors. Moreover, the pattern of hybridization reveals co-expression of both genes within the same cells. Even in the fetal liver, which supersedes the yolk sac as the major site of murine fetal erythropoiesis, there is a brief co-expression of zeta and alpha genes followed by the exclusive expression of the adult alpha genes. These data indicate an important difference in hematopoietic ontogeny between mouse and that of human, where zeta expression precedes that of alpha. In addition to resolving the embryonic expression of these globin genes, our results suggest that the embryonic alpha-like globin gene zeta may be physiologically redundant, even during the earliest stages of embryonic development.

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Methylation of α-type embryonic globin gene απrepresses transcription in primary erythroid cells

Blood ◽

10.1182/blood-2002-02-0457 ◽

2002 ◽

Vol 100 (12) ◽

pp. 4217-4222 ◽

Cited By ~ 22

Author(s):

Rakesh Singal ◽

Jane M. vanWert ◽

Larry Ferdinand

Keyword(s):

Globin Gene ◽

Gene Promoter ◽

Luciferase Reporter ◽

Globin Genes ◽

Erythroid Cells ◽

Histone H4 ◽

Luciferase Reporter Gene ◽

Cpg Dinucleotides ◽

Expression Construct ◽

Globin Promoter

The inverse relationship between expression and methylation of β-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian α-type globin genes. The embryonicαπ-globin promoter was unmethylated, andαπ-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the απ promoter associated with loss of expression of απ-globin gene was seen during development in primary erythroid cells. A 315-bpαπ-globin promoter region was cloned in an expression construct (απpGL3E) containing a luciferase reporter gene and SV40 enhancer. The απpGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of απpGL3E plasmid andαπ-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bpαπ-globin gene promoter fragment formed amethyl cytosine-binding proteincomplex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with theαπ-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian α-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.

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“Definitive” Erythropoiesis Has Distinct Developmental Origins and Globin Expression Patterns in the Mammalian Embryo.

Blood ◽

10.1182/blood.v114.22.2539.2539 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2539-2539

Author(s):

Kathleen E. McGrath ◽

Jenna M Frame ◽

George Fromm ◽

Anne D Koniski ◽

Paul D Kingsley ◽

...

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Globin Gene ◽

Yolk Sac ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Low Levels ◽

Globin Gene Expression

Abstract Abstract 2539 Poster Board II-516 A transient wave of primitive erythropoiesis begins at embryonic day 7.5 (E7.5) in the mouse as yolk sac-derived primitive erythroid progenitors (EryP-CFC) generate precursors that mature in the circulation and expand in numbers until E12.5. A second wave of erythroid progenitors (BFU-E) originates in the yolk sac beginning at E8.25 that generate definitive erythroid cells in vitro. These BFU-E colonize the newly forming liver beginning at E10.5, prior to the initial appearance there of adult-repopulating hematopoietic stem cells (HSCs) between E11.5-12.5. This wave of definitive erythroid yolk sac progenitors is proposed to be the source of new blood cells required by the growing embryo after the expansion of primitive erythroid cells has ceased and before HSC-derived hematopoiesis can fulfill the erythropoietic needs of the embryo. We utilized multispectral imaging flow cytometry both to distinguish erythroid lineages and to define specific stages of erythroid precursor maturation in the mouse embryo. Consistent with this model, we found that small numbers of definitive erythrocytes first enter the embryonic circulation beginning at E11.5. All maturational stages of erythroid precursors were observed in the E11.5 liver, consistent with these first definitive erythrocytes having rapidly completed their maturation in the liver. The expression of βH1 and εy-beta globin genes is thought to be limited to primitive erythroid cells. Surprisingly, examination of globin gene expression by in situ hybridization revealed high levels of βH1-, but not εy-globin, transcripts in the parenchyma of E11.5-12.5 livers. RT-PCR analysis of globin mRNAs confirmed the expression of βH1- and adult β1-, but not εy-globin, in E11.5 liver-derived definitive (ckit+, Ter119lo) proerythroblasts sorted by flow cytometry to remove contaminating primitive (ckit-, Ter119+) erythroid cells. A similar pattern of globin gene expression was found in individual definitive erythroid colonies derived from E9.5 yolk sac and from early fetal liver. In vitro differentiation of definitive erythroid progenitors from E9.5 yolk sac revealed a maturational “switch” from βH1- and β1-globins to predominantly β1-globin. βH1-globin transcripts were not observed in proerythroblasts from bone marrow or E16.5 liver or in erythroid colonies from later fetal liver. ChIP analysis revealed that hyperacetylated domains encompass all beta globin genes in primitive erythroid cells but only the adult β1- and β2-globin genes in E16.5 liver proerythroblasts. Consistent with their unique gene expression, E11.5 liver proerythroblasts have hyperacetylated domains encompassing the βh1-, β1- and β2-, but not εy-globin genes. We also examined human globin transgene expression in mice carrying a single copy of the human beta globin locus. Because of the overlapping presence and changing proportion of primitive and definitive erythroid cells during development, we analyzed sorted cell populations whose identities were confirmed by murine globin gene expression. We confirmed that primitive erythroid cells express higher levels of γ- than ε-globin and little β-globin. E11.5 proerythroblasts and cultured E9.5 progenitors express γ- and β-, but not ε-globin. E16.5 liver proerythroblasts express β- and low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. In summary, two forms of definitive erythropoiesis emerge in the murine embryo, each with distinct globin expression patterns and chromatin modifications of the β-globin locus. While both lineages predominantly express adult globins, the first, yolk sac-derived lineage uniquely expresses low levels of the embryonic βH1-globin gene as well as the human γ-globin transgene. The second definitive erythroid lineage, found in the later fetal liver and postnatal marrow, expresses only adult murine globins as well as low levels of the human γ-globin transgene only in the fetus. Our studies reveal a surprising complexity to the ontogeny of erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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Developmental stage–specific epigenetic control of human β-globin gene expression is potentiated in hematopoietic progenitor cells prior to their transcriptional activation

Blood ◽

10.1182/blood-2003-05-1540 ◽

2003 ◽

Vol 102 (12) ◽

pp. 3989-3997 ◽

Cited By ~ 45

Author(s):

Stefania Bottardi ◽

Angélique Aumont ◽

Frank Grosveld ◽

Eric Milot

Keyword(s):

Transgenic Mice ◽

Progenitor Cells ◽

Globin Gene ◽

Histone H3 ◽

Hematopoietic Progenitor Cells ◽

Globin Genes ◽

Erythroid Cells ◽

Hematopoietic Progenitor ◽

Epigenetic Control ◽

In Erythroid Cells

Abstract To study epigenetic regulation of the human β-globin locus during hematopoiesis, we investigated patterns of histone modification and chromatin accessibility along this locus in hematopoietic progenitor cells (HPCs) derived from both humans and transgenic mice. We demonstrate that the developmentally related activation of human β-like globin genes in humans and transgenic mice HPCs is preceded by a wave of gene-specific histone H3 hyperacetylation and K4 dimethylation. In erythroid cells, expression of β-like globin genes is associated with histone hyperacetylation along these genes and, surprisingly, with local deacetylation at active promoters. We also show that endogenous mouse β major and human β-like genes are subject to different epigenetic control mechanisms in HPCs. This difference is likely due to intrinsic properties of the human β-globin locus since, in transgenic mice, this locus is epigenetically regulated in the same manner as in human HPCs. Our results suggest that a defined pattern of histone H3 acetylation/dimethylation is important for specific activation of human globin promoters during development in human and transgenic HPCs. We propose that this transient acetylation/dimethylation is involved in gene-specific potentiation in HPCs (ie, before extensive chromatin remodeling and transcription take place in erythroid cells).

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In Vivo and In Vitro Analysis of an Aγ-Globin Gene Silencer in Human β-Yac Transgenic Mice.

Blood ◽

10.1182/blood.v104.11.1220.1220 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1220-1220

Author(s):

Susanna Harju ◽

Kenneth R. Peterson

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Fetal Liver ◽

Globin Gene ◽

Yolk Sac ◽

Luciferase Reporter ◽

Globin Genes ◽

Mobility Shift ◽

Gene Switching ◽

Globin Gene Expression

Abstract Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching and is best exemplified by the ε-globin gene. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between −730 to −378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A 5.4 Kb marked Aγ-globin gene (Aγm) inserted between LCR 5′HS1 and the ε-globin gene in a β-YAC (Aγm 5′ ε β-YAC) was silenced in transgenic mice during adult definitive erythropoiesis, even in the absence of an adult β-globin gene. In contrast, when a marked β-globin gene (βm) was inserted in this same location in another β-YAC (βm 5′ ε β-YAC), the βm-globin gene was expressed throughout ontogeny. From these data we concluded that: 1) any gene located near the LCR will be strongly expressed throughout ontogeny, unless some gene-specific silencing mechanism exists, 2) competition between the γ- and β-globin genes for interaction with the LCR is not the exclusive mechanism controlling γ- to β-globin gene switching, and 3) that the Aγm-globin gene was autonomously silenced. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC via homologous recombination to produce a Δ1s Aγm 5′ ε β-YAC. This YAC was microinjected and six founders were obtained. Four transgenic lines were established carrying at least one full-length β-globin locus and two were established that lacked the adult β-globin gene. All founders containing an intact β-globin gene expressed the Δ1s Aγm-globin during adult erythropoiesis (45% – 122% relative to human β-globin expression). In one line examined in detail, the Δ1s Aγm-globin gene was expressed in the embryonic yolk sac, fetal liver, and adult blood. ε-globin gene expression was not detected in the embryonic yolk sac and expression of the normally located γ-globin genes was not observed at any developmental stage. β-globin gene expression was observed in the fetal liver and adult blood, although its expression was decreased. To further delineate the function of the Δ1s fragment, transient transfection assays to test silencer function and protein-DNA interaction assays were performed. Silencer activity of the352 bp Δ1s fragment was examined using a series of pGL2 luciferase reporter plasmids that were synthesized to include the Δ1s fragment; these were electroporated into various cells. Electrophoretic mobility shift assays (EMSAs) and DNAse I footprinting were employed to begin assessment of protein binding within the Δ1s fragment. A 50 bp DNA fragment spanning −713 to −664 of the Δ1s element was used in EMSAs; DNA binding activity was observed in K562 nuclear extracts. These preliminary data suggest that the −730 to −378 γ-globin gene silencer binds a repressor protein complex.

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Human β-Globin Locus Control Region Hypersensitive Site Specificity for Globin Gene Activation during Erythropoiesis.

Blood ◽

10.1182/blood.v106.11.3630.3630 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3630-3630

Author(s):

Kenneth R. Peterson ◽

Halyna Fedosyuk ◽

Susanna Harju

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Globin Gene ◽

Gene Activation ◽

Gene Promoter ◽

Yolk Sac ◽

Site Specificity ◽

Globin Genes ◽

Definitive Erythropoiesis ◽

Globin Gene Expression

Abstract Although the human β-globin locus control region (LCR) functions as a holocomplex within an active chromatin hub, we provide evidence that within the aggregate hypersensitive site (HS) activation domain of the holocomplex, the individual HSs still mediate preferential activation of the globin genes during development. A 2.9 Kb deletion of 5′HS3 (Δ5′HS3) or a 234 bp deletion of the 5′HS3 core (Δ5′HS3c) in a 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) abrogate ε-globin gene expression during primitive erythropoiesis in β-YAC transgenic mice, suggesting that 5′HS3 sequences of the LCR are involved directly in ε-globin gene activation. The reduction of ε-globin gene transcription in Δ5′HS3 or Δ5′HS3c β-YAC transgenics can be explained by two hypotheses. The first is site-specificity. The interaction between the LCR and the ε-globin gene promoter involves specific sequences of 5′HS3 and specific sequences of the ε-globin gene promoter. When 5′HS3 or its core is deleted, these interactions do not take place and ε-globin gene transcription is diminished. The second hypothesis is change in conformation of the LCR. Normally, in the embryonic stage, the LCR achieves a three-dimensional conformation that favors interaction with the first gene in the complex, the ε-globin gene. When 5′HS3 is deleted, an alternate conformation is assumed that decreases the chance that there will be an interaction between the LCR and the ε-globin gene. However, the LCR interacts with the next gene in order, the γ-globin gene. In Δ5′HS3c β-YAC mice, γ-globin gene expression is normal during primitive erythropoiesis, but is extinguished in the fetal stage of definitive erythropoiesis. These data suggest that a conformational change occurs in the Δ5′HS3c LCR during the switch from embryonic to definitive erythropoiesis, from one that supports γ-globin gene expression to one that does not. Alternately, the embryonic trans-acting environment may allow the mutant LCR to interact with and activate the γ-globin genes, but the fetal trans-acting environment may not support this interaction in the absence of the 5′HS3 core. To distinguish between these possibilities, β-YAC lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (βm), coupled to either an intact LCR, a 2.9 Kb 5′HS3 deletion or a 234 bp 5′HS3 core deletion. Δ5′HS3c Δε::βm β-YAC mice expressed βm-globin throughout development beginning at day 10 in the yolk sac. γ-globin was expressed in the embryonic yolk sac, but not in the fetal liver. Some wild-type β-globin was expressed in addition to βm-globin in adult mice. The γ-globin phenotype is consistent with published data on Δ5′HS3c β-YAC mice. Although ε-globin was not expressed in Δ5′HS3c β-YAC mice, βm-globin was expressed in Δ5′HS3c Δε::βm β-YAC embryos, demonstrating that the 5′HS3 core was necessary for ε-globin expression during embryonic erythropoiesis, but not for βm-globin expression. A similar phenotype was observed in Δ5′HS3 Δε::βm β-YAC mice, except βm-globin expression was higher in the day 10 yolk sac and γ-globin expression continued into the fetal liver stage of definitive erythropoiesis consistent with results published on Δ5′HS3 β-YAC mice. These data support a site specificity model of LCR HS-globin gene interaction.

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Statistical Evidence for the Existence of a Regulatory Mechanism That Balances α-Globin and AHSP Expression in Erythroid Cells.

Blood ◽

10.1182/blood.v110.11.1775.1775 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1775-1775

Author(s):

Maria Elena Fabucci ◽

Katija Jelicic ◽

Elena Alfani ◽

Anna Rita F. Migliaccio

Keyword(s):

Gene Expression ◽

Regulatory Mechanism ◽

Globin Gene ◽

Statistical Analyses ◽

Globin Genes ◽

Erythroid Cells ◽

Globin Mrna ◽

Donor Variability ◽

In Erythroid Cells

Abstract Alpha haemoglobin stabilizing protein (AHSP) is encoded by a gene abundantly expressed in erythroid cells whose function is to chaperone α-chains in the process of haemoglobin assembly (Yu et al, JCI2007; 117:1856). The central role of the excess of α-chains in the pathogenesis of β-thalassemia and the AHSP ability to limit the toxicity of excessive α-globin suggest that increases of AHSP expression might ameliorate the clinical phenotype of β-thalassemia. To clarify the relationship between AHSP and globin gene expression, we measured the levels of mRNA for these genes in erythroblasts generated in vitro from the blood of 30 normal donors and 8 β-thalassemic patients. Normal erythroblasts presented a marked donor-to-donor variability in the expression levels of all the genes analysed. Inter-quartile range (IQR) analyses indicates that the gene whose expression has the highest variability is α-globin (IQR=31.5), followed by β-globin (IQR=8.74), AHSP (IQR=2.82) and γ-globin (IQR=0.86). The IQR value for the α/non-α globin ratio (1.91) is higher than that of the γ/γ+β ratio (0.11), an indication of the existence of donor variegation in the levels of unbalance between expression of α- and non-α globin genes in cells from different donors. The extent of this variegation is even more apparent by the high IQR level of the α-(non-α) expression difference (IQR=38.6). β-thalassemic erythroblasts expressed normal levels of α- and γ-globin, significantly (P<.05) lower levels of β-globin mRNA and, surprisingly, high levels (by 10-fold) of AHSP mRNA. Subject-variability in gene expression was also observed for β-thalassemic erythroblasts. In this case, the gene whose expression had the highest variability is AHSP (IQR=42.8), followed by α-globin (IQR=11.75), β-globin (IQR=3.32), and γ-globin (IQR=1.74). The IQR for the α/non-α globin ratio (7.2) is higher than that of the γ/γ+β ratio (0.67) also for β-thalassemic erythroblasts. The difference between the variances of the excess of α-expression [α-(non-α)] in β-thalassemic and normal erythroblasts is significant by F test (P=.0023). Statistical analyses of these results indicates that, as expected, the levels of α-globin mRNA are positively correlated to those of the non-α globin genes in normal erythroblasts (R2=.93, P<.001) but not in β-thalassemic cells (R2=.22, P<.24). In contrast, the levels of α-globin mRNA are positively correlated with those of AHSP both in normal (R2=.86, P<.0001) and β-thalassemic (R2=.66, P<.05) erythroblasts. Moreover, in spite of the fact that expression of α-globin is correlated, at least in normal erythroblasts, with that of γ+β mRNA, no correlation is found between levels of AHSP mRNA and those of γ+β mRNA. No correlation is also observed between levels of AHSP mRNA and the α/non-α ratio. In contrast, the levels of AHSP mRNA are correlated with the levels of excess of α-globin mRNA in normal erythroblasts (R2=0.86, P<.0001) and the fact that are not correlated in β-thalassemic cells (R2=.45, P=.066) might be due to the limited experimental points available for analyses. In conclusion, this statistical analyses provides evidence for the existence of a regulatory mechanism that balances expression of AHSP with that of excess of α-globin mRNA in erythroid cells. It is suggested that this regulatory mechanism may represent a target for eventual gene modifiers of the β-thalassemic trait. MEF is the recipient of a Marie Curie training Network Fellowship from EU.

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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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Structural and Functional Cross-Talk between a Distant Enhancer and the ɛ-Globin Gene Promoter Shows Interdependence of the Two Elements in Chromatin

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7600 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7600-7609 ◽

Cited By ~ 17

Author(s):

Jennifer C. McDowell ◽

Ann Dean

Keyword(s):

Globin Gene ◽

Gene Promoter ◽

Transcription Activation ◽

Tata Box ◽

Globin Genes ◽

Hypersensitive Site ◽

Nuclease Digestion ◽

Dnase I Hypersensitive Site ◽

Globin Promoter ◽

Gata Motif

ABSTRACT We investigated the requirements for enhancer-promoter communication by using the human β-globin locus control region (LCR) DNase I-hypersensitive site 2 (HS2) enhancer and the ɛ-globin gene in chromatinized minichromosomes in erythroid cells. Activation of globin genes during development is accompanied by localized alterations of chromatin structure, and CACCC binding factors and GATA-1, which interact with both globin promoters and the LCR, are believed to be critical for globin gene transcription activation. We found that an HS2 element mutated in its GATA motif failed to remodel the ɛ-globin promoter or activate transcription yet HS2 nuclease accessibility did not change. Accessibility and transcription were reduced at promoters with mutated GATA-1 or CACCC sites. Strikingly, these mutations also resulted in reduced accessibility at HS2. In the absence of a globin gene, HS2 is similarly resistant to nuclease digestion. In contrast to observations in Saccharomyces cerevisiae, HS2-dependent promoter remodeling was diminished when we mutated the TATA box, crippling transcription. This mutation also reduced HS2 accessibility. The results indicate that the ɛ-globin promoter and HS2 interact both structurally and functionally and that both upstream activators and the basal transcription apparatus contribute to the interaction. Further, at least in this instance, transcription activation and promoter remodeling by a distant enhancer are not separable.

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Developmental- and differentiation-specific patterns of human γ- and β-globin promoter DNA methylation

Blood ◽

10.1182/blood-2007-01-068635 ◽

2007 ◽

Vol 110 (4) ◽

pp. 1343-1352 ◽

Cited By ~ 43

Author(s):

Rodwell Mabaera ◽

Christine A. Richardson ◽

Kristin Johnson ◽

Mei Hsu ◽

Steven Fiering ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Methylation ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Cells ◽

Promoter Dna Methylation ◽

Globin Promoter ◽

Promoter Dna

AbstractThe mechanisms underlying the human fetal-to-adult β-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human γ- and β-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at −162 of the γ promoter and −126 of the β promoter are hypomethylated in ABM and FL, respectively. We also studied γ-globin promoter methylation during in vitro differentiation of erythroid cells. The γ promoters are initially hypermethylated in CD34+ cells. The upstream γ promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient γ-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human γ- and β-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human β-globin locus gene switching.

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