scholarly journals Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 861-864 ◽  
Author(s):  
N Raich ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos ◽  
T Enver
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Negative Element ◽  
Cis Acting

The human epsilon-globin gene displays normal developmental regulation in transgenic mice; it is expressed only in embryonic and in definitive erythroid cells. We show here that deletion of a negative element located between -182 and -467 bp upstream of the epsilon-globin gene cap site results in continuation of epsilon gene expression in the definitive erythroblasts of the fetal liver and in the red blood cells of adult transgenic mice. These data provide direct in vivo evidence that cis acting silencing elements are involved in the developmental control of the epsilon-globin gene.

scholarly journals Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 861-864 ◽  
Author(s):  
N Raich ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos ◽  
T Enver
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Negative Element ◽  
Cis Acting

Abstract The human epsilon-globin gene displays normal developmental regulation in transgenic mice; it is expressed only in embryonic and in definitive erythroid cells. We show here that deletion of a negative element located between -182 and -467 bp upstream of the epsilon-globin gene cap site results in continuation of epsilon gene expression in the definitive erythroblasts of the fetal liver and in the red blood cells of adult transgenic mice. These data provide direct in vivo evidence that cis acting silencing elements are involved in the developmental control of the epsilon-globin gene.

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 Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids.

1995 ◽  
Vol 15 (8) ◽  
pp. 3969-3978 ◽  
Author(s):  
S J Stanworth ◽  
N A Roberts ◽  
J A Sharpe ◽  
J A Sloane-Stanley ◽  
W G Wood
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Somatic Cell Hybrids ◽  
Stable Conformation ◽  
Cell Hybrids ◽  
Beta Gene

Somatic cell hybrids generated from transgenic mouse cells have been used to examine the developmental regulation of human gamma-to-beta-globin gene switching. In hybrids between mouse erythroleukemia (MEL) cells and transgenic erythroblasts taken at various stages of development, there was regulated expression of the human fetal gamma and adult beta genes, reproducing the in vivo pattern prior to fusion. Hybrids formed from embryonic blood cells produced predominantly gamma mRNA, whereas beta gene expression was observed in adult hybrids and a complete range of intermediate patterns was found in fetal liver hybrids. The adult environment of the MEL cells, therefore, did not appear to influence selective transcription from this gene complex. Irradiation of the embryonic erythroid cells prior to fusion resulted in hybrids containing only small fragments of donor chromosomes, but the pattern of gene expression did not differ from that of unirradiated hybrids. This finding suggests that continued expression of trans-acting factors from the donor erythroblasts is not necessary for continued expression of the human gamma gene in MEL cells. These results contrast with the lack of developmental regulation of the cluster after transfection of naked DNA into MEL cells and suggest that epigenetic processes established during normal development result in the gene cluster adopting a developmental stage-specific, stable conformation which is maintained through multiple rounds of replication and transcription in the MEL cell hybrids. On prolonged culture, hybrids that initially expressed only the gamma transgene switched to beta gene expression. The time period of switching, from approximately 10 to > 40 weeks, was similar to that seen previously in human fetal erythroblast x MEL cell hybrids but in this case bore no relationship to the time of in vivo switching. It seems unlikely, therefore, that switching in these hybrids is regulated by a developmental clock.

Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells

2021 ◽  
Author(s):  
Andrew D. Beale ◽  
Priya Crosby ◽  
Utham K. Valekunja ◽  
Rachel S. Edgar ◽  
Johanna E. Chesham ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Human Subjects ◽  
Developmental Regulation ◽  
Physiological Role ◽  
Cell Types ◽  
The Body

AbstractCellular circadian rhythms confer daily temporal organisation upon behaviour and physiology that is fundamental to human health and disease. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body. Being naturally anucleate, RBC circadian rhythms share key elements of post-translational, but not transcriptional, regulation with other cell types. The physiological function and developmental regulation of RBC circadian rhythms is poorly understood, however, partly due to the small number of appropriate techniques available. Here, we extend the RBC circadian toolkit with a novel biochemical assay for haemoglobin oxidation status, termed “Bloody Blotting”. Our approach relies on a redox-sensitive covalent haem-haemoglobin linkage that forms during cell lysis. Formation of this linkage exhibits daily rhythms in vitro, which are unaffected by mutations that affect the timing of circadian rhythms in nucleated cells. In vivo, haemoglobin oxidation rhythms demonstrate daily variation in the oxygen-carrying and nitrite reductase capacity of the blood, and are seen in human subjects under controlled laboratory conditions as well as in freely-behaving humans. These results extend our molecular understanding of RBC circadian rhythms and suggest they serve an important physiological role in gas transport.

Changes in Globin Gene Methylation and Covalent Histone Modifications of Chromatin Associated with the ε-, γ-, and β-Globin Promoters of the Baboon (P. Anubis) during Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1206-1206
Author(s):  
Donald Lavelle ◽  
Kestas Vaitkus ◽  
Maria Hankewych ◽  
Mahipal Singh ◽  
Joseph DeSimone
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Rna Pol Ii ◽  
Cpg Sites ◽  
Globin Promoter ◽  
Globin Gene Expression ◽  
Covalent Histone Modifications

Abstract The pattern of globin gene expression during development is conserved in all simian primates, but not in prosimians or other species. Therefore knowledge of the mechanisms regulating globin gene expression in animal models such as the baboon (P. anubis) is directly applicable to human. This investigation addressed the role of chromatin structure in developmental regulation of globin gene expression. DNA methylation of the ε- and γ-gene promoters and covalent histone modifications in chromatin associated with the ε- γ- and β-globin gene promoters have been investigated in 40d fetal primitive nucleated yolk sac-derived RBCs, and definitive erythroid precursor cells from fetal liver (40d to 56d), fetal BM (154d to 160d), and BM from phlebotomized adults. The methylation status of 3 CpG sites in the ε-globin promoter and 5 CpG sites in the γ-globin promoter was analyzed by sequencing 10 cloned PCR products of each sample following bisulfite modification. The ε-globin promoter was unmethylated in 40d primitive yolk-sac derived RBCs. Moderate methylation of the ε-globin promoter was observed in 40d fetal liver (33%: 50%) and was increased in fetal liver samples obtained 2 weeks later in gestation (54d: 76.6%, 56d: 79.1%) to levels observed in late term fetal BM ( 154d: 80%, 156d: 96.6%, 160 d: 93.1%) and adult BM (84.1%; n=2). Methylation of the γ-globin promoter was lowest in 40d primitive RBC (0%) and early fetal liver (40d: 3.1%, 54d: 0%, 56d: 7.1%) and was moderately increased in fetal BM (154d: 38.6%, 156d: 20%, 160d: 30%) compared to adult BM ( 67.3%; n=3). Levels of ac-H3, ac-H4, dimethyl H3 lys4 (H3-dimeK4), dimethyl H3 lys79 (H3-meK79), dimethyl H3 lys36 (H3-meK36), and RNA pol II bound to the ε-, γ-, and β-globin promoters were determined by immunoprecipitation of formaldehyde-fixed, sheared chromatin (ChIP) followed by real time PCR. The amount of RNA pol II, ac-H3, and ac-H4 associated with each globin promoter correlated with developmental-specific gene expression and differed from the pattern of H3-meK79 and H3-meK4 associated with these promoters during development. The amount of H3-meK79 and H3-dimeK4 bound to the the ε- and γ-globin promoters in 40d primitive RBC and fetal liver erythroid precursors (54 and 56d) was 5 times greater than to the β-globin promoter, while similar levels of each (< 2 fold difference) were associated with all three promoters in fetal and adult bone marrow cells. In contrast, the highest level of H3-meK36 was associated with developmentally silenced genes. The amount of H3-meK36 bound to the ε promoter was 2–3 fold higher than to the γ and β promoters in fetal liver (54 and 56d). Similar levels (<2 fold difference) of H3-meK36 were associated with the γ and ε promoters in late term fetal and adult BM and were 2–6 fold greater than bound to the β promoter. We conclude that the chromatin cofiguration of the β-globin locus undergoes distinctive changes associated with both gene activation and silencing during development. Changes in the levels of H3-dimeK4 and H3-meK79 may reflect generalized domain opening, while high levels of ac-H3 and ac-H4 are bound to the promoters of activated genes. In contrast, gene silencing is correlated with increased DNA methylation and enrichment of H3-meK36 bound to the promoters. Thus the baboon model offers unique opportunities to study developmental regulation of globin gene expression.

The -566 Aγ-Globin HPFH Mutation Disrupts Temporal Repression of Fetal Hemoglobin Synthesis During Fetal Liver Definitive Erythropoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2019-2019
Author(s):  
Kenneth R Peterson ◽  
Halyna Fedosyuk ◽  
Flavia C Costa
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Wild Type ◽  
Repressor Complex ◽  
Definitive Erythropoiesis ◽  
Gata Motif ◽  
Globin Gene Expression

Abstract Abstract 2019 Poster Board I-1041 Hereditary persistence of fetal hemoglobin (HPFH) is a condition associated with continued fetal hemoglobin (HbF) production in adults, where normally only very low levels of HbF are found. Sickle cell disease (SCD) patients are phenotypically normal if they carry a compensatory HPFH mutation due to the high levels of HbF. Understanding the molecular mechanisms leading to reactivation or derepression of γ-globin gene expression will lead to the development of new or better therapies to treat SCD patients. In our long-established and highly-characterized model system, transgenic mice carrying wild-type human β-globin locus yeast artificial chromosomes (β-YACs) express predominantly γ-globin and a lesser amount of γ-globin in the primitive erythroid cells of the yolk sac, mostly β-globin and some γ-globin in the definitive erythroid cells of the fetal liver and nearly exclusively β-globin in the adult definitive red blood cells, as measured both at the transcript and protein levels. We recently identified a novel Aγ-globin gene silencer motif located at -566 relative to the mRNA CAP site in a GATA motif. Repression is mediated by binding a GATA-1-FOG-1-Mi2 protein complex. Since our initial studies of this GATA-1 repressor complex were performed using β-YAC transgenic mice in which a second copy of the Aγ-globin gene was introduced between the locus control region (LCR) and the γ-globin gene, our first goal was to test if this mutation was functional at the normally-located Aγ-globin globin gene. β-YAC transgenic mice were produced with the T>G HPFH point mutation at the -566 GATA site of this gene. These mice display a mild HPFH phenotype during adult definitive erythropoiesis; γ-globin gene expression levels were increased approximately 3% compared to wild-type β-YAC mice. Expression of γ-globin is also elevated relative to wild-type β-YAC controls during primitive erythropoiesis in the embryonic yolk sac and definitive erythropoiesis in the fetal liver. Chromatin immunoprecipitation (ChIP) experiments using day E12 to E18 post-conception fetal liver samples from wild type β-YAC transgenic mice demonstrate that GATA-1 is recruited to this GATA silencer first at day E16, followed by recruitment of FOG-1 and Mi2 at day E17. In addition, ChIP experiments performed with day E18 samples from the -566 HPFH mice demonstrate that this point mutation disrupts the recruitment of GATA-1 to this site at a developmental stage when it normally binds as a repressor in wild-type β-YAC transgenic samples. GATA-2 does not bind at the -566 GATA motif when γ-globin is actively transcribed. Thus, GATA-2/GATA-1 competition does not play a role in the function of this silencer or the mechanism of HPFH at this site. In addition, BCL11A does not appear to be a component of this GATA-1 repressor complex. Taken together our data indicate that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that the presence of the -566 Aγ-globin HPFH mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

Lapdesf 1 a Compound Hybrid of Hydroxyrea and Thalidomide Increases Gamma Globin Expression in the Human CD34+ Cultures and Reduces Chemotactic Activity in Neutrophils

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2124-2124
Author(s):  
Sheley Gambero ◽  
Carolina Lanaro ◽  
Flavia Rubia Pallis ◽  
Carla Fernanda Franco-Penteado ◽  
Lidia Moreira Lima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Cell Cultures ◽  
Blood Cells ◽  
Mononuclear Cells ◽  
Globin Gene ◽  
Chemotactic Activity ◽  
Significant Difference ◽  
Globin Gene Expression

Abstract Abstract 2124 Sickle cell anemia (SCA), a disorder in which the inheritance of the gene codifies abnormal hemoglobin S (HbS), leads to Hb polymerization, causing a series of cellular alterations in the red cell. Fetal hemoglobin (HbF) is a modulator of clinical and hematologic features. Higher HbF levels are associated with a reduced rate of acute painful episodes, less frequent acute chest syndromes and protection against morbidity and mortality. Previous results showed that Lapdesf1(2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate, a novel compound that links hydroxyurea and thalidomide's phtalimide, increases HbF and has an anti-inflammatory effect in sickle mice. The aim of this study to evaluate whether Lapdesf1 induces HbF gene expression in CD34+ cell cultures. We also evaluated the chemotaxis and production of reactive oxygen species (ROS) in neutrophils treated in vitro with Lapdesf1.CD34+cell cultures from 8 healthy volunteers were treated with Lapdesf1 on day 9 and the gama-globin (γ-globin) gene expression was evaluated on day 13 by Real Time PCR. We used, as a positive control, Hydroxyurea (HU), thalidomide and both together. Neutrophils, platelets, mononuclear cells and red blood cells were isolated from peripheral blood samples of healthy controls and patients with SCA (Transfusion-independent patients, not on hydroxyurea treatment). ROS measurement was performed by the incubation with 2'-7'-dichlorofluorecin diacetate (DCFH) and analyzed by flow cytometry. Spontaneous and IL-8-induced neutrophils chemotaxis were assessed using a 96-well chemotaxis chamber assay (ChemoTXNeuroprobe). Data were analyzed statistically using ANOVA followed by Dunnett's test, where a P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethics Committee of the Faculty of Medical Sciences of University of Campinas (UNICAMP).Lapdesf1 (5μM) increased γ-globin gene expression, compared with that of the control (1.85 ± 0.54 vs 0.66 ± 0.16, P<0.05, n=8). HU and thalidomide also increased at 100μM (1.81 ± 0.13; 1.85 ± 0.06, P<0.05, n=3), however treatment with HU and thalidomide together did not increase γ-globin gene expression (0.99 ± 0.04, P<0.05, n=3). There was no significant difference in the ROS production in platelets, red blood cells, mononuclear cells and neutrophils, treated with Lapdesf1. In addition, no significant differences were observed in the chemotaxis of controls and SCA neutrophils. For IL-8-induced chemotaxis, treatment with Lapdesf1 reduced the chemotactic activity at 300 and 600μM (3.94 ± 1.17; 3.86 ± 1.06 respectivelyvs 21.76 ± 6.06, P<0.05, n=3) in controls and 300μM (4.96 ± 0.59 vs 17.54 ± 7.12, P<0.05, n=3) in SCA compared with IL-8 induced control. Our results showed that the Lapdesf1 is capable of inducing CD34+ cell γ-globin gene expression at a low concentration and reducing chemotactic activity. Even though further studies are needed, these results suggest that Lapdesf1 may be a promising drug candidate that may provide multiple beneficial actions in the treatment of sickle cell disease symptoms and offering an alternative drug therapy. This work was supported by FAPESP, INCT and CNPq. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes.

1990 ◽  
Vol 10 (3) ◽  
pp. 1116-1125 ◽  
Author(s):  
C Perez-Stable ◽  
F Costantini
Keyword(s):  
Blood Cells ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Upstream Region ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Cis Acting ◽  
Beta Globin Gene ◽  
Gamma Globin

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.

Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes

1990 ◽  
Vol 10 (3) ◽  
pp. 1116-1125
Author(s):  
C Perez-Stable ◽  
F Costantini
Keyword(s):  
Blood Cells ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Upstream Region ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Cis Acting ◽  
Beta Globin Gene ◽  
Gamma Globin

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.

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