Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes

1993 ◽  
Vol 13 (4) ◽  
pp. 2298-2308
Author(s):  
Q Zhang ◽  
P M Reddy ◽  
C Y Yu ◽  
C Bastiani ◽  
D Higgs ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Site Directed Mutagenesis ◽  
Erythroid Cell ◽  
Nuclear Factor ◽  
Sequence Motifs ◽  
Alpha Globin ◽  
Globin Promoter

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

scholarly journals Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes.

1993 ◽  
Vol 13 (4) ◽  
pp. 2298-2308 ◽  
Author(s):  
Q Zhang ◽  
P M Reddy ◽  
C Y Yu ◽  
C Bastiani ◽  
D Higgs ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Site Directed Mutagenesis ◽  
Erythroid Cell ◽  
Nuclear Factor ◽  
Sequence Motifs ◽  
Alpha Globin ◽  
Globin Promoter

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

scholarly journals Identification of a major positive regulatory element located 5' to the human zeta-globin gene

Blood ◽  
1995 ◽  
Vol 85 (9) ◽  
pp. 2587-2597 ◽  
Author(s):  
DE Sabath ◽  
KM Koehler ◽  
WQ Yang ◽  
K Patton ◽  
G Stamatoyannopoulos
Keyword(s):  
Point Mutation ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Luciferase Expression ◽  
Globin Mrna ◽  
Mobility Shift ◽  
Alpha Globin ◽  
Globin Promoter

The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5′ to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5′ to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.

Sequences Downstream of the Erythroid Promoter Promote High-Level Expression of the Human α-Spectrin Gene by Providing Boundary Activity and Positive Regulatory Elements.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1572-1572
Author(s):  
Patrick G. Gallagher ◽  
Douglas G. Nilson ◽  
Jolinta Lin ◽  
David M. Bodine
Keyword(s):  
Regulatory Element ◽  
Globin Gene ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Globin Mrna ◽  
Intron 1 ◽  
Exon 1 ◽  
Globin Promoter ◽  
High Level ◽  
Positive Regulatory Element

Abstract Characterization of the regulatory elements that control α-spectrin (ASp) gene expression is important for understanding the pathogenesis of ASp-linked hemolytic anemia. Our previous studies demonstrated that the ASp promoter directs low levels of expression, and, addition of a downstream region of noncoding exon 1 and intron 1 containing 2 GATA-1 sites conferred a 10-fold increase in activity in transient transfection assays. Transgenic (TG) mouse lines with the ASp promoter, the Asp promoter-exon 1-intron 1, or ASp promoter-exon 1-intron 1 with mutations of both splice sites linked to the human Aγ-globin gene as reporter were created. In reticultocytes, no expression was detected in any of the 8 lines transmitting the ASp promoter-Aγ-globin transgene. TG mice with the ASp promoter-exon1-intron 1 demonstrated significant levels of Aγ-globin gene expression in reticulocytes, with levels of Aγ-globin mRNA of ~0.4% of mouse α-globin mRNA/transgene copy #. This expression was nearly position independent, as 22/24 lines expressed the transgene. Using a FACS-based assay, γ-globin protein was present in 100% of erythrocytes. Expression levels comparable to the Asp promoter-exon 1-intron 1 TG were detected in 9/9 lines with the mutated splice sites, indicating splicing did not contribute to changes in expression. DNaseI hypersensitive site (HS) mapping identified a broad, erythroid-specific HS across exon 1 and intron 1. The presence of a DNaseI HS site suggested the presence of a positive regulatory element or a chromatin modification such as a boundary element. Analysis of a positive regulatory element in vivo was sought by stably transfecting the following luciferase (luc) plasmids into K562 cells: ASp promoter, ASp promoter-exon 1-intron 1, ASp promoter-exon 1, ASp promoter-intron 1, and ASp promoter-exon 1-intron 1 with both GATA-1 sites mutated. Clones with copy # ≤5 were analyzed; ≥9 independent clones/line were analyzed. Normalized luc activity of the ASp promoter-exon 1-intron 1 was significantly higher than the ASp promoter in stably transfected cells, 86±15 v 28±3 (p<0.001). Mutation of both GATA-1 sites in the exon 1-intron 1 plasmid reduced activity to background. Normalized luc activity from the promoter-exon was 46±6; from the promoter-intron 101±31, suggesting the intron functions as a positive regulatory element. A barrier assay was performed by flanking a β-globin promoter-EGFP gene using wild type (WT) exon 1, exon 1 with the GATA site abolished, or WT intron 1, and stably transfecting the plasmids into K562 cells. The WT exon 1 and mutant exon 1 expressed GFP in 10/12 and 7/8 lines, respectively, indicating a barrier function for exon 1 independent of GATA-1 activity. Only 1/9 lines expressed EGFP when the cassette was flanked by the ASp intron and 0/8 expressed EGFP when there were no sequences flanking the β-globin promoter. TG mouse lines with the Asp promoter-exon 1 or the Asp promoter-intron 1 linked to the Aγ-globin gene were created. 1/5 TG lines with ASp promoter-exon 1 expressed at low levels and 3/7 TG lines with ASp promoter-intron 1 expressed at levels comparable to the ASp promoter-exon 1-intron 1. These results demonstrate that 2 elements downstream of the ASp promoter are required for high-level, erythroid-specific expression. Exon 1 has barrier activity and intron 1 functions as a positive regulatory element. This is an excellent candidate region for mutations associated with ASp-linked inherited hemolytic anemia.

scholarly journals Erythroid Cell-Specific α-Globin Gene Regulation by the CP2 Transcription Factor Family

2005 ◽  
Vol 25 (14) ◽  
pp. 6005-6020 ◽  
Author(s):  
Ho Chul Kang ◽  
Ji Hyung Chae ◽  
Yeon Ho Lee ◽  
Mi-Ae Park ◽  
June Ho Shin ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Splice Variants ◽  
Globin Gene ◽  
293T Cell ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Transcriptional Activation Domain ◽  
Dna Binding Activity ◽  
Globin Promoter ◽  
Globin Gene Regulation

ABSTRACT We previously demonstrated that ubiquitously expressed CP2c exerts potent erythroid-specific transactivation of α-globin through an unknown mechanism. This mechanism is reported here to involve specific CP2 splice variants and protein inhibitor of activated STAT1 (PIAS1). We identify a novel murine splice isoform of CP2, CP2b, which is identical to CP2a except that it has an additional 36 amino acids encoded by an extra exon. CP2b has an erythroid cell-specific transcriptional activation domain, which requires the extra exon and can form heteromeric complexes with other CP2 isoforms, but lacks the DNA binding activity found in CP2a and CP2c. Transcriptional activation of α-globin occurred following dimerization between CP2b and CP2c in erythroid K562 and MEL cells, but this dimerization did not activate the α-globin promoter in nonerythroid 293T cells, indicating that an additional erythroid factor is missing in 293T cells. PIAS1 was confirmed as a CP2 binding protein by the yeast two-hybrid screen, and expression of CP2b, CP2c, and PIAS1 in 293T cell induced α-globin promoter activation. These results show that ubiquitously expressed CP2b exerts potent erythroid cell-specific α-globin gene expression by complexing with CP2c and PIAS1.

Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene

1988 ◽  
Vol 8 (10) ◽  
pp. 4270-4281
Author(s):  
C G Kim ◽  
K M Barnhart ◽  
M Sheffery
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Affinity Purification ◽  
Globin Gene ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Polyacrylamide Gels ◽  
Dodecyl Sulfate ◽  
Alpha Globin ◽  
Globin Promoter

Three erythroid cell factors that bind the murine alpha-globin promoter were enriched more than 1,000-fold by conventional and DNA sequence affinity chromatography. Visualization of enriched polypeptides revealed simple patterns suggesting that each binding activity was purified. Two of the purified proteins, alpha-CP1 and alpha-CP2, have been shown previously to interact with distinct binding sites that overlap in the alpha-globin CCAAT box. Affinity purification of alpha-CP1 revealed seven polypeptides with Mrs raging from 27,000 to 38,000. In contrast, purified alpha-CP2 was made up of a polypeptide doublet with Mrs of 64,000 and 66,000. The third purified binding activity, alpha-IRP, interacted with sequences that formed an inverted repeat (IR) between the alpha-globin CCAAT and TATAA boxes. Affinity-purified alpha-IRP was made up of a single polypeptide with an Mr of 85,000. We confirmed that the purified polypeptides corresponded to alpha-CP1-, alpha-CP2-, and alpha-IRP-binding activities by UV cross-linking experiments (alpha-CP2 and alpha-IRP) or by renaturation of binding activity after elution of polypeptides from sodium dodecyl sulfate-polyacrylamide gels (alpha-CP1 and alpha-CP2). The apparent complexity of the polypeptides accounting for alpha-CP1 binding activity prompted a further physical characterization of this factor. Sedimentation of affinity-purified alpha-CP1 in glycerol gradients containing 100 mM KCl showed that all seven polypeptides migrated as a complex that cosedimented with alpha-CP1-binding activity. In contrast, when sedimented in glycerol gradients containing 500 mM KCl, alpha-CP1 dissociated into at least two components. Under these conditions, alpha-CP1-binding activity was reduced or lost. Activity was reconstituted, however, by combining fractions that were enriched in the two components. These results were confirmed by experiments in which we showed that alpha-CP1-binding activity can be recovered only by combining distinct sets of polypeptides that were isolated and renatured from sodium dodecyl sulfate-polyacrylamide gels. Our results suggest that the seven polypeptides visualized after affinity purification of alpha-CP1 interact to form a heterotypic complex (or set of complexes) required for alpha-CP1-binding activity.

SATB1 family protein expressed during early erythroid differentiation modifies globin gene expression

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3330-3339 ◽  
Author(s):  
Jie Wen ◽  
Suming Huang ◽  
Heather Rogers ◽  
Liliane A. Dickinson ◽  
Terumi Kohwi-Shigematsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Family Protein ◽  
Globin Promoter ◽  
Globin Gene Expression

AbstractSpecial AT-rich binding protein 1 (SATB1) nuclear protein, expressed predominantly in T cells, regulates genes through targeting chromatin remodeling during T-cell maturation. Here we show SATB1 family protein induction during early human adult erythroid progenitor cell differentiation concomitant with ϵ-globin expression. Erythroid differentiation of human erythroleukemia K562 cells by hemin simultaneously increases γ-globin and down-regulates SATB1 family protein and ϵ-globin gene expression. Chromatin immunoprecipitation using anti-SATB1 anti-body shows selective binding in vivo in the β-globin cluster to the hypersensitive site 2 (HS2) in the locus control region (LCR) and to the ϵ-globin promoter. SATB1 overexpression increases ϵ-globin and decreases γ-globin gene expression accompanied by histone hyperacetylation and hypomethylation in chromatin from the ϵ-globin promoter and HS2, and histone hypoacetylation and hypermethylation associated with the γ-globin promoter. In K562 cells SATB1 family protein forms a complex with CREB-binding protein (CBP) important in transcriptional activation. In cotransfection experiments, increase in ϵ-promoter activity by SATB1 was amplified by CBP and blocked by E1A, a CBP inhibitor. Our results suggest that SATB1 can up-regulate the ϵ-globin gene by interaction with specific sites in the β-globin cluster and imply that SATB1 family protein expressed in the erythroid progenitor cells may have a role in globin gene expression during early erythroid differentiation. (Blood. 2005;105:3330-3339)

scholarly journals The major regulatory element upstream of the alpha-globin gene has classical and inducible enhancer activity

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 1058-1066 ◽  
Author(s):  
S Ren ◽  
XN Luo ◽  
GF Atweh
Keyword(s):  
Messenger Rna ◽  
Phorbol Esters ◽  
Regulatory Element ◽  
Globin Gene ◽  
Expression System ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Enhancer Activity ◽  
Alpha Globin ◽  
Functional Features

Abstract A major positive regulatory element has recently been identified 40 kb upstream from the human zeta 2-globin gene. This regulatory element increases the expression of a linked alpha-globin gene in mouse erythroleukemia cells and in transgenic mice. This element has been shown to share many of the structural and functional features of the locus control region (LCR) of the beta-globin gene cluster. We have examined the activity of a small fragment from this regulatory domain (alpha LCR) in a transient expression system. We show that this element is active as an enhancer in the erythroid environment of K562 cells. It is somewhat less effective as an enhancer in the nonerythroid environment of HeLa cells. This alpha LCR fragment does not exhibit promoter specificity because it can activate both the promoter of its endogenous target gene and the heterologous promoter of the SV40 early genes. Although the major activity of this element is mediated by its interaction with the promoter of the alpha-globin gene, some increase in activity is seen when structural elements from the 5′ end of the alpha-globin gene are included with the target promoter. In addition, we show that the enhancing activity of the alpha LCR is potentiated by hemin-induction of K562 cells. Whereas phorbol esters that induce megakaryocytic differentiation of K562 cells markedly decrease alpha- globin messenger RNA accumulation, they do not seem to have a negative effect on the activity of the alpha LCR. These studies suggest a role for the alpha LCR in the basal activity of the alpha-globin gene in erythroid cells and in its increased expression seen with erythroid differentiation. The mechanism of negative regulation of alpha-globin gene expression in phorbol-differentiated K562 cells does not appear to be mediated through the action of the alpha LCR.

scholarly journals Induction of γ-Globin by Histone Deacetylase Inhibitors

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 2075-2083 ◽  
Author(s):  
Patricia G. McCaffrey ◽  
David A. Newsome ◽  
Eitan Fibach ◽  
Minoru Yoshida ◽  
Michael S.-S. Su
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylase ◽  
Trichostatin A ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Proximal Promoter ◽  
Erythroid Cell ◽  
Globin Promoter ◽  
Ccaat Box

Abstract The short-chain fatty acid butyrate has been shown to elevate fetal hemoglobin (HbF ) by inducing expression of the γ-globin gene. Regulation of gene expression by butyrate is thought to proceed via inhibition of the enzyme histone deacetylase, leading to elevated levels of core histone acetylation which affect chromatin structure and transcription rates. To determine whether changes in histone acetylation are critical for the regulation of the γ-globin gene, we tested three potent and specific inhibitors of histone deacetylase, the cyclic tetrapeptides trapoxin and Helminthsporium carbonum toxin (HC toxin), and the antifungal antibiotic trichostatin A for their ability to induce fetal hemoglobin expression in erythroid cells. These compounds induced fetal hemoglobin in both primary erythroid cell cultures and human erythroleukemia (K562) cells. A butyrate-responsive element spanning the duplicated CCAAT box region of the γ-globin promoter has been identified in transient transfection assays using a reporter construct in K562 cells, and we show that the same promoter region is required for response to trapoxin and trichostatin. Mutational analysis of the γ-globin promoter indicates that the distal CCAAT box and 3′ flanking sequence (CCAATAGCC) is critical for activation by butyrate, trapoxin, and trichostatin, whereas the proximal element (CCAATAGTC) plays a less important role. These results show that inhibition of histone deacetylase can lead to transcriptional activation of γ-globin promoter reporter gene constructs through proximal promoter elements, and suggest that butyrate induces γ-globin expression via such changes in histone acetylation.

scholarly journals A novel developmental regulatory motif required for stage-specific activation of the epsilon-globin gene and nuclear factor binding in embryonic erythroid cells.

1994 ◽  
Vol 14 (6) ◽  
pp. 3763-3771 ◽  
Author(s):  
W L Trepicchio ◽  
M A Dyer ◽  
M H Baron
Keyword(s):  
Transcriptional Activation ◽  
Cultured Cells ◽  
Regulatory Element ◽  
Globin Gene ◽  
Point Mutations ◽  
Gene Promoter ◽  
Specific Gene ◽  
Nuclear Factor ◽  
Erythroid Cells ◽  
Conserved Sequence

Members of the human beta-globin gene family are expressed at discrete stages of development and therefore provide an important model system for examining mechanisms of temporal gene regulation. We have previously shown that expression of the embryonic beta-like globin gene (epsilon) is mediated by a complex array of positive and negative upstream control elements. Correct developmental stage- and tissue-specific gene expression is conferred by synergistic interactions between a positive regulatory element (termed epsilon-PRE II) which is active only in embryonic erythroid cells and at least two other regulatory domains upstream of the epsilon-globin gene promoter. A nuclear factor highly enriched in cultured embryonic erythroid cells and in mouse embryonic yolk sac binds to a novel, evolutionarily conserved sequence within epsilon-PRE II. We show here that binding of this factor to the conserved element within epsilon-PRE II is critical for transcriptional activity. Point mutations that interfere with protein binding to epsilon-PRE II abolish transcriptional activation of the constitutive epsilon-globin promoter. Adult erythroid nuclei (from cultured cells or adult mouse liver) also contain a factor that binds to this region, but the complex formed migrates more rapidly during nondenaturing electrophoresis, suggesting either that distinct proteins bind to epsilon-PRE II or that a single protein is differentially modified in these cells in a way that modulates its activity. Several lines of evidence suggest that the binding factors in embryonic and adult erythroid cells are distinguished by posttranscriptional differences.

scholarly journals The major regulatory element upstream of the alpha-globin gene has classical and inducible enhancer activity

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 1058-1066
Author(s):  
S Ren ◽  
XN Luo ◽  
GF Atweh
Keyword(s):  
Messenger Rna ◽  
Phorbol Esters ◽  
Regulatory Element ◽  
Globin Gene ◽  
Expression System ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Enhancer Activity ◽  
Alpha Globin ◽  
Functional Features

A major positive regulatory element has recently been identified 40 kb upstream from the human zeta 2-globin gene. This regulatory element increases the expression of a linked alpha-globin gene in mouse erythroleukemia cells and in transgenic mice. This element has been shown to share many of the structural and functional features of the locus control region (LCR) of the beta-globin gene cluster. We have examined the activity of a small fragment from this regulatory domain (alpha LCR) in a transient expression system. We show that this element is active as an enhancer in the erythroid environment of K562 cells. It is somewhat less effective as an enhancer in the nonerythroid environment of HeLa cells. This alpha LCR fragment does not exhibit promoter specificity because it can activate both the promoter of its endogenous target gene and the heterologous promoter of the SV40 early genes. Although the major activity of this element is mediated by its interaction with the promoter of the alpha-globin gene, some increase in activity is seen when structural elements from the 5′ end of the alpha-globin gene are included with the target promoter. In addition, we show that the enhancing activity of the alpha LCR is potentiated by hemin-induction of K562 cells. Whereas phorbol esters that induce megakaryocytic differentiation of K562 cells markedly decrease alpha- globin messenger RNA accumulation, they do not seem to have a negative effect on the activity of the alpha LCR. These studies suggest a role for the alpha LCR in the basal activity of the alpha-globin gene in erythroid cells and in its increased expression seen with erythroid differentiation. The mechanism of negative regulation of alpha-globin gene expression in phorbol-differentiated K562 cells does not appear to be mediated through the action of the alpha LCR.

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