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.

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.

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 A DNA-bending protein interacts with an essential upstream regulatory element of the human embryonic beta-like globin gene.

1996 ◽  
Vol 16 (3) ◽  
pp. 829-838 ◽  
Author(s):  
M A Dyer ◽  
R Naidoo ◽  
R J Hayes ◽  
C J Larson ◽  
G L Verdine ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Globin Gene ◽  
Gene Promoter ◽  
Developmental State ◽  
Biochemical Properties ◽  
Erythroid Cell ◽  
Control Element ◽  
Specific Gene ◽  
Globin Genes ◽  
Specific Expression

The mammalian beta-like globin gene family has served as an important model system for analysis of tissue- and developmental state-specific gene regulation. Although the activities of a number of regulatory proteins have been implicated in the erythroid cell-specific transcription of globin genes, the mechanisms that restrict their expression to discrete stages of development are less well understood. We have previously identified a novel regulatory element (PRE II) upstream from the human embryonic beta-like globin gene (epsilon) that synergizes with other sequences to confer tissue- and stage-specific expression on a minimal epsilon-globin gene promoter in cultured embryonic erythroid cells. Binding of an erythroid nuclear protein (PRE II-binding factor [PRE-IIBF]) to the PRE II control element is required for promoter activation. Here we report on some of the biochemical properties of PREIIBF, including the characterization of its specificity and affinity for DNA. The embryonic and adult forms of PREIIBF recognize their cognate sequences with identical specificities, supporting our earlier conclusion that they are very similar proteins. PREIIBF binds DNA as a single polypeptide with an Mr of approximately 80,000 to 85,000 and introduces a bend into the target DNA molecule. These results suggest a mechanism by which PREIIBF may contribute to the regulation of the embryonic beta-like globin gene within the context of a complex locus.

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 Nuclear factor I (NF I) binds to an NF I-type site but not to the CCAAT site in the human alpha-globin gene promoter.

1992 ◽  
Vol 267 (12) ◽  
pp. 8478-8484 ◽  
Author(s):  
H Zorbas ◽  
T Rein ◽  
A Krause ◽  
K Hoffmann ◽  
E.L. Winnacker
Keyword(s):  
Globin Gene ◽  
Gene Promoter ◽  
Nuclear Factor ◽  
Factor I ◽  
Alpha Globin ◽  
Nuclear Factor I ◽  
Type Site

Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells

1994 ◽  
Vol 14 (5) ◽  
pp. 3108-3114
Author(s):  
M H Baron ◽  
S M Farrington
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cultured Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Targeted Mutagenesis ◽  
Erythroid Cell ◽  
Erythroid Cells

The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion.

scholarly journals 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

1998 ◽  
Vol 18 (11) ◽  
pp. 6634-6640 ◽  
Author(s):  
Denise E. Sabatino ◽  
Amanda P. Cline ◽  
Patrick G. Gallagher ◽  
Lisa J. Garrett ◽  
George Stamatoyannopoulos ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fetal Liver ◽  
Globin Gene ◽  
Gene Promoter ◽  
Yolk Sac ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
Globin Promoter ◽  
In Erythroid Cells

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.

scholarly journals Transcriptional role of a conserved GATA-1 site in the human epsilon-globin gene promoter.

1991 ◽  
Vol 11 (5) ◽  
pp. 2558-2566 ◽  
Author(s):  
Q H Gong ◽  
J Stern ◽  
A Dean
Keyword(s):  
Globin Gene ◽  
Point Mutations ◽  
Gene Promoter ◽  
Globin Genes ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Nuclear Extracts ◽  
Protein Dna Interactions ◽  
Globin Promoter

The epsilon-globin gene is the first of the human beta-like globin genes to be expressed during development. We have analyzed protein-DNA interactions in the epsilon-globin promoter region by DNase I footprinting and electrophoretic mobility shift experiments using nuclear extracts from K562 human erythroid cells and from nonerythroid HeLa cells. A restricted set of ubiquitous proteins, including Sp1, bound to regions of the promoter including the CACCC and CCAAT sites. Three interactions, at positions -213, -165, and +3 relative to the transcription start site, were erythroid specific and corresponded to binding of GATA-1, a transcription factor highly restricted to the erythroid lineage. Interestingly, the GATA-1 site at -165 has been conserved in the promoters of 10 mammalian embryonic globin genes. Point mutations demonstrate that GATA-1 binding to this site is necessary for interaction with an erythroid-specific enhancer but that in the absence of an enhancer, GATA-1 does not increase transcription.

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