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.

Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements

1993 ◽  
Vol 13 (12) ◽  
pp. 7457-7468
Author(s):  
W L Trepicchio ◽  
M A Dyer ◽  
M H Baron
Keyword(s):  
Developmental Regulation ◽  
Regulatory Element ◽  
Globin Gene ◽  
Negative Control ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Specific Expression ◽  
Systematic Analysis ◽  
Specific Regulation

The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene.

scholarly journals Developmental regulation of the human embryonic beta-like globin gene is mediated by synergistic interactions among multiple tissue- and stage-specific elements.

1993 ◽  
Vol 13 (12) ◽  
pp. 7457-7468 ◽  
Author(s):  
W L Trepicchio ◽  
M A Dyer ◽  
M H Baron
Keyword(s):  
Developmental Regulation ◽  
Regulatory Element ◽  
Globin Gene ◽  
Negative Control ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Specific Expression ◽  
Systematic Analysis ◽  
Specific Regulation

The stage-specific regulation of mammalian embryonic globin genes has been an experimentally elusive problem, in part because of the developmentally early timing of their expression. We have carried out a systematic analysis of truncation and internal deletion mutations within the 5'-flanking region of the human embryonic beta-like globin gene (epsilon) in erythroid and nonerythroid cell lines. Within a 670-bp region upstream from the constitutive promoter are multiple positive and negative control elements. Of these, a positive regulatory element (epsilon-PRE II) which is active only in embryonic erythroid cells is of particular interest. Remarkably, although it is inactive on its own, in the presence of other sequences located further upstream, it confers tissue- and developmental stage-specific expression on a constitutive epsilon-globin or heterologous promoter. The activity of epsilon-PRE II is also modulated by another positive regulatory domain located further downstream to direct erythroid cell-specific, but little or no embryonic stage-specific, transcription. A nuclear factor highly enriched in embryonic erythroid cells binds specifically within a 19-bp region of epsilon-PRE II. Nuclei from adult erythroid cells also contain a factor that binds to this region but forms a complex of faster electrophoretic mobility. We speculate that interactions between epsilon-PRE II and other upstream control elements play an important role in the developmental regulation of the human embryonic beta-like globin gene.

Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells

1987 ◽  
Vol 7 (1) ◽  
pp. 398-402
Author(s):  
T Rutherford ◽  
A W Nienhuis
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Promoters ◽  
Globin Genes ◽  
Specific Expression ◽  
Tissue Specific ◽  
Promoter Sequences ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.

scholarly journals The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 855-860
Author(s):  
M Donovan-Peluso ◽  
S Acuto ◽  
D O'Neill ◽  
A Hom ◽  
A Maggio ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Globin Gene ◽  
K562 Cells ◽  
Specific Gene ◽  
Fetal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Specific Expression ◽  
Enhancer Element ◽  
Gamma Globin

We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3′ enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3′ enhancer is erythroid-specific but not developmental stage- or gene-specific.

scholarly journals p45NFE2 Is a Negative Regulator of Erythroid Proliferation Which Contributes to the Progression of Friend Virus-Induced Erythroleukemias

2001 ◽  
Vol 21 (1) ◽  
pp. 73-80 ◽  
Author(s):  
You-Jun Li ◽  
Rachel R. Higgins ◽  
Brian J. Pak ◽  
Ramesh A. Shivdasani ◽  
Paul A. Ney ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Globin Gene ◽  
Negative Regulator ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Globin Genes ◽  
Friend Virus ◽  
Wild Type

ABSTRACT In previous studies, we identified a common site of retroviral integration designated Fli-2 in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia cell lines. Insertion of F-MuLV at the Fli-2 locus, which was associated with the loss of the second allele, resulted in the inactivation of the erythroid cell- and megakaryocyte-specific genep45 NFE2 . Frequent disruption ofp45 NFE2 due to proviral insertion suggests a role for this transcription factor in the progression of Friend virus-induced erythroleukemias. To assess this possibility, erythroleukemia was induced by F-MuLV inp45 NFE2 mutant mice. Sincep45 NFE2 homozygous mice mostly die at birth, erythroleukemia was induced in +/− and +/+ mice. We demonstrate that +/− mice succumb to the disease moderately but significantly faster than +/+ mice. In addition, the spleens of +/− mice were significantly larger than those of +/+ mice. Of the 37 tumors generated from the +/− and +/+ mice, 10 gave rise to cell lines, all of which were derived from +/− mice. Establishment in culture was associated with the loss of the remaining wild-typep45 NFE2 allele in 9 of 10 of these cell lines. The loss of a functional p45NFE2 in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-typep45 NFE2 in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression ofp45 NFE2 in these cells also slows tumor growth in vivo. These results indicate thatp45 NFE2 functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.

scholarly journals The HMG Domain Protein SSRP1/PREIIBF Is Involved in Activation of the Human Embryonic β-Like Globin Gene

1998 ◽  
Vol 18 (5) ◽  
pp. 2617-2628 ◽  
Author(s):  
Michael A. Dyer ◽  
Patrick J. Hayes ◽  
Margaret H. Baron
Keyword(s):  
Developmental Stage ◽  
Regulatory Element ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Erythroid Cell ◽  
Expression Cloning ◽  
Erythroid Cells ◽  
Specific Expression ◽  
Binding Factor

ABSTRACT The human embryonic β-like globin (ɛ-globin) gene is expressed in primitive erythroid cells of the yolk sac during the first few weeks of development. We have previously shown that developmental stage-specific expression of the ɛ-globin gene is mediated by multiple positive and negative regulatory elements upstream of the start of transcription. Of particular interest is one positive regulatory element, PRE II, that works together with other elements (PRE I and PRE V) to confer developmental stage- and/or tissue-specific expression on a minimal promoter. An ∼85- to 90-kDa PRE II binding factor (PREIIBF) was identified in the nuclei of erythroid cells and shown to bind specifically to a novel 19-bp region within PRE II; binding of this protein to PRE II resulted in bending of the target DNA and was required for promoter activation. In this report, we present the cDNA expression cloning of PREIIBF. The cDNA encodes a previously identified member of the HMG domain family of DNA binding proteins termed SSRP1. By a number of biochemical and immunological criteria, recombinant SSRP1 appears to be identical to the PREII binding factor from erythroid nuclei. A hallmark of HMG domain proteins is their ability to bend their target DNAs; therefore, as we speculated previously, DNA bending by SSRP1/PREIIBF may contribute to the mechanism by which PRE II synergizes with other regulatory elements located upstream and downstream. In contrast with reports from other investigators, we demonstrate that SSRP1 binds DNA with clear sequence specificity. Moreover, we show that SSRP1/PREIIBF lacks a classical activation domain but that binding by this protein to PRE II is required for activation of a minimal promoter in stable erythroid cell lines. These studies provide the first evidence that SSRP1 plays a role in transcriptional regulation. SSRP1/PREIIBF may serve an architectural function by helping to coordinate the assembly of a multiprotein complex required for stage-specific regulation of the human ɛ-globin gene.

scholarly journals Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells.

1987 ◽  
Vol 7 (1) ◽  
pp. 398-402 ◽  
Author(s):  
T Rutherford ◽  
A W Nienhuis
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Promoters ◽  
Globin Genes ◽  
Specific Expression ◽  
Tissue Specific ◽  
Promoter Sequences ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.

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.

scholarly journals The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 855-860 ◽  
Author(s):  
M Donovan-Peluso ◽  
S Acuto ◽  
D O'Neill ◽  
A Hom ◽  
A Maggio ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Globin Gene ◽  
K562 Cells ◽  
Specific Gene ◽  
Fetal Life ◽  
Globin Genes ◽  
Beta Globin ◽  
Specific Expression ◽  
Enhancer Element ◽  
Gamma Globin

Abstract We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3′ enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3′ enhancer is erythroid-specific but not developmental stage- or gene-specific.

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