scholarly journals A single beta-globin locus control region element (5' hypersensitive site 2) is sufficient for developmental regulation of human globin genes in transgenic mice.

1992 ◽  
Vol 12 (5) ◽  
pp. 2057-2066 ◽  
Author(s):  
B J Morley ◽  
C A Abbott ◽  
J A Sharpe ◽  
J Lida ◽  
P S Chan-Thomas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Control Region ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Beta Globin ◽  
Hypersensitive Site

The beta-globin gene complex is regulated by an upstream locus control region (LCR) which is responsible for high-level, position-independent, erythroid-cell-specific expression of the genes in the cluster. Its role in the developmental regulation of beta-like globin gene transcription remains to be established. We have examined the effect of a single LCR element, hypersensitive site 2 (HS2), on the developmental regulation of the human fetal gamma and adult beta genes in transgenic mice. In mice bearing HS2A gamma beta and HS2G gamma A gamma-117 delta beta human globin gene constructs, switching from gamma- to beta-gene expression begins at about day 13.5 of gestation and is largely completed shortly after birth. The larger construct also demonstrates a switch in G gamma- to A gamma-gene expression during the gamma-to-beta switch similar to that observed during normal human development. We conclude that HS2 alone is sufficient for developmental regulation of the human beta-globin genes.

A single beta-globin locus control region element (5' hypersensitive site 2) is sufficient for developmental regulation of human globin genes in transgenic mice

1992 ◽  
Vol 12 (5) ◽  
pp. 2057-2066
Author(s):  
B J Morley ◽  
C A Abbott ◽  
J A Sharpe ◽  
J Lida ◽  
P S Chan-Thomas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Control Region ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Beta Globin ◽  
Hypersensitive Site

The beta-globin gene complex is regulated by an upstream locus control region (LCR) which is responsible for high-level, position-independent, erythroid-cell-specific expression of the genes in the cluster. Its role in the developmental regulation of beta-like globin gene transcription remains to be established. We have examined the effect of a single LCR element, hypersensitive site 2 (HS2), on the developmental regulation of the human fetal gamma and adult beta genes in transgenic mice. In mice bearing HS2A gamma beta and HS2G gamma A gamma-117 delta beta human globin gene constructs, switching from gamma- to beta-gene expression begins at about day 13.5 of gestation and is largely completed shortly after birth. The larger construct also demonstrates a switch in G gamma- to A gamma-gene expression during the gamma-to-beta switch similar to that observed during normal human development. We conclude that HS2 alone is sufficient for developmental regulation of the human beta-globin genes.

Direct interaction of NF-E2 with hypersensitive site 2 of the β-globin locus control region in living cells

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 334-339 ◽  
Author(s):  
E. Camilla Forsberg ◽  
Karen M. Downs ◽  
Emery H. Bresnick
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Hypersensitive Site ◽  
Intact Cells ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression

The human β-globin locus control region (LCR) confers high-level, tissue-specific expression to the β-globin genes. Tandem Maf recognition elements (MAREs) within the hypersensitive site 2 (HS2) subregion of the LCR are important for the strong enhancer activity of the LCR. Multiple proteins are capable of interacting with these sites in vitro, including the erythroid cell- and megakaryocyte-specific transcription factor, NF-E2. The importance of NF-E2 for β-globin gene expression is evident in murine erythroleukemia cells lacking the p45 subunit of NF-E2. These CB3 cells have a severe defect in - and β-globin gene transcription, which can be restored by expression of NF-E2. However, mice nullizygous for p45 express nearly normal levels of β-globin. Thus, either a redundant factor(s) exists in mice that can functionally replace NF-E2, or NF-E2 does not function through the LCR to regulate β-globin gene expression. To address this issue, we asked whether NF-E2 binds directly to the tandem MAREs of HS2 in intact cells. Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver. The specific immunoisolation of HS2 sequences was dependent on the presence of p45 and on intact MAREs within HS2. These results support a direct role for NF-E2 in the regulation of β-globin gene expression through activation of the LCR.

Correct Function of the Locus Control Region May Require Passage Through a Nonerythroid Cellular Environment

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 703-712 ◽  
Author(s):  
George Vassilopoulos ◽  
Patrick A. Navas ◽  
Evangelia Skarpidi ◽  
Kenneth R. Peterson ◽  
Chris H. Lowrey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Globin Mrna ◽  
L Cells ◽  
Correct Function ◽  
Globin Gene Expression

Abstract The function of the β-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb β-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a β-globin locus (β-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb β-YAC that encompasses the entire β-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). ThePpo-155 β-YAC was used to directly lipofect MEL 585 cells. In 7 β-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, ɛ + γ + β) per copy of integrated β-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the β-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked β-YAC DNA. To test whether passage of the β-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(β-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human β-globin mRNA (ie, ɛ + γ + β) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

Direct interaction of NF-E2 with hypersensitive site 2 of the β-globin locus control region in living cells

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 334-339 ◽  
Author(s):  
E. Camilla Forsberg ◽  
Karen M. Downs ◽  
Emery H. Bresnick
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Hypersensitive Site ◽  
Intact Cells ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
Globin Gene Expression

Abstract The human β-globin locus control region (LCR) confers high-level, tissue-specific expression to the β-globin genes. Tandem Maf recognition elements (MAREs) within the hypersensitive site 2 (HS2) subregion of the LCR are important for the strong enhancer activity of the LCR. Multiple proteins are capable of interacting with these sites in vitro, including the erythroid cell- and megakaryocyte-specific transcription factor, NF-E2. The importance of NF-E2 for β-globin gene expression is evident in murine erythroleukemia cells lacking the p45 subunit of NF-E2. These CB3 cells have a severe defect in - and β-globin gene transcription, which can be restored by expression of NF-E2. However, mice nullizygous for p45 express nearly normal levels of β-globin. Thus, either a redundant factor(s) exists in mice that can functionally replace NF-E2, or NF-E2 does not function through the LCR to regulate β-globin gene expression. To address this issue, we asked whether NF-E2 binds directly to the tandem MAREs of HS2 in intact cells. Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver. The specific immunoisolation of HS2 sequences was dependent on the presence of p45 and on intact MAREs within HS2. These results support a direct role for NF-E2 in the regulation of β-globin gene expression through activation of the LCR.

Correct Function of the Locus Control Region May Require Passage Through a Nonerythroid Cellular Environment

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 703-712 ◽  
Author(s):  
George Vassilopoulos ◽  
Patrick A. Navas ◽  
Evangelia Skarpidi ◽  
Kenneth R. Peterson ◽  
Chris H. Lowrey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Globin Mrna ◽  
L Cells ◽  
Correct Function ◽  
Globin Gene Expression

The function of the β-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb β-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a β-globin locus (β-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb β-YAC that encompasses the entire β-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). ThePpo-155 β-YAC was used to directly lipofect MEL 585 cells. In 7 β-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, ɛ + γ + β) per copy of integrated β-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the β-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked β-YAC DNA. To test whether passage of the β-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(β-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human β-globin mRNA (ie, ɛ + γ + β) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

scholarly journals Antagonistic Regulation of β-Globin Gene Expression by Helix-Loop-Helix Proteins USF and TFII-I

2006 ◽  
Vol 26 (18) ◽  
pp. 6832-6843 ◽  
Author(s):  
Valerie J. Crusselle-Davis ◽  
Karen F. Vieira ◽  
Zhuo Zhou ◽  
Archana Anantharaman ◽  
Jörg Bungert
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Control Region ◽  
Adult Stage ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Globin Gene Expression ◽  
In Erythroid Cells

ABSTRACT The human β-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the β-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased β-globin gene expression, while diminishing TFII-I activity increased β-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult β-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the β-globin gene locus and play important roles in restricting β-globin gene expression to the adult stage of erythropoiesis.

scholarly journals Human beta-globin locus control region: analysis of the 5' DNase I hypersensitive site HS 2 in transgenic mice.

1991 ◽  
Vol 88 (5) ◽  
pp. 1626-1630 ◽  
Author(s):  
J. J. Caterina ◽  
T. M. Ryan ◽  
K. M. Pawlik ◽  
R. D. Palmiter ◽  
R. L. Brinster ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Control Region ◽  
Locus Control Region ◽  
Dnase I ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Region Analysis ◽  
Dnase I Hypersensitive Site

scholarly journals Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1399-1401 ◽  
Author(s):  
Q Li ◽  
G Stamatoyannopoulos
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Reporter Expression ◽  
Chromatin Insulator ◽  
Hypersensitive Sites ◽  
Globin Gene Expression ◽  
Beta Gene

Abstract The human beta locus control region (LCR) consists of five DNAse I hypersensitive sites (HS), four of which are erythroid specific and one, the further upstream located 5′HS5, is constitutive. To characterize the function of 5′HS5 we analyzed globin gene expression of various constructs containing HS3 as an enhancer, HS5, and the beta gene as a reporter. Expression was analyzed in stably transfected MEL cells. We found that the enhancing effect of hypersensitive site 3 is blocked when the HS5 is interposed between HS3 and the beta globin gene. These data suggest that the human 5′HS5 has the properties of a chromatin insulator.

scholarly journals Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

1996 ◽  
Vol 16 (11) ◽  
pp. 6055-6064 ◽  
Author(s):  
Q H Gong ◽  
J C McDowell ◽  
A Dean
Keyword(s):  
Control Region ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Locus Control Region ◽  
Dnase I ◽  
Beta Globin ◽  
Hypersensitive Site

Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free of nucleosomes. The restricted structural change observed upon transcriptional activation may indicate that the LCR need only make a specific contact with the proximal gene promoter to activate transcription.

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