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 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

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.

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.

scholarly journals The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene

Development ◽  
1996 ◽  
Vol 122 (12) ◽  
pp. 3991-3999 ◽  
Author(s):  
R. Tewari ◽  
N. Gillemans ◽  
A. Harper ◽  
M. Wijgerde ◽  
G. Zafarana ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Developmental Expression ◽  
Chromatin Conformation ◽  
Lacz Gene ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Hypersensitive Sites

The beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5′HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been described. Expression of the beta-like globin genes is absolutely dependent on the presence of the LCR. The developmental expression pattern of the genes in the cluster is achieved through competition of the promoters for the activating function of the LCR. Transgenic mice experiments suggest that subtle changes in the transcription factor environment lead to the successive silencing of the embryonic epsilon-globin and fetal gamma-globin promoters, resulting in the almost exclusive transcription of the beta-globin gene in adult erythropoiesis. In this paper, we have asked the question whether the LCR and its individual hypersensitive sites 5′HS1-4 can activate a basic promoter in the absence of any other globin sequences. We have employed a minimal promoter derived from the mouse Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The results show that the muLCR and 5′HS3 direct erythroid-specific, embryonic expression of this construct, while 5′HS1, 5′HS2 and 5′HS4 are inactive at any stage of development. Expression of the muLCR and 5′HS3 transgenes is repressed during fetal stages of development. The transgenes are in an inactive chromatin conformation and the lacZ gene is not transcribed, as shown by in situ hybridization. These data are compatible with the hypothesis that the LCR requires the presence of an active promoter to adopt an open chromatin conformation and with models proposing progressive heterochromatization during embryogenesis. The results suggest that the presence of a beta-globin gene is required for LCR function as conditions become more stringent during development.

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.

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.

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.

scholarly journals NF-E2 disrupts chromatin structure at human beta-globin locus control region hypersensitive site 2 in vitro.

1996 ◽  
Vol 16 (10) ◽  
pp. 5634-5644 ◽  
Author(s):  
J A Armstrong ◽  
B M Emerson
Keyword(s):  
Control Region ◽  
Chromatin Structure ◽  
Locus Control Region ◽  
Dnase I ◽  
Hematopoietic Cell ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites

The human beta-globin locus control region (LCR) is responsible for forming an active chromatin structure extending over the 100-kb locus, allowing expression of the beta-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HS1 to -4). DNase I hypersensitive sites are thought to represent nucleosome-free regions of DNA which are bound by trans-acting factors. Of the four hypersensitive sites only HS2 acts as a transcriptional enhancer. In this study, we examine the binding of an erythroid protein to its site within HS2 in chromatin in vitro. NF-E2 is a transcriptional activator consisting of two subunits, the hematopoietic cell-specific p45 and the ubiquitous DNA-binding subunit, p18. NF-E2 binds two tandem AP1-like sites in HS2 which form the core of its enhancer activity. In this study, we show that when bound to in vitro-reconstituted chromatin, NF-E2 forms a DNase I hypersensitive site at HS2 similar to the site observed in vivo. Moreover, NF-E2 binding in vitro results in a disruption of nucleosome structure which can be detected 200 bp away. Although NF-E2 can disrupt nucleosomes when added to preformed chromatin, the disruption is more pronounced when NF-E2 is added to DNA prior to chromatin assembly. Interestingly, the hematopoietic cell-specific subunit, p45, is necessary for binding to chromatin but not to naked DNA. Interaction of NF-E2 with its site in chromatin-reconstituted HS2 allows a second erythroid factor, GATA-1, to bind its nearby sites. Lastly, nucleosome disruption by NF-E2 is an ATP-dependent process, suggesting the involvement of energy-dependent nucleosome remodeling factors.

Reduced beta-Globin Gene Expression in Adult Mice Containing Deletions of Locus Control Region 5' HS-2 or 5' HS-3a

1998 ◽  
Vol 850 (1 COOLEY'S ANEM) ◽  
pp. 45-53 ◽  
Author(s):  
TIMOTHY J. LEY ◽  
BRUCE HUG ◽  
STEVEN FIERING ◽  
ELLIOT EPNER ◽  
M. A. BENDER ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Adult Mice ◽  
Globin Gene Expression
Sign in / Sign up

Export Citation Format

Share Document