Activation of the beta-globin promoter by the locus control region correlates with binding of a novel factor to the CAAT box in murine erythroleukemia cells but not in K562 cells

1993 ◽  
Vol 13 (11) ◽  
pp. 6969-6983
Author(s):  
N L Delvoye ◽  
N M Destroismaisons ◽  
L A Wall
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
Locus Control Region ◽  
Model Systems ◽  
Beta Globin ◽  
Wild Type ◽  
Globin Promoter ◽  
Murine Erythroleukemia

Four distinct factors in extracts from murine erythroleukemia (MEL) cells interacted with the human beta-globin gene promoter CAAT box: CP1, GATA-1, and two novel factors, denoted a and b, one of which is highly inducible in the MEL system. GATA-1 binding to the CAAT element was very unstable (half-life < 1 min), whereas bindings of a, b, and CP1 were comparatively stable, with half-lives of 18, 19, and 3.5 min, respectively. Stable transfections of MEL cells showed that in the presence of the beta-globin locus control region (LCR), the wild-type CAAT box, a mutant which bound to GATA-1 with increased stability over the normal sequences, and a mutant which bound a, b, and CP1 specifically could all stimulate transcription greater than ninefold over that induced by a null CAAT mutation in both uninduced and terminally differentiated MEL cells. A mutant which bound the a and b factors specifically gave only a twofold stimulation of promoter activity, and this lower activity correlated with a decrease in the stability of binding of the b protein. On the other hand, CP1 binding alone did not stimulate transcription. Taken together, these results suggest that in the context of the wild-type beta-globin CAAT element the b factor stimulates transcription directed by the LCR in MEL cells, although the LCR can also function through more stable GATA-1-binding sequences. However, in K562 cells, the wild-type beta-globin CAAT box alone was unable to stimulate gene expression directed by the LCR and high levels of transcription were obtained only upon inclusion of more upstream beta-globin promoter sequences. In contrast, a construct containing only the A gamma-globin CAAT box region did give high expression levels in K562 cells. Thus, there is a fundamental difference in the way the LCR functions in these two model systems in terms of its requirements at the promoter level.

scholarly journals Activation of the beta-globin promoter by the locus control region correlates with binding of a novel factor to the CAAT box in murine erythroleukemia cells but not in K562 cells.

1993 ◽  
Vol 13 (11) ◽  
pp. 6969-6983 ◽  
Author(s):  
N L Delvoye ◽  
N M Destroismaisons ◽  
L A Wall
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
Locus Control Region ◽  
Model Systems ◽  
Beta Globin ◽  
Wild Type ◽  
Globin Promoter ◽  
Murine Erythroleukemia

Four distinct factors in extracts from murine erythroleukemia (MEL) cells interacted with the human beta-globin gene promoter CAAT box: CP1, GATA-1, and two novel factors, denoted a and b, one of which is highly inducible in the MEL system. GATA-1 binding to the CAAT element was very unstable (half-life < 1 min), whereas bindings of a, b, and CP1 were comparatively stable, with half-lives of 18, 19, and 3.5 min, respectively. Stable transfections of MEL cells showed that in the presence of the beta-globin locus control region (LCR), the wild-type CAAT box, a mutant which bound to GATA-1 with increased stability over the normal sequences, and a mutant which bound a, b, and CP1 specifically could all stimulate transcription greater than ninefold over that induced by a null CAAT mutation in both uninduced and terminally differentiated MEL cells. A mutant which bound the a and b factors specifically gave only a twofold stimulation of promoter activity, and this lower activity correlated with a decrease in the stability of binding of the b protein. On the other hand, CP1 binding alone did not stimulate transcription. Taken together, these results suggest that in the context of the wild-type beta-globin CAAT element the b factor stimulates transcription directed by the LCR in MEL cells, although the LCR can also function through more stable GATA-1-binding sequences. However, in K562 cells, the wild-type beta-globin CAAT box alone was unable to stimulate gene expression directed by the LCR and high levels of transcription were obtained only upon inclusion of more upstream beta-globin promoter sequences. In contrast, a construct containing only the A gamma-globin CAAT box region did give high expression levels in K562 cells. Thus, there is a fundamental difference in the way the LCR functions in these two model systems in terms of its requirements at the promoter level.

scholarly journals Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.

1995 ◽  
Vol 9 (24) ◽  
pp. 3083-3096 ◽  
Author(s):  
J Bungert ◽  
U Dave ◽  
K C Lim ◽  
K H Lieuw ◽  
J A Shavit ◽  
...  
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Synergistic Regulation ◽  
Gene Switching

scholarly journals A novel deletion of approximately 27 kb including the beta-globin gene and the locus control region 3'HS-1 regulatory sequence: beta zero- thalassemia or hereditary persistence of fetal hemoglobin?

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 822-827 ◽  
Author(s):  
AJ Dimovski ◽  
V Divoky ◽  
AD Adekile ◽  
E Baysal ◽  
JB Wilson ◽  
...  
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Locus Control Region ◽  
Regulatory Sequence ◽  
Beta Globin ◽  
Gamma Chain ◽  
Beta Globin Gene ◽  
Gamma Globin

Abstract A novel deletion of approximately 27 kb with the 5′ breakpoint 1.5 to 2.2 kb upstream of the beta-globin gene, and the 3′ breakpoint approximately 24 kb downstream of the beta-globin gene, has been found in five members of two families from Southeast Asia (Vietnam and Cambodia). Six members of another family from China, previously reported from our laboratory, have also been shown to carry this deletion. The patients presented with mild hypochromia and microcytosis, a hemoglobin (Hb) A2 level of approximately 4.0%, and a markedly increased, heterocellularly distributed, Hb F level (14.0 to 26.0%). In vitro globin-chain synthesis showed a mild imbalance with appreciable gamma-chain compensation (alpha/beta + gamma ratio of 1.46). The 3′ end of this deletion includes the 3′HS-1, and we hypothesize that removal of this region results in the loss of its gamma-globin gene-silencing effect, which causes a markedly elevated Hb F level with a modest increase in Hb A2 levels, unlike the situation in other deletional beta zero-thalassemias. The possible influence of particular sequence variations in the locus control region 5′HS-2 and the G gamma promoter, present on the chromosome with this deletion, on the overall gamma-globin gene should also be considered.

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 Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region.

1992 ◽  
Vol 6 (6) ◽  
pp. 928-938 ◽  
Author(s):  
C G Kim ◽  
E M Epner ◽  
W C Forrester ◽  
M Groudine
Keyword(s):  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Beta Globin ◽  
Beta Globin Gene

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 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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