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Query: UNIPROT:P06889 (Mol)
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The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.
Mol Cell Biol 2001 May
PMID:Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement. 1128 3

There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.
Mol Genet Genomics 2002 Jan
PMID:Subcellular localization of the homocitrate synthase in Penicillium chrysogenum. 1181 Feb 44

The beta-globin locus control region (LCR) is a powerful regulatory element required for high-level globin gene expression. We have generated transgenic mouse lines carrying a beta-globin locus yeast artificial chromosome lacking the LCR to determine if the LCR is required for globin gene activation. beta-Globin gene expression was analyzed by RNase protection, but no detectable levels of epsilon-, gamma- and beta-globin gene transcripts were produced at any stage of development. These findings suggest that the presence of the LCR is a minimum requirement for globin gene expression. Next, we tested whether the LCR is necessary to activate globin gene expression in a gamma-globin promoter mutant that causes hereditary persistence of fetal hemoglobin (HPFH). beta-YAC transgenic mice carrying the -117 HPFH mutation and a HS3 core deletion that specifically abolishes gamma-globin gene expression during definitive erythropoiesis were produced to test whether the -117 (A)gamma promoter is activated in the absence of interaction with the LCR. In four transgenic mouse lines, gamma-globin gene expression was absent in adult erythrocytes, suggesting that an interaction between the gamma-globin gene promoter and the LCR is required for gamma gene activation even when the promoter contains an HPFH mutation.
Hum Mol Genet 2002 Apr 15
PMID:Activation of the beta-like globin genes in transgenic mice is dependent on the presence of the beta-locus control region. 1197 71

High-level beta-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1-HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb beta-globin locus yeast artificial chromosome and measuring the activity of beta-globin genes in GT6m beta-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the beta-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of epsilon- and gamma-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, gamma-globin gene expression was significantly reduced while beta-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal epsilon- and gamma-globin gene expression during embryonic erythropoiesis and for gamma-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression.
Hum Mol Genet 2003 Nov 15
PMID:Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes. 1450 28

The locus control region (LCR) is the most important cis-element in the regulation of beta-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of beta-LCR. To examine the effect of HS2, HS3, and HS2-HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human beta-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by beta-globin promoter and under the control of HS2, HS3, and HS2-HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of beta-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2-HS3), the two cis-elements showed a marked synergy in regulating beta-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer beta-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same beta-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of beta-globin gene in the developmental stages and the switch of gamma-->beta-globin genes. Our results indicate that the mechanism of gamma-->beta switch could be best explained by the "divided model."
Blood Cells Mol Dis
PMID:Effects of human locus control region elements HS2 and HS3 on human beta-globin gene expression in transgenic mouse. 1463 53

Using recombinase-mediated cassette exchange to test multiple transgenes at the same site of integration, we demonstrate a novel chromatin context-dependent silencer activity of the beta-globin locus control region (LCR). This silencer activity requires DNase I hypersensitive sites HS2 and HS3 but not HS4. After silencing, the silenced cassettes adopt a typical closed chromatin conformation (histone H3 and H4 deacetylation, histone H3-K4 methylation, DNA methylation, and replication in late S phase). In the absence of the LCR at the same site of integration, the chromatin remains decondensed. We demonstrate that the LCR is necessary but not sufficient to trigger these chromatin changes. We also provide evidence that this novel silencing activity is caused by transcriptional interference triggered by activation of transcription in the flanking sequences by the LCR.
Mol Cell Biol 2005 May
PMID:The human beta-globin locus control region can silence as well as activate gene expression. 1587 Feb 61

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.
Mol Cell Biol 2005 Aug
PMID:Synergistic and additive properties of the beta-globin locus control region (LCR) revealed by 5'HS3 deletion mutations: implication for LCR chromatin architecture. 1605 15

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.
J Mol Biol 2007 Jan 05
PMID:Cooperativeness of the higher chromatin structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of the LCR. 1705 66

Terminal deoxynucleotidyl transferase (TdT) expression is controlled at the transcriptional level, however, the TdT core promoter combining D, D', an initiator (Inr) and downstream basal elements (DBE) does not recapitulate the whole complex regulation of TdT expression. We hypothesized that important cis-regulatory elements of the gene are located outside of the TdT promoter. In an attempt to identify these elements, we performed DNase I hypersensitivity assays over 24kb including a 10kb region located upstream of the transcription start site (+1) and a 14kb region spanning exons and introns I to VI. Hypersensitive sites (HS) HS1 and HS2 were localized 8.5 and 8kb upstream of the transcription start site, respectively, and were exclusively detected in TdT+ cell types. HS3, HS4 and HS5 were mapped at positions -7, -3.4 and -3kb, respectively, and detected in both TdT negative and positive cells. HS6, HS7 and HS8 were detected immediately upstream of the TdT promoter. HS10 and HS11 were localized in the first and third intron of the gene. Luciferase reporter assays revealed that HS1, HS2 and HS3 synergize with the TdT promoter to activate transcription in a TdT+ pre-T cell line but not in a TdT+ pro-B cell line. In summary novel cis-regulatory elements have been identified in the 5' region of the TdT locus that synergize with the promoter to activate gene expression and our results suggest these elements may be more active in T cells.
Mol Immunol 2008 Feb
PMID:Identification of a new cis-regulatory element of the terminal deoxynucleotidyl transferase gene in the 5' region of the murine locus. 1785 98

Our data on 114 Iranian individuals with thalassemia intermedia phenotype revealed homozygous or compound heterozygous beta-globin mutations to be the predominant disease factor in 86.2% of cases. However, 8.2% of these individuals were found to be heterozygous or wild type for beta-globin mutations. In search for determinants outside of the beta-globin gene, which could be responsible for the unexpected thalassemia intermedia phenotype in these subjects, we screened the alpha-globin genes, the 5'HS3 and 5'HS4 regions of the beta-globin LCR, and the NF-E2 transcription factor for sequence variations in selected individuals. The -3.7 deletion was the only alpha-globin mutation detected, and no alterations were found in 5'HS3 and NF-E2. Sequence analysis of the 5'HS4 LCR core region identified three known SNPs in a single patient, who required irregular blood transfusions. The A/G polymorphism in the 5'HS4 palindromic region was also observed to be variable. Family studies were carried out on a female G/G homozygous patient, who received irregular blood transfusions. Her father, who had the same heterozygous IVSII-1 beta-globin mutation but the A/G genotype at the 5'HS4 palindromic site, presented with mild anemia and no requirement for blood transfusions. This suggests an impact of SNPs in the 5'HS4 LCR core region on the thalassemia phenotype and offers an interesting subject for further investigations in the Iranian population.
Blood Cells Mol Dis 2011 Mar 15
PMID:Analyzing 5'HS3 and 5'HS4 LCR core regions and NF-E2 in Iranian thalassemia intermedia patients with normal or carrier status for beta-globin mutations. 2123 98


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