The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Richard A Flavell - One of the best experts on this subject based on the ideXlab platform.
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hypersensitive site 6 of the th2 Locus Control Region is essential for th2 cytokine expression
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Adam Williams, Charalampos G Spilianakis, Soo Seok Hwang, Stephanie C Eisenbarth, Richard A FlavellAbstract:The T helper type 2 (Th2) cytokine genes Il4, Il5, and Il13 are contained within a 140-kb Region of mouse chromosome 11 and their expression is Controlled by a Locus Control Region (LCR) embedded within this Locus. The LCR is composed of a number of DNase I–hypersensitive sites (HSs), which are believed to encompass the regulatory core of the LCR. To determine the function of these sites, mutant mice were generated in which combinations of these HSs had been deleted from the endogenous LCR, and the effect on Th2 cytokine expression was assessed through the use of in vivo and in vitro models. These experiments revealed that, although all of the hypersensitive sites analyzed are important for appropriate LCR function, some sites are more important than others in regulating cytokine expression. Interestingly, each LCR mutation showed contrasting effects on cytokine expression, in some cases with mutants displaying opposing phenotypes between in vitro cultures and in vivo immunizations. These studies indicated that Rad50 hypersensitive site 6 was the singularly most important HS for Th2 cytokine expression, displaying consistent reductions in cytokine levels in all models tested. Furthermore analysis of chromatin modifications revealed that deletion of Rad50 hypersensitive site 6 impacted epigenetic modifications at the promoters of the Il4, Il5, and Il13 genes as well as other regulatory sites within the Th2 Locus.
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demethylation of a specific hypersensitive site in the th2 Locus Control Region
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Patrick E Fields, Richard A FlavellAbstract:A growing body of literature has examined and implicated DNA methylation as a critical epigenetic modification in T helper (Th) cell differentiation. The absence of DNA methyltransferases or methyl-binding proteins derepresses many cytokine loci, allowing their ectopic expression, while methylation of specific CpG residues is sufficient to prevent expression. Here, we characterize demethylation events of the Th2 cytokine Locus Control Region (LCR). rad50 hypersensitive site 7 (RHS7), a hypersensitive site within this LCR, becomes demethylated in a STAT6-dependent manner and only in cells stimulated under type 2 conditions. Robust demethylation appears to require signaling contributions from both IL-4 receptor, via STAT6, and CD28, but it cannot be effected by GATA3. Finally, RHS7 is demethylated independently of cell division, consistent with an “active,” rather than passive, mechanism. Taken together, these findings firmly connect RHS7 demethylation and Th2 LCR activation in the type 2 differentiation program.
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hypersensitive site 7 of the t h 2 Locus Control Region is essential for expressing t h 2 cytokine genes and for long range intrachromosomal interactions
Nature Immunology, 2005Co-Authors: Charalampos G Spilianakis, Richard A FlavellAbstract:Several regulatory Regions are important for the expression of genes encoding T helper type 2 (TH2) cytokines, including TH2-specific DNase I hypersensitivity sites in the TH2 cytokine Locus Control Region. Among these sites, Rad50 hypersensitive site 7 (RHS7) shows rapid TH2-specific demethylation after antigenic stimulation. To investigate the function of RHS7 in TH2 cell differentiation, we have generated RHS7-deficient mice. CD4+ T cells and mast cells showed a notable reduction in TH2 cytokine expression in vitro and TH2 responses in vivo were considerably impaired in RHS7-deficient mice. Deletion of RHS7 did not affect the expression of a linked Rad50 gene, but it did reduce long-range intrachromosomal interactions between the Locus Control Region and promoters of the TH2 cytokine genes. Our findings show that RHS7 is essential for the proper regulation of TH2 cytokine gene expression.
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hypersensitive site 7 of the th2 Locus Control Region is essential for expressing th2 cytokine genes and for long range intrachromosomal interactions
Nature Immunology, 2005Co-Authors: Gap Ryol Lee, Richard A Flavell, Charalampos G SpilianakisAbstract:Several regulatory Regions are important for the expression of genes encoding T helper type 2 (TH2) cytokines, including TH2-specific DNase I hypersensitivity sites in the TH2 cytokine Locus Control Region. Among these sites, Rad50 hypersensitive site 7 (RHS7) shows rapid TH2-specific demethylation after antigenic stimulation. To investigate the function of RHS7 in TH2 cell differentiation, we have generated RHS7-deficient mice. CD4+ T cells and mast cells showed a notable reduction in TH2 cytokine expression in vitro and TH2 responses in vivo were considerably impaired in RHS7-deficient mice. Deletion of RHS7 did not affect the expression of a linked Rad50 gene, but it did reduce long-range intrachromosomal interactions between the Locus Control Region and promoters of the TH2 cytokine genes. Our findings show that RHS7 is essential for the proper regulation of TH2 cytokine gene expression.
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th2 specific chromatin remodeling and enhancer activity in the th2 cytokine Locus Control Region
Immunity, 2004Co-Authors: Patrick E Fields, Gap Ryol Lee, Sean T Kim, Victor Bartsevich, Richard A FlavellAbstract:We recently identified a 3' Region of the rad50 gene possessing strong enhancer activity as well as activity consistent with function as a Locus Control Region (LCR) for the flanking Th2 cytokine genes. In this study, we identify several functional elements within this Region by examining chromatin changes as well as activity in transgenic mice. We find within this Region four DNase I hypersensitive clusters, three of which are highly conserved and predominantly expressed in Th2 cells. Histone acetylation of this Region is elevated in Th2 cells. Further, one of the hypersensitive sites (RHS7) is rapidly demethylated in Th2, but not Th1, cells. In transgenic mice, these hypersensitive sites impart strong, Th2-specific enhancer activity as well as copy number-dependent expression of the reporter gene, recapitulating LCR function. We postulate that these sites function alone or in combination with other regulatory elements to coordinate gene expression in the Th2 cytokine Locus.
Ross C Hardison - One of the best experts on this subject based on the ideXlab platform.
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functional and binding studies of hs3 2 of the beta globin Locus Control Region
Gene, 2002Co-Authors: Joseph M Molete, Webb Miller, Hanna M Petrykowska, Martin Sigg, Ross C HardisonAbstract:The distal Locus Control Region (LCR) is required for high-level expression of the complex of genes (HBBC) encoding the b-like globins of mammals in erythroid cells. Several major DNase hypersensitive sites (HSs 1‐5) mark the LCR. Sequence conservation and direct experimental evidencehave implicated sequences within andbetween the HScores infunction ofthe LCR. In this reportwe confirm the mapping of a minor HS between HS3 and HS4, called HS3.2, and show that sequences including it increase the number of random integration sites at which a drug resistance gene is expressed. We also show that nuclear proteins including GATA1 and Oct1 bind specifically to sequences within HS3.2. However, the protein Pbx1, whose binding site is the best match to one highly conserved sequence, does not bind strongly. GATA1 and Oct1 also bind in the HS cores of the LCR and to promoters in HBBC. Their binding to this minor HS suggests that they may be used in assembly of a large complex containing multiple regulatory sequences. q 2002 Elsevier Science B.V. All rights reserved.
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sequences flanking hypersensitive sites of the β globin Locus Control Region are required for synergistic enhancement
Molecular and Cellular Biology, 2001Co-Authors: Joseph M Molete, Webb Miller, Yongqing Feng, Eric E Bouhassira, Hanna M Petrykowska, Ross C HardisonAbstract:The major distal regulatory sequence for the b-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.
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a negative cis element regulates the level of enhancement by hypersensitive site 2 of the β globin Locus Control Region
Journal of Biological Chemistry, 2001Co-Authors: Laura Elnitski, Webb Miller, Constance T Noguchi, Ross C HardisonAbstract:Abstract The core of DNase hypersensitive site (HS) 2 from the β-globin Locus Control Region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5′ and 3′ ends of the core. Analysis of constructs with and without the 5′ negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5′ end of the HS2 core map the negative element within a 20-base pair Region containing two conserved sequences.
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description and targeted deletion of 5 hypersensitive site 5 and 6 of the mouse β globin Locus Control Region
Blood, 1998Co-Authors: M A Bender, Ross C Hardison, Steven Fiering, Elliot Epner, Andreas Reik, Jennie Close, Agnes Telling, Mark GroudineAbstract:The most upstream hypersensitive site (HS) of the β-globin Locus Control Region (LCR) in humans (5′ HS 5) and chickens (5′ HS 4) can act as an insulating element in some gain of function assays and may demarcate a β-globin domain. We have mapped the most upstream HSs of the mouse β-globin LCR and sequenced this Region. We find that mice have a Region homologous to human 5′ HS 5 that is associated with a minor HS. In addition we map a unique HS upstream of 5′ HS 5 and refer to this novel site as mouse 5′ HS 6. We have also generated mice containing a targeted deletion of the Region containing 5′ HS 5 and 6. We find that after excision of the selectable marker in vivo, deletion of 5′ HS 5 and 6 has a minimal effect on transcription and does not prevent formation of the remaining LCR HSs. Taken together these findings suggest that the most upstream HSs of the mouse β-globin LCR are not necessary for maintaining the β-globin Locus in an active configuration or to protect it from a surrounding repressive chromatin environment.
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phylogenetic footprinting of hypersensitive site 3 of the β globin Locus Control Region
Blood, 1997Co-Authors: David A Shelton, Webb Miller, Ross C Hardison, Lauren Stegman, Jeffery H Bock, Jerry L Slightom, Morris Goodman, Deborah L GumucioAbstract:Hypersensitive site 3 (HS3) of the β-like globin Locus Control Region has been implicated as an important regulator of the β-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding Regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two γ genes (galago γ is embryonic; human γ is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of γ in adult life).
Frank Grosveld - One of the best experts on this subject based on the ideXlab platform.
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hs5 of the human β globin Locus Control Region a developmental stage specific border in erythroid cells
The EMBO Journal, 2003Co-Authors: Albert W K Wai, Sara Pruzina, Sjaak Philipsen, Nynke Gillemans, Selina Raguzbolognesi, Gaetano Zafarana, Dies Meijer, Frank GrosveldAbstract:Elements with insulator/border activity have been characterized most extensively in Drosophila melanogaster. In vertebrates, the first example of such an element was provided by a hypersensitive site of the chicken beta-globin Locus, cHS4. It has been proposed that the homologous site in humans, HS5, functions as a border of the human beta-globin Locus. Here, we have characterized HS5 of the human beta-globin Locus Control Region. We have examined its tissue-specificity and assessed its insulating properties in transgenic mice using a lacZ reporter assay. Most importantly, we have tested its enhancer blocking activity in the context of the full beta-globin Locus. Our results show that HS5 is erythroid-specific rather than ubiquitous in human tissues. Furthermore, HS5 does not fulfil the criteria of a general in vivo insulator in the transgene protection assay. Finally, a HS5 conditional deletion from the complete Locus demonstrates that HS5 has no discernable activity in adult erythroid cells. Surprisingly, HS5 functions as an enhancer blocker in embryonic erythroid cells. We conclude that HS5 is a developmental stage-specific border in erythroid cells.
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altered dna binding specificity mutants of eklf and sp1 show that eklf is an activator of the β globin Locus Control Region in vivo
Genes & Development, 1998Co-Authors: Nynke Gillemans, Frank Grosveld, Rita Tewari, Fokke Lindeboom, Robbert J Rottier, Ton De Wit, Mark Wijgerde, Sjaak PhilipsenAbstract:The Locus Control Region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for Locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3.
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erythroid kruppel like factor eklf is active in primitive and definitive erythroid cells and is required for the function of 5 hs3 of the β globin Locus Control Region
The EMBO Journal, 1998Co-Authors: Rita Tewari, Frank Grosveld, Nynke Gillemans, Mark Wijgerde, Beatriz Nuez, Marieke Von Lindern, Sjaak PhilipsenAbstract:Disruption of the gene for transcription factor EKLF (erythroid Kruppel-like factor) results in fatal anaemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid-specific genes, including the embryonic epsilon- and fetal gamma-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the beta-gene promoter, even though it is already present in embryonic red cells. Here, we show that a beta-globin gene linked directly to the Locus Control Region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF-/- embryos. Thus, embryonic beta-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the Locus Control Region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5'HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5'HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from gamma- to beta-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in beta-globin gene activation.
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a dominant chromatin opening activity in 5 hypersensitive site 3 of the human beta globin Locus Control Region
The EMBO Journal, 1996Co-Authors: James Ellis, Sjaak Philipsen, Kian Tanun, Alex Harper, D Michalovich, Nikos Yannoutsos, Frank GrosveldAbstract:Single-copy human beta-globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the Locus Control Region (LCR). Here we show that the 6.5 kbp microLocus LCR cassette reproducibly directs full expression from independent single-copy beta-globin transgenes. By testing individual DNase I-hypersensitive sites (HS) present in the microLocus cassette, we demonstrate that the 1.5 kbp 5'HS2 enhancer fragment does not direct beta-globin expression from single-copy transgenes. In contrast, the 1.9 kbp 5'HS3 fragment directs beta-globin expression in five independent single-copy transgenic mouse lines. Moreover, the 5'HS3 core element and beta-globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5'HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5'HS2. We postulate that the different elements of the LCR form a 'holocomplex' that interacts with the individual globin genes.
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transcriptional activation by hypersensitive site three of the human β globin Locus Control Region in murine erythroleukemia cells
Biochimica et Biophysica Acta, 1994Co-Authors: Sara Pruzina, M Antoniou, J Hurst, Frank Grosveld, Sjaak PhilipsenAbstract:In this paper we describe a complete deletional analysis of hypersensitive site three (HS3) of the human beta-globin Locus Control Region (LCR). The previously defined core fragment consists of 6 footprinted Regions, with multiple binding sites for the erythroid-specific factor GATA-1 and G-rich motifs that can interact with ubiquitous factors such as Sp1 and TEF-2. We show in this paper that the 5' half of this fragment is the most important for activity in murine erythroleukemia (MEL) cells. A fragment containing footprints 1-4 can stimulate transcription of a linked human beta-globin gene to levels of about 40% of that obtained with footprints 1-6. Constructs containing either footprints 1-3 or 3-6 cannot be distinguished from the beta-globin gene alone. We further show that binding sites for the erythroid-specific factor NF-E2 can co-operatively interact with parts of the HS3 core fragment, and that HS3 requires elements upstream from -103 in the human beta-globin promoter for full activity. The importance of these results is discussed in the context of the regulation of the genes in the human beta-globin cluster.
Sjaak Philipsen - One of the best experts on this subject based on the ideXlab platform.
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hs5 of the human β globin Locus Control Region a developmental stage specific border in erythroid cells
The EMBO Journal, 2003Co-Authors: Albert W K Wai, Sara Pruzina, Sjaak Philipsen, Nynke Gillemans, Selina Raguzbolognesi, Gaetano Zafarana, Dies Meijer, Frank GrosveldAbstract:Elements with insulator/border activity have been characterized most extensively in Drosophila melanogaster. In vertebrates, the first example of such an element was provided by a hypersensitive site of the chicken beta-globin Locus, cHS4. It has been proposed that the homologous site in humans, HS5, functions as a border of the human beta-globin Locus. Here, we have characterized HS5 of the human beta-globin Locus Control Region. We have examined its tissue-specificity and assessed its insulating properties in transgenic mice using a lacZ reporter assay. Most importantly, we have tested its enhancer blocking activity in the context of the full beta-globin Locus. Our results show that HS5 is erythroid-specific rather than ubiquitous in human tissues. Furthermore, HS5 does not fulfil the criteria of a general in vivo insulator in the transgene protection assay. Finally, a HS5 conditional deletion from the complete Locus demonstrates that HS5 has no discernable activity in adult erythroid cells. Surprisingly, HS5 functions as an enhancer blocker in embryonic erythroid cells. We conclude that HS5 is a developmental stage-specific border in erythroid cells.
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altered dna binding specificity mutants of eklf and sp1 show that eklf is an activator of the β globin Locus Control Region in vivo
Genes & Development, 1998Co-Authors: Nynke Gillemans, Frank Grosveld, Rita Tewari, Fokke Lindeboom, Robbert J Rottier, Ton De Wit, Mark Wijgerde, Sjaak PhilipsenAbstract:The Locus Control Region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for Locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3.
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erythroid kruppel like factor eklf is active in primitive and definitive erythroid cells and is required for the function of 5 hs3 of the β globin Locus Control Region
The EMBO Journal, 1998Co-Authors: Rita Tewari, Frank Grosveld, Nynke Gillemans, Mark Wijgerde, Beatriz Nuez, Marieke Von Lindern, Sjaak PhilipsenAbstract:Disruption of the gene for transcription factor EKLF (erythroid Kruppel-like factor) results in fatal anaemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid-specific genes, including the embryonic epsilon- and fetal gamma-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the beta-gene promoter, even though it is already present in embryonic red cells. Here, we show that a beta-globin gene linked directly to the Locus Control Region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF-/- embryos. Thus, embryonic beta-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the Locus Control Region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5'HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5'HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from gamma- to beta-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in beta-globin gene activation.
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role of dna sequences outside the cores of dnase hypersensitive sites hss in functions of the β globin Locus Control Region domain opening and synergism between hs2 and hs3
Journal of Biological Chemistry, 1996Co-Authors: John D Jackson, Webb Miller, Sjaak Philipsen, Hanna M Petrykowska, Ross C HardisonAbstract:The roles of each DNase hypersensitive site (HS), and the DNA sequences between them, in the activity of the Locus Control Region of the mammalian b-globin gene domain were examined by placing human and rabbit restriction fragments containing the cores of HS2, HS3, HS4, and HS5, along with varying amounts of flanking DNA, upstream of a hybrid e-globin-luciferase reporter gene and testing for effects on expression both prior to and after integration into the chromosomes of K562 cells, a human erythroid cell line. Prior to integration, fragments containing HS2 enhanced expression to the greatest extent, and the modest enhancement by some fragments containing HS3 correlated with the presence of a well-conserved binding site for AP1/NFE2. The stronger effects of larger Locus Control Region DNA fragments in clones of stably transfected cells indicates a role for sequences outside the HS cores after integration into the genome. The strong effect of a 1.9-kilobase HindIII fragment containing HS3 after, but not prior to, integration argues for the presence of a chromatin domain-opening activity. Use of a rabbit DNA fragment containing both HS2 and HS3 demonstrated a synergistic interaction between the two HSs when their natural context and spacing are preserved.
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a dominant chromatin opening activity in 5 hypersensitive site 3 of the human beta globin Locus Control Region
The EMBO Journal, 1996Co-Authors: James Ellis, Sjaak Philipsen, Kian Tanun, Alex Harper, D Michalovich, Nikos Yannoutsos, Frank GrosveldAbstract:Single-copy human beta-globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the Locus Control Region (LCR). Here we show that the 6.5 kbp microLocus LCR cassette reproducibly directs full expression from independent single-copy beta-globin transgenes. By testing individual DNase I-hypersensitive sites (HS) present in the microLocus cassette, we demonstrate that the 1.5 kbp 5'HS2 enhancer fragment does not direct beta-globin expression from single-copy transgenes. In contrast, the 1.9 kbp 5'HS3 fragment directs beta-globin expression in five independent single-copy transgenic mouse lines. Moreover, the 5'HS3 core element and beta-globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5'HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5'HS2. We postulate that the different elements of the LCR form a 'holocomplex' that interacts with the individual globin genes.
Webb Miller - One of the best experts on this subject based on the ideXlab platform.
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functional and binding studies of hs3 2 of the beta globin Locus Control Region
Gene, 2002Co-Authors: Joseph M Molete, Webb Miller, Hanna M Petrykowska, Martin Sigg, Ross C HardisonAbstract:The distal Locus Control Region (LCR) is required for high-level expression of the complex of genes (HBBC) encoding the b-like globins of mammals in erythroid cells. Several major DNase hypersensitive sites (HSs 1‐5) mark the LCR. Sequence conservation and direct experimental evidencehave implicated sequences within andbetween the HScores infunction ofthe LCR. In this reportwe confirm the mapping of a minor HS between HS3 and HS4, called HS3.2, and show that sequences including it increase the number of random integration sites at which a drug resistance gene is expressed. We also show that nuclear proteins including GATA1 and Oct1 bind specifically to sequences within HS3.2. However, the protein Pbx1, whose binding site is the best match to one highly conserved sequence, does not bind strongly. GATA1 and Oct1 also bind in the HS cores of the LCR and to promoters in HBBC. Their binding to this minor HS suggests that they may be used in assembly of a large complex containing multiple regulatory sequences. q 2002 Elsevier Science B.V. All rights reserved.
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sequences flanking hypersensitive sites of the β globin Locus Control Region are required for synergistic enhancement
Molecular and Cellular Biology, 2001Co-Authors: Joseph M Molete, Webb Miller, Yongqing Feng, Eric E Bouhassira, Hanna M Petrykowska, Ross C HardisonAbstract:The major distal regulatory sequence for the b-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.
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a negative cis element regulates the level of enhancement by hypersensitive site 2 of the β globin Locus Control Region
Journal of Biological Chemistry, 2001Co-Authors: Laura Elnitski, Webb Miller, Constance T Noguchi, Ross C HardisonAbstract:Abstract The core of DNase hypersensitive site (HS) 2 from the β-globin Locus Control Region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5′ and 3′ ends of the core. Analysis of constructs with and without the 5′ negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5′ end of the HS2 core map the negative element within a 20-base pair Region containing two conserved sequences.
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phylogenetic footprinting of hypersensitive site 3 of the β globin Locus Control Region
Blood, 1997Co-Authors: David A Shelton, Webb Miller, Ross C Hardison, Lauren Stegman, Jeffery H Bock, Jerry L Slightom, Morris Goodman, Deborah L GumucioAbstract:Hypersensitive site 3 (HS3) of the β-like globin Locus Control Region has been implicated as an important regulator of the β-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding Regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two γ genes (galago γ is embryonic; human γ is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of γ in adult life).
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conserved e boxes function as part of the enhancer in hypersensitive site 2 of the beta globin Locus Control Region role of basic helix loop helix proteins
Journal of Biological Chemistry, 1997Co-Authors: Laura Elnitski, Webb Miller, Ross C HardisonAbstract:The human beta-globin gene cluster is regulated in part by a distal Locus Control Region that is required for opening a chromatin domain in erythroid cells and enhancing expression of the beta-like globin genes at the correct developmental stages. One part of the Locus Control Region, called hypersensitive site 2 (HS2), functions as a strong enhancer. Matches to the consensus binding sites for basic helix-loop-helix (bHLH) proteins (E boxes) are well conserved within the HS2 core. We show that mutations of the HS2 core that alter an invariant E box cause a 3.5-fold reduction in enhancement of expression of an epsilon-globin reporter gene in transiently transfected K562 cells, both before and after induction. Mutations of the HS2 core that alter a less-highly conserved E box cause a more modest reduction in enhancement. Footprint analysis shows binding of erythroid nuclear proteins in vitro to the invariant E box as well as an adjacent CAC/GTG box. Probes containing the E box Regions form sequence-specific complexes with proteins from both K562 and MEL nuclear extracts; these are disrupted by the same mutations that decrease enhancement. Some of these latter complexes contain known bHLH proteins, as revealed by specific loss of individual complexes when treated with antibodies against TAL1 and USF. Interaction between the E boxes and the bHLH proteins, as well as other binding proteins, could account for the role of these sites in enhancement by HS2.
