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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.
Punam Malik - One of the best experts on this subject based on the ideXlab platform.
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a novel combination of chicken Hypersensitive Site 4 insulator elements improves titers and restores full insulator activity
Blood, 2009Co-Authors: Paritha Arumugam, Fabrizia Urbinati, Chinavenmeni S Velu, Tomoyasu Higashimoto, Leighton H Grimes, Punam MalikAbstract:Abstract 3566 Poster Board III-503 Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. A prototypic insulator, the 1.2Kb chicken Hypersensitive Site-4 (cHS4) element utilizes CTCF and USF-1/2 motifs in the proximal 250bp. This smaller “core” cHS4 element provides enhancer blocking activity and reduces position effects. However, the cHS4 core sequences alone do not insulate viral vectors effectively. Two copies of the core, while effective in plasmid based systems, are unstable in viral vectors. In contrast, the full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers (Urbinati et al, Mol Ther 2009). Therefore, we performed a structure-function analysis of the full-length cHS4 in the context of self inactivating lentivirus-vectors to identify minimal insulator elements required for optimal insulation. Specifically, we analyzed transgene expression in the clonal progeny of primary murine hematopoietic stem cells using the secondary bone marrow transplant assay, and analyzed epigenetic changes in cHS4 and the transgene promoter in vitro in clonal integrants. As expected, the full length cHS4 insulator reduced clonal variegation in transgene expression, reduced position effects and blocked silencing-associated epigenetic modifications over the insulator core and the transgene promoter. However, while either the 5′ cHS4 250bp core or the 3′ cHS4 400bp sequences similarly effected only lower clonal variegation in transgene expression, when they were combined these 650bp sequences recapitulated the activity of the full length 1.2kb insulator, with minimal impact on viral titer. The distal 3′ 400bp fragment contains no consensus Sites for USF or CTCF. However, ChIP analysis on proviruses carrying only the 3′ 400bp showed that it binds CTCF. USF-1 binding to the 3′ 400bp, however, only occurred when both the 5′ 250bp core and the 3′ 400bp fragment were present in the proviruses. Indeed, the silencing associated epigenetic marks over the 3′ 400bp region were blocked only when the vector carried both these ends of cHS4 insulator sequences, suggesting that USF-1 bridges the 5′ core and the 3′ 400bp to confer full insulator activity. Furthermore, the 650bp sequences or the full length insulator had the maximal reduction in clonal dominance in the in vitro immortalization assay of lineage negative primary murine hematopoietic cells (Arumugam et al, Mol Ther. In press). Our studies confirm and extend earlier observations on the 5′ 250bp insulator core and identify a new “core-like” insulator activity in the 3′ end of cHS4. The specific elements in the 3′ 400bp sequences that promote interaction with the 5′ 250bp sequences would be important to determine, and may be present in other insulators in and across the genome/s. In the meanwhile, new vector systems flanked by this optimized ‘650bp’ cHS4 sequences, can provide excellent insulation of the transgene without significant loss in viral titers and have important safety and efficacy implications for gene therapy. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors. Disclosures: No relevant conflicts of interest to declare.
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the 3 region of the chicken Hypersensitive Site 4 insulator has properties similar to its core and is required for full insulator activity
PLOS ONE, 2009Co-Authors: Paritha Arumugam, Fabrizia Urbinati, Chinavenmeni S Velu, Tomoyasu Higashimoto, Leighton H Grimes, Punam MalikAbstract:Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken Hypersensitive Site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the "core", which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5' core with a novel 3' 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors.
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the 3 end of the chicken Hypersensitive Site 4 insulator has properties similar to the 5 insulator core and is necessary in conjunction with the core for full insulator activity
Blood, 2008Co-Authors: Paritha Arumugam, Fabrizia Urbinati, Chinavenmeni S Velu, Leighton H Grimes, Punam MalikAbstract:Genetic correction of hematologic defects is currently impeded by inefficient vector technology. We find that vectors that insulate the correcting transgene from position effects and genotoxicity compromise viral titers. Here we present an improved vector system which utilizes a modified insulator element, without sacrificing viral titers. Specifically, our genetic and epigenetic analysis of the 1.2kb chicken β-globin Hypersensitive Site-4 (cHS4) insulator reveal heretofore unknown activities in regions of the chicken β-globin insulator element outside the canonical and well studied 250bp 5′ “core” element. Previously, the core insulator activity was mapped to CTCF and USF-1/2 binding Sites, located only in the 5′ 250bp core. However, we find that the 5′ 250bp core alone is ineffective at shielding from position effects when it flanks transgenes, and gammaretrovirus/lentivirus vectors. In contrast, the entire 1.2kb cHS4 efficiently insulates, but significantly lowers titers of lentiviral vectors. To identify insulating activities which might be appended to the 5′ 250bp core to properly insulate transgene expression cassettes without sacrificing viral titers, we performed a structure-function analysis of the cHS4 insulator placed within the 3′LTR of a lentivirus containing the regulatory and coding sequences of human b-globin (Table 1). We compared single-copy clonal progeny of mouse erythroleukemia cells (MEL) and primary transduced and transplanted hematopoietic stem cells for position effects. Additionally, we studied repressive and activating histone marks over the transgene promoter and cHS4 in the different proviruses. Our data indicate that while all vectors containing the core reduced the coefficient of variation (CV) of human b-globin (HbA) expression, several constructs suggested that cHS4 sequences in the most 3′ 400bp (furthest from the core) may be critical to full length insulator activity. We next analyzed HbA expression in vector-corrected thalassemia mice, and generated single copy secondary CFU-S, the gold standard for studying chromatin position effects (Table 2). While all vectors containing the cHS4 core provided some ‘insulator’ activity when compared to the uninsulated vector control (conceivably by reducing the CV/clonal variegation) the full length 1.2kb insulator vector provided maximum shielding from position effects, with nearly 2.5-fold higher HbA expression compared to the uninsulated vector. These data were confirmed in secondary CFU-S. Epigenetic analyses of the vector b-globin promoter revealed that transcriptionally repressive histone modifications were decreased, and activating histone modifications increased when the last 400bp sequences of cHS4 were present. Notably, vectors carrying only the 3′ 400bp sequences of cHS4 reduced clonal variegation in MEL cells and secondary CFU-S, but did not increase HbA expressing cells both in vitro and in vivo (Table 2). However, full insulator activity was restored in MEL clones when both the 5′ 250bp core was combined with the 3′ 400bp element. The addition of the 3′ 400bp element to the core was accompanied with a significant enrichment of active histone marks and minimal repressive histone marks in the provirus as seen with the 1.2Kb insulator. These data consolidate the known insulating activity of the 5′ 250bp core element with a novel 3′ 400bp element which (together) constitutes a new insulated vector system with excellent insulating properties and viral titers. Our data have important implications in the design of gene therapy vectors, where optimal insulator activity can be achieved with a minimal reduction in viral titers. Table 1. Single copy MEL clones showing effect of insulator sequences on position effects in β-globin carrying lentiviruses Table 2. Effect of insulator sequences on the differentiated progeny of transduced and transplanted thalassemia hematopoietic stem cells
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improved human β globin expression from self inactivating lentiviral vectors carrying the chicken Hypersensitive Site 4 chs4 insulator element
Molecular Therapy, 2007Co-Authors: Paritha I Arumugam, Jessica Scholes, Natalya Perelman, Ping Xia, Jiingkuan Yee, Punam MalikAbstract:Effective gene therapy for β-thalassemia major (β-TM) requires consistent, high expression of human β-globin (hβ-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hβ/hγ-globin genes and large elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hβ/hγ-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken Hypersensitive Site-4 (cHS4) chromatin insulator element and compared them with their “un-insulated” counterparts. We observed a consistent twofold-higher hβ expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hβ expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hβ-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hβ-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hβ expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.
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67 the chicken Hypersensitive Site chs4 insulator element reduces position effects thereby increasing expression from globin lentiviral vectors
Molecular Therapy, 2006Co-Authors: Paritha I Arumugam, Jessica Scholes, Natalya Perelman, Ping Xia, Jiingkuan Yee, Alexander Zarzuela, Punam MalikAbstract:Lentiviral vectors (LV) carrying the human |[beta]|-globin gene (h|[beta]|) and locus control region (LCR) have changed the field of gene therapy for hemoglobinopathies. However, their random integration into host cells results in variable h|[beta]| expression from chromatin position effects. We analyzed the role of a 1.2 kb chicken |[beta]|-globin locus Hypersensitive Site 4 insulator element (cHS4) in a self-inactivating (SIN) LV. The BGM vector (carrying h|[beta]|/LCR) was compared to an analogous vector BGMI, with cHS4 such that it flanks the provirus upon integration. Both vectors additionally carried the mutant P140K methyl guanine methyl transferase (MGMTP140K) cDNA to enrich for genetically modified cells. First, murine erythroleukemia (MEL) cells were transduced at <5% transduction efficiency to generate single-copy clones and transduced clones identified by PCR ('Unselected' clones) and analyzed for h|[beta]| expression by FACS and RNase Protection Assay (RPA). 'Unselected' BGMI+-MEL clones had a higher proportion of h|[beta]|+ cells (68|[plusmn]|3%) compared to BGM+ clones (36|[plusmn]|9%, n=24; P<0.001) and a reduced coefficient of variation (CV) of h|[beta]| expression: 168|[plusmn]|20 vs. 327|[plusmn]|64 in BGM (P<0.02). RPA showed a 2-fold increase in h|[beta]|/total m|[alpha]|-globin mRNA: BGMI 45|[plusmn]|6% vs. BGM 21|[plusmn]|7%, n=24, P<0.01. Next, MEL cells were selected with BG/BCNU and single-copy clones isolated ('Selected' clones). 'Selected' BGMI+-MEL clones also had a higher proportion of h|[beta]|+ cells compared to BGM+ clones (80|[plusmn]|15% vs. 72|[plusmn]|20%, n=86, P<0.03), with reduced CV (140|[plusmn]|2.8 vs. 170|[plusmn]|13, P<0.01) and higher h|[beta]|-mRNA (BGMI+ 83.6%|[plusmn]|40 vs. 49.6%|[plusmn]|25, n=24, P<0.02). Two additional vectors termed BG and BGI (with cHS4) were constructed with the h|[beta]|/LCR but no MGMTP140K cassette. RPA showed that BGI+ MEL pools expressed 85% h|[beta]|/total m|[alpha]|-globin/ vector copy as compared to 38% from BG. BG and BGI single copy MEL clones showed similar results. In in vivo studies lethally irradiated normal mice were transplanted with BGM and BGMI- transduced thalassemia hematopoietic stem cells (MOI 20). Engraftment and vector copy number in both groups were similar (66%|[plusmn]|15% vs. 68%|[plusmn]|10%, and 0.21 vs. 0.17 in BGMI and BGM groups, respectively, n=12). While, 18|[plusmn]|4% of RBC expressed h|[beta]| in the BGMI group, only 4%|[plusmn]|1.5% expressed h|[beta]| in the BGM group (P<0.001). There was a 4-fold increase in chimeric hemoglobin (m|[alpha]|- h|[beta]|) in BGMI mice (13%|[plusmn]|4% vs. 3%|[plusmn]|2% in BGM mice, P<0.001). Secondary colony forming units-spleen (CFU-S) derived from these mice showed increased numbers of h|[beta]|+ cells in BGMI (21%|[plusmn]|4% vs. 8%|[plusmn]|4% BGM, n=30, P<0.03) with reduced CV (698|[plusmn]|91 vs. 987|[plusmn]|99 in BGM, P<0.04). Taken together, 'insulated' SIN-h|[beta]|/LCR LV increased the probability of expression of integrants and reduced chromatin position effects, resulting in consistent and higher expression of h|[beta]|. The enhancer blocking effect of the cHS4, although not tested here, would further improve the bio-safety of these lineage- specific, SIN LV.
Philippe Leboulch - One of the best experts on this subject based on the ideXlab platform.
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Retroviral transfer of a human beta-globin/delta-globin hybrid gene linked to beta locus control region Hypersensitive Site 2 aimed at the gene therapy of sickle cell disease.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Ken Julian Takekoshi, Karen Westerman, Irving M. London, Philippe LeboulchAbstract:Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I Hypersensitive Site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients.
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retroviral transfer of a human beta globin delta globin hybrid gene linked to beta locus control region Hypersensitive Site 2 aimed at the gene therapy of sickle cell disease
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Ken Julian Takekoshi, Karen Westerman, Irving M. London, Philippe LeboulchAbstract:Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I Hypersensitive Site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients.
Charalampos G Spilianakis - 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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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.
Vincent Murray - One of the best experts on this subject based on the ideXlab platform.
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chromatin structure at the flanking regions of the human beta globin locus control region dnase i Hypersensitive Site 2 proposed nucleosome positioning by dna binding proteins including gata 1
Biochimica et Biophysica Acta, 2004Co-Authors: Neil P Davies, John Freebody, Vincent MurrayAbstract:Abstract The human beta-globin locus control region DNase I Hypersensitive Site-2 (LCR HS-2) is erythroid-specific and is located 10.9 kb upstream of the epsilon-globin gene. Most studies have only examined the core region of HS-2. However, previous studies in this laboratory indicate that positioned nucleosomes are present at the 5′- and 3′-flanking regions of HS-2. In addition, footprints were observed that indicated the involvement of DNA-binding proteins in positioning the nucleosome cores. A consensus GATA-1 Site exists in the region of the 3′-footprint. In this study, using an electrophoretic mobility shift assay (EMSA) and DNase I footprinting, we confirmed that GATA-1 binds in vitro at the 3′-end of HS-2. An additional GATA-1 Site was found to bind GATA-1 in vitro at a Site positioned 40 bp upstream. At the 5′-end of HS-2, DNase I footprinting revealed a series of footprints showing a marked correlation with the in vivo footprints. EMSA indicated the presence of several erythroid-specific complexes in this region including GATA-1 binding. Sequence alignment for 12 mammalian species in HS-2 confirmed that the highest conservation to be in the HS-2 core. However, a second level of conservation extends from the core to the Sites of the proposed positioning proteins at the HS-2 flanking regions, before declining rapidly. This indicates the importance of the HS-2 flanking regions and supports the proposal of nucleosome positioning proteins in these regions.
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chromatin structure at the 3 boundary of the human β globin locus control region Hypersensitive Site 2
The International Journal of Biochemistry & Cell Biology, 2001Co-Authors: Aeri Kim, Vincent MurrayAbstract:Abstract Chromatin structure was examined at the 3′-boundary region of the human β-globin locus control region Hypersensitive Site-2 (LCR HS-2) using several footprinting agents. Erythroid K562 cells (possessing HS-2) were damaged by the footprinting agents: hedamycin, bleomycin and four nitrogen mustard analogues. Purified DNA and non-erythroid HeLa cells (lacking HS-2) were also damaged as controls for comparison with K562 cells. The comparison between intact cells and purified DNA showed several protected regions in K562 cells. A large erythroid-specific protected region of 135 bp was found at the boundary of HS-2. The length of this protected region (135 bp) was close to that of DNA contained in a nucleosome core (146 bp). Another two protected regions were found upstream of the protected region. A 16-bp erythroid-specific footprint co-localised with a GATA-1 motif—this indicated that the GATA-1 protein could be involved in positioning the nucleosome. Further upstream, a 100-bp footprint coincided with an AT-rich region. Thus our footprinting results suggest that the 3′-boundary of LCR HS-2 is flanked by a positioned nucleosome and that an erythroid-specific protein binds to the sequence adjacent to the nucleosome and acts to position the nucleosome at the boundary of the Hypersensitive Site.
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A large “footprint” at the boundary of the human β-globin locus control region Hypersensitive Site-2
The international journal of biochemistry & cell biology, 2000Co-Authors: Aeri Kim, Vincent MurrayAbstract:Abstract The 5′-boundary region of the human β-globin locus control region Hypersensitive Site-2 (HS-2) was examined for protein–DNA interactions. The HS-2 is an erythroid specific DNase I Hypersensitive Site that extends for approximately 600 bp. Erythroid K562 cells and non-erythroid HeLa cells were damaged by bleomycin and hedamycin — these agents are able to “footprint” nucleosome cores and proteins bound to DNA. The fragments generated by DNA damage were amplified by the ligation-mediated polymerase chain reaction with primers specific for the 5′-boundary region of HS-2 and examined at base pair resolution on DNA sequencing gels. The intensity of damage in intact cells was compared with that in purified DNA. The comparison between intact cells and purified DNA revealed a protected region of 226 bp with bleomycin and 182 bp with hedamycin in K562 cells. The length of the protected region was consistent with the presence of a nucleosome core. We postulate that an erythroid-specific protein binds next to the positioned nucleosome at the boundary of HS-2 to prevent sliding of the nucleosome into the Hypersensitive Site — this would also account for the large size of the protected region. HeLa cells (lacking a Hypersensitive Site in the β-globin cluster) did not have an area of protection in this region.
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protein dna interactions in the human β globin locus control region Hypersensitive Site 2 as revealed by four nitrogen mustards
Nucleic Acids Research, 1997Co-Authors: Mark D Temple, William A. Denny, Murray J Cairns, Vincent MurrayAbstract:Four nitrogen mustards have been used in this study to examine protein-DNA interactions in intact human cells, specifically at the locus control region Hypersensitive Site-2 (LCR HS-2) of the human beta-globin locus. Three of these nitrogen mustards are DNA-targeted by attachment of an acridine or amsacrine intercalating chromophore, while the fourth (chlorambucil) is a non-targeted mustard. The ligation-mediated PCR technique was used to determine the Sites of damage at base pair resolution on DNA sequencing gels. A densitometric comparison was made between DNA damaged in intact erythroid K562 cells and in purified DNA. The intensity of DNA damage Sites in the LCR HS-2 were found to differ significantly between intact K562 cells and purified DNA. At the NF-E2/AP-1 motif, pronounced damage protection was observed in DNA derived from drug treated cells. The nuclear factor- erythroid 2 (NF-E2) protein factor is thought to bind at this NF-E2/AP-1 motif in K562 cells. Other Sites of protection and enhancement that corresponded to known transcription factor binding Sites were also detected. These nitrogen mustards are therefore very effective compounds for detection of transcription factor binding to DNA in intact cells and are superior to other commonly used agents. The sequence selectivity of the compounds was determined using plasmid DNA and compared to that found in intact cells. The acridine-based nitrogen mustard had a preference for forming adducts at guanine bases, while the two amsacrine-based nitrogen mustards and chlorambucil formed adducts at both guanine and adenine bases.