scholarly journals Acetylation of a Specific Promoter Nucleosome Accompanies Activation of the ɛ-Globin Gene by β-Globin Locus Control Region HS2

2001 ◽  
Vol 21 (4) ◽  
pp. 1155-1163 ◽  
Author(s):  
Chang-Yun Gui ◽  
Ann Dean
Keyword(s):  
Control Region ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Trichostatin A ◽  
Globin Gene ◽  
Chromatin Modification ◽  
Locus Control Region ◽  
Micrococcal Nuclease ◽  
Nuclease Sensitivity

ABSTRACT On stably replicating episomes, transcriptional activation of the ɛ-globin promoter by the β-globin locus control region HS2 enhancer is correlated with an increase in nuclease sensitivity which is limited to the TATA-proximal nucleosome (N1). To elucidate what underlies this increase in nuclease sensitivity and the link between chromatin modification and gene expression, we examined the nucleoprotein composition and histone acetylation status of transcriptionally active and inactive promoters. Micrococcal nuclease digestion of active promoters in nuclei released few nucleosome-like nucleoprotein complexes containing N1 sequences in comparison to results with inactive promoters. We also observed that N1 DNA fragments from active promoters are of a subnucleosomal length. Nevertheless, chromatin immunoprecipitation experiments indicate that histones H3 and H4 are present on N1 sequences from active promoters, with H3 being dramatically hyperacetylated compared with that from inactive promoters and vector sequences. Strikingly, H3 in the adjacent upstream nucleosome (N2) does not appear to be differentially acetylated in active and inactive promoters, indicating that the nucleosome modification of the promoter that accompanies transactivation by HS2 is highly directed and specific. However, global acetylation of histones in vivo by trichostatin A did not activate transcription in the absence of HS2, suggesting that HS2 contributes additional activities necessary for transactivation. N1 sequences from active promoters also contain reduced levels of linker histone H1. The detection of a protected subnucleosomal sized N1 DNA fragment and the recovery of N1 DNA sequences in immunoprecipitations using anti-acetylated H3 and H4 antibodies argue that N1 is present, but in an altered conformation, in the active promoters.

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.

scholarly journals The 87-kD A gamma-globin enhancer-binding protein is a product of the HOXB2(HOX2H) locus

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1420-1427 ◽  
Author(s):  
PK Sengupta ◽  
DE Lavelle ◽  
J DeSimone
Keyword(s):  
Fusion Protein ◽  
Developmental Stage ◽  
Control Region ◽  
Dna Sequences ◽  
Nuclear Protein ◽  
Globin Gene ◽  
Locus Control Region ◽  
Specific Factor ◽  
Regulatory Sequences ◽  
Gamma Globin

Abstract Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta- globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

scholarly journals Locus control region elements HS1 and HS4 enhance the therapeutic efficacy of globin gene transfer in β-thalassemic mice

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4175-4178 ◽  
Author(s):  
Leszek Lisowski ◽  
Michel Sadelain
Keyword(s):  
Gene Transfer ◽  
Control Region ◽  
Therapeutic Option ◽  
Globin Gene ◽  
Locus Control Region ◽  
Thalassemia Major ◽  
Hematopoietic Stem ◽  
Promising Alternative ◽  
High Level

Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with β-thalassemia major. In this approach, high level, erythroid-specific globin transgene expression should correct ineffective erythropoiesis and hemolytic anemia following the delivery of only 1 to 2 vector copies per cell. The generation of vectors that provide high-level globin expression and require low vector copy (VC) integration is therefore essential for both safety and efficacy. We show here the major roles played by 2 lesser-known locus control region elements, termed HS1 and HS4. Partial deletions within HS4 markedly reduce in vivo globin expression requiring multiple VC per cell to correct the anemia. Most strikingly, addition of HS1 to HS2-3-4 increases globin expression by 52%, yielding 9 g Hb/VC in β-thalassemic mice. Thus, while vectors encoding HS2-3-4 provide curative levels of hemoglobin at 1 to 2 copies per cell, adding HS1 is a promising alternative strategy if upcoming clinical trials prove higher levels of expression to be necessary.

scholarly journals The 87-kD A gamma-globin enhancer-binding protein is a product of the HOXB2(HOX2H) locus

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1420-1427
Author(s):  
PK Sengupta ◽  
DE Lavelle ◽  
J DeSimone
Keyword(s):  
Fusion Protein ◽  
Developmental Stage ◽  
Control Region ◽  
Dna Sequences ◽  
Nuclear Protein ◽  
Globin Gene ◽  
Locus Control Region ◽  
Specific Factor ◽  
Regulatory Sequences ◽  
Gamma Globin

Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta- globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

The Importance of the Human β-Globin Locus Control Region HS1 and HS4 Elements for Therapeutic β-Globin Gene Expression in β-Thalassemic Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 272-272 ◽  
Author(s):  
Leszek Lisowski ◽  
Michel Sadelain
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Transgene Expression ◽  
Transcriptional Control ◽  
Globin Gene ◽  
Locus Control Region ◽  
Thalassemia Major ◽  
Flanking Region ◽  
Globin Promoter

Abstract Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with β-thalassemia major. In this approach, high level, erythroid specific transgene expression is needed to correct ineffective erythropoiesis and hemolytic anemia following the delivery of few copies of therapeutic vector per cell. Several groups have successfully treated mouse models of severe hemoglobinopathies utilizing lentiviral vectors encoding β- or γ-globin genes placed under the transcriptional control of the human β-globin promoter and the HS2, HS3 and HS4 elements of the β-globin locus control region. The HS2 and HS3 elements are the most powerful and the best characterized single elements within the LCR. The relative importance of HS1 and HS4 is less well defined. We show here the major roles played by HS1 and HS4, which although not seen in MEL cells, are striking in β-thalassemic mice. The effect of HS1 element was tested in vectors, derived from the previously published TNS9 vector, that harbor different globin promoters (either 265, 615 or 1555bp in length). Addition of HS1 to vectors containing the 615bp or 1555bp promoters had no effect on average transgene expression per vector copy (VC) and even decreased average transgene expression from 38±3% (n=32 MEL cell pools) to 26±2% (n=23) of endogenous β-globin levels (p<0.001) in the context of the 265bp promoter. In vivo, however, addition of HS1 had a dramatic effect on globin expression. Transgene expression increased from 27±6% of the endogenous β-globin mRNA to 41±9% for vectors harboring the HS1 element (p<0.001), after normalization to vector copy number. On the Hb level, the vectors without HS1 element provided 4–6g/dl/VC, while addition of HS1 increased this value to 9g/dl/VC. To evaluate the effect of HS4 on gene expression, we created panel of vectors with truncations of 3′ or 5′ flanking regions of HS4. In vectors harboring LCR HS1-4, the 5′ truncation significantly decreased mean in vivo globin expression from 26±2.5% to 20±2% of the endogenous β-globin (p<0.001). A similar effect was observed for 3′ or 5′ truncations in vectors lacking HS1 element. The 5′ flanking region of HS4 was also replaced with an unrelated DNA spacer, the same size fragment of HS3 flanking region, or the human IFN-β S/MAR element. Only the addition of the S/MAR element rescued the function of HS4, restoring average globin expression to the level of vectors encoding the full HS4 element, which suggests that this region may contain a functional S/MAR element. This analysis underscores the importance of carefully analyzing the size and relative positioning of transcriptional control elements within tissue-specific vectors, as well as the critical importance of assessing these elements in animal models of disease. Based on this analysis, we are proceeding to a phase I clinical trial in subjects with β-thalassemia major, utilizing the TNS9.3 vector, which harbors the 615bp human β-globin promoter and HS2–3–4, providing curative levels of hemoglobin at 1 to 2 copies per cell. Addition of HS1 is a promising alternative strategy if higher levels of expression are eventually needed.

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.

Deletions within the Mouse β-Globin Locus Control Region Preferentially Reduce βmin Globin Gene Expression

Genomics ◽  
2000 ◽  
Vol 63 (3) ◽  
pp. 417-424 ◽  
Author(s):  
Raouf Alami ◽  
M.A. Bender ◽  
Yong-Qing Feng ◽  
Steven N. Fiering ◽  
Bruce A. Hug ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Globin Gene Expression

scholarly journals Locus control region-A gamma transgenic mice: a new model for studying the induction of fetal hemoglobin in the adult

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1326-1333 ◽  
Author(s):  
P Constantoulakis ◽  
B Josephson ◽  
L Mangahas ◽  
T Papayannopoulou ◽  
T Enver ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Control Region ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Locus Control Region ◽  
In Vivo Studies ◽  
Regulatory Sequences ◽  
Mouse Development ◽  
New Model ◽  
Gamma Globin

Abstract All pharmacologic agents that induce fetal hemoglobin (Hb) have been discovered with in vivo studies of humans, macaques, and baboons. We tested whether transgenic mice carrying human fetal (gamma) globin genes provide a model for studying the pharmacologic induction of HbF in the adult. In initial studies, phenylhydrazine-induced hemolytic anemia, 5-azacytidine, butyrate, or combinations of these treatments failed to activate the human gamma-globin gene in a transgenic mouse line carrying a 4.4-kb G gamma globin gene construct that is expressed only in the embryonic stage of mouse development. Subsequently, adult mice carrying the human A gamma gene linked to the locus control region (LCR) regulatory sequences and expressing heterocellularly HbF (about 25%, gamma-positive cells) were used. Treatments with erythropoietin, 5- azacytidine, hydroxyurea, or butyrate resulted in induction of gamma gene expression as documented by measurement of F-reticulocytes, the gamma/gamma + beta biosynthetic ratio and the level of steady state gamma mRNA. Administration of erythropoietin or butyrate to transgenic mice carrying a muLCR-beta (human) globin construct, failed to increase human beta-globin expression. These results suggest that the muLCR-A gamma transgenic mice provide a new model for studying the induction of fetal Hb in the adult.

scholarly journals A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897 ◽  
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

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