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.

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-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 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

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 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.

Position independence and proper developmental control of gamma-globin gene expression require both a 5' locus control region and a downstream sequence element

1994 ◽  
Vol 14 (9) ◽  
pp. 6087-6096
Author(s):  
Q Li ◽  
J A Stamatoyannopoulos
Keyword(s):  
Transgenic Mice ◽  
Control Region ◽  
Locus Control Region ◽  
Regulatory Sequences ◽  
Globin Genes ◽  
Sequence Element ◽  
Position Effects ◽  
Developmental Control ◽  
Developmental Patterns ◽  
Gamma Globin

We have analyzed the expression of human gamma-globin genes during development in F2 progeny of transgenic mice carrying two types of constructs. In the first type, gamma-globin genes were linked individually to large (approximately 4-kb) sequence fragments spanning locus control region (LCR) hypersensitive site 2 (HS2) or HS3. These LCR fragments contained not only the core HS elements but also extensive evolutionarily conserved flanking sequences. The second type of construct contained tandem gamma- and beta-globin genes linked to identical HS2 or HS3 fragments. We show that gamma-globin expression in transgenic mice carrying HS2 gamma or HS3 gamma constructs is highly sensitive to position effects and that such effects override the cis regulatory elements present in these constructs to produce markedly different developmental patterns of gamma-globin expression in lines carrying the same transgene. In contrast, gamma-globin expression in both HS2 gamma beta and HS3 gamma beta mice is sheltered from position effects and the developmental patterns of gamma-globin expression in lines carrying the same transgene are identical and display stage-specific regulation. The results suggest that cis regulatory sequences required for proper developmental control of fetal globin expression in the presence of an LCR element reside downstream from the gamma genes.

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.

scholarly journals Sheltering of gamma-globin expression from position effects requires both an upstream locus control region and a regulatory element 3' to the A gamma-globin gene.

1997 ◽  
Vol 17 (1) ◽  
pp. 240-247 ◽  
Author(s):  
J A Stamatoyannopoulos ◽  
C H Clegg ◽  
Q Li
Keyword(s):  
Transgenic Mice ◽  
Control Region ◽  
Regulatory Element ◽  
Globin Gene ◽  
Locus Control Region ◽  
Regulatory Elements ◽  
Sequence Information ◽  
Hindiii Site ◽  
Position Effects ◽  
Gamma Globin

Integration position-independent expression of human globin transgenes in transgenic mice requires the presence of regulatory elements from the beta-globin locus control region (LCR) in the transgene construct. However, several recent studies have suggested that, while clearly necessary, such elements are not by themselves sufficient to realize this effect. In the case of the human fetal gamma-globin genes, previous results have indicated that additional regulatory information required for sheltering of gamma-globin transgene expression from position effects may reside downstream from the A gamma gene. To investigate this possibility, we established 17 lines of transgenic mice carrying constructs comprising a micro-LCR (microLCR) element, an A gamma-globin gene fragment, and a variable length of 3' sequence information beyond the A gamma 3' HindIII site. gamma-Globin expression during development was studied in 170 individual F2 progeny from these lines. We find that gamma-globin expression becomes sheltered from position effects when the normally position-sensitive microLCR-A gamma construct is extended by 600 bp beyond the 3' HindIII site to include a previously identified regulatory sequence (the A gamma-globin enhancer), the functional significance of which in vivo had heretofore been unclear. The results suggest that the mechanism whereby an upstream LCR achieves sheltering of globin gene expression from position effects involves cooperation with a gene-proximal regulatory element distinct from the promoter region.

New Concepts in Genome Regulation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-47-SCI-47
Author(s):  
Ann Dean ◽  
Jongjoo Lee ◽  
Ryan Dale ◽  
Ivan Krivega
Keyword(s):  
Sickle Cell Disease ◽  
Long Range ◽  
Control Region ◽  
Sickle Cell ◽  
Globin Gene ◽  
Locus Control Region ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Cell Disease ◽  
Gamma Globin

Abstract Manipulating gene regulation to favor gamma-globin transcription over beta-globin transcription has been a goal of research in erythropoiesis for decades because of its relevance to amelioration of the pathophysiology of sickle cell disease and beta-thalassemia. A fundamental unanswered question in biology is how the unique pattern of gene expression, the transcriptome, of the many different individual mammalian cell types arises from the same genome blueprint and changes during development and differentiation. There is a growing appreciation that genome organization and the folding of chromosomes is a key determinant of gene transcription. Within this framework, enhancers function to increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding, or looping. These long range interactions are orchestrated by cell type specific proteins and protein complexes that bind to enhancers and promoters and stabilize their interaction with each other. We have been studying LDB1, a member of an erythroid protein complex containing GATA1, TAL1 and LMO2. The LDB1complex activates erythroid genes through occupancy of virtually all erythroid enhancers. LDB1engages in homo- and heterotypic interactions with proteins occupying the promoters of erythroid genes to bring them into proximity with their enhancers. We find that enhancer long range looping activity can be redirected. Both targeting of the beta-globin locus control region to the gamma-globin gene in adult erythroid cells by the tethering of LDB1 or epigenetic unmasking of a silenced gamma-globin gene lead to increased locus control region (LCR)/gamma-globin contact frequency and reduced LCR/beta-globin contact. The outcome of these manipulations is robust, pan-cellular gamma-globin transcription activation with a concomitant reduction in beta-globin transcription. These examples suggest that chromosome looping can be considered a therapeutic target for gene activation or gene silencing to ameliorate genetic diseases such as sickle cell disease and beta-thalassemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Human β-Globin Locus Control Region HS5Contains CTCF- and Developmental Stage-Dependent Enhancer-BlockingActivity in ErythroidCells

2003 ◽  
Vol 23 (24) ◽  
pp. 8946-8952 ◽  
Author(s):  
Keiji Tanimoto ◽  
Akiko Sugiura ◽  
Akane Omori ◽  
Gary Felsenfeld ◽  
James Douglas Engel ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Control Region ◽  
Yeast Artificial Chromosome ◽  
Globin Gene ◽  
Artificial Chromosome ◽  
Locus Control Region ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Globin Genes ◽  
Mechanistic Basis

ABSTRACT The human β-globin locus contains five developmentally regulatedβ -type globin genes. All five genes depend on the locus control region (LCR), located at the 5′ end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human β-globin yeast artificial chromosome transgenic mice and observed reduced expression of all theβ -type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancer-blocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the ε-globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the β-globin gene.

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

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.

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