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PubMed1997

Analysis of enhancer function of the HS-40 core sequence of the human alpha-globin cluster.

HS-40 is the major regulatory element of the human alpha-globin locus, located 40 kb upstream of the zeta-globin gene. To test for potential interactions between HS-40 and the beta- or the gamma-globin gene promoters in stable transfection assays, the HS-40 core sequence was cloned upstream of either the beta promoter or the gamma promoter driving the neomycin phosphotransferase gene and enhancer activity was measured using a colony assay. In K562 or in MEL cells, enhancer activity of HS-40 was higher than that of the individual core sequences of the DNase I hypersensitive sites (HS) of the beta-globin locus control region (LCR), and approximately 60% of the enhancer activity of a 2.5 kb microLCR, which contains the core elements of DNase I hypersensitive sites 1-4. In contrast to the synergistic interaction between the DNase I hypersensitive sites of beta locus LCR, combination of HS-40 with these DNase I hypersensitive sites failed to display cooperativity in K562 cells and inhibited enhancer function in MEL cells. Inhibition of enhancer function was also observed when two copies of the HS-40 were arranged tandemly. We conclude that the core element of HS-40 (i) is a powerful enhancer of gamma- and beta-globin gene expression, (ii) in contrast to other classical enhancers, acts best as a single copy, (iii) does not cooperate with the regulatory elements of the beta-globin locus control region.

Animals↗
PubMed2006

Antagonistic regulation of beta-globin gene expression by helix-loop-helix proteins USF and TFII-I.

The human beta-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the beta-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased beta-globin gene expression, while diminishing TFII-I activity increased beta-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult beta-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the beta-globin gene locus and play important roles in restricting beta-globin gene expression to the adult stage of erythropoiesis.

Animals↗
PubMed1995

Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the Krüppel family proteins Sp1 and EKLF.

An unresolved aspect of current understanding of erythroid cell-specific gene expression relates to how a limited number of transcriptional factors cooperate to direct high-level expression mediated by cis-regulatory elements separated over large distances within globin loci. In this report, we provide evidence that GATA-1, the major erythroid transcription factor, activates transcription in a synergistic fashion with two Krüppel family factors, the ubiquitous protein Sp1 and the erythroid-restricted factor EKLF (erythroid Krüppel-like factor), which recognize GC and/or GT/CACC motifs. Binding sites for both GATA-1 and these Krüppel proteins (especially Sp1) are found in close association in the promoters and enhancers of numerous erythroid cell-expressed genes and appear to cooperate in directing their expression. We have shown that GATA-1 interacts physically with Sp1 and EKLF and that interactions are mediated through their respective DNA-binding domains. Moreover, we show that GATA-1 and Sp1 synergize from a distance in constructs designed to mimic the architecture of globin locus control regions and downstream globin promoters. Finally, the formation of GATA-1-SP1 complexes was demonstrated in vivo by the ability of Sp1 to recruit GATA-1 to a promoter in the absence of GATA-binding sites. These experiments provide the first evidence for functionally important protein-protein interactions involved in erythroid cell-specific expression and suggest a mechanism by which DNA loops between locus control regions and globin promoters (or enhancers) might be formed or stabilized.

Animals↗
PubMed1994

Recombinant human hemoglobins designed for gene therapy of sickle cell disease.

Two human hemoglobins designed to inhibit the polymerization of sickle hemoglobin (Hb S; alpha 2 beta S2) have been produced. Mutations that disrupt the ability of Hb S to form polymers were introduced into the normal human beta-globin gene by site-specific mutagenesis. These mutations affect the axial and lateral contacts in the sickle fiber. The recombinant hemoglobin designated anti-sickling hemoglobin 1 (Hb AS1) contains the mutations beta 22 glutamic acid to alanine and beta 80 asparagine to lysine. Hb AS2 has the same beta 22 glutamic acid to alanine mutation combined with beta 87 threonine to glutamine. Human alpha- and beta AS-globin genes were separately fused downstream of beta-globin locus control region sequences and these constructs were coinjected into fertilized mouse eggs. Transgenic mouse lines that synthesize high levels of each anti-sickling hemoglobin were established and anti-sickling hemoglobins were purified from hemolysates and characterized. Both AS hemoglobins bind oxygen cooperatively and the oxygen affinities of these molecules are in the normal range. Delay time experiments demonstrate that Hb AS2 is a potent inhibitor of Hb S polymerization; therefore, locus control region beta AS2-globin gene constructs may be suitable for future gene therapy of sickle cell disease.

2,3-Diphosphoglycerate↗
PubMed1995

Thymic epithelial neoplasms in transgenic mice expressing SV40 T antigen under the control of an erythroid-specific enhancer.

The beta-globin locus control region is known to be a powerful erythroid-specific enhancer. In an attempt to produce immortalized erythroid percursor cells, transgenic mice have been generated with the simian virus (SV) 40 T antigen gene under the control of the locus control region. As previously reported, transgenic mice did not develop erythroleukaemia, but rather succumbed to insulinomas and poorly differentiated rhabdomyosarcomas. This paper describes additional mice containing this transgene that developed thymomas of the mixed epithelial/lymphocytic type, in which only the epithelial component expressed the T antigen. Epithelial cell lines have been established from these tumours. This system may be useful in future studies on the pathogenesis of thymomas and the function of thymic epithelial cells.

Animals↗
PubMed1996

Long-range disruption of gene expression by a selectable marker cassette.

Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in "knockout" mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the beta-like globin gene cluster. The insertion of PGK-Neo into the granzyme B gene, the most 5' gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the beta-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3' to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these-long distance effects are unknown, the expression of PGK-Neo can be "captured" by the regulatory domain into which it is inserted. These results suggest that the PGK-Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Animals↗
PubMed1997

Characterization of the chicken beta-globin insulator.

Insulators, first identified in Drosophila, are DNA sequence elements that shield a promoter from nearby regulatory elements. We have previously reported that a DNA sequence at the 5' end of the chicken beta-globin locus can function as an insulator. It is capable of shielding a reporter gene from the activating effects of a nearby mouse beta-globin locus control region element in the human erythroleukemic cell line K562. In this report, we show that most of the insulating activity lies in a 250-bp CpG island (core element), which contains the constitutive DNase I-hypersensitive site (5'HS4). DNA binding assays with the core sequence reveal a complex protein binding pattern. The insulating activity of the core element is multiplied when tandem copies are used. Although CpG islands are often associated with promoters of housekeeping genes, we find little evidence that the core element is a promoter. Furthermore, the insulator differs from a promoter in its ability to block the locus control region effect directionally.

Animals↗
PubMed1994

Distinct negative regulatory mechanisms involved in the repression of human embryonic epsilon- and fetal G gamma-globin genes in transgenic mice.

A current model for human beta-globin gene switching proposes that the stage-specific activation of embryonic and fetal globin genes requires the interaction of the beta-globin locus control region with proximal promoter elements. Subsequent repression in fetal and adult stages likely involves negative regulatory promoter elements and factors. To begin addressing these negative regulatory mechanisms, the regulation of human fetal G gamma-globin promoter fused to the SV40 T antigen gene was analyzed in transgenic mice. The results showed correct developmental expression in erythroid tissue, but lower levels of expression were also detected in non-erythroid tissue. Thus, the 5'-flanking G gamma-globin promoter sequence contains stage-specific erythroid elements but is lacking nonerythroid-specific negative elements. In contrast, the human embryonic epsilon-globin gene was only expressed in nonerythroid tissue of transgenic embryos, suggesting the presence of an erythroid-specific negative element(s). With the locus control region, complete repression of epsilon-globin RNA in fetal liver was observed in epsilon-globin genes without the previously characterized silencer, suggesting the presence of additional negative elements. Overall, this transgenic study suggests that distinct negative regulatory mechanisms function in the repression of embryonic and fetal globin genes.

Animals↗
PubMed1999

The gamma-globin promoter has a major role in competitive inhibition of beta-globin gene expression in early erythroid development.

The human gamma-globin gene competitively inhibits beta-globin gene expression in early erythroid development. To identify the gamma-globin gene sequences required for this effect, transgenic mice and stable transfection analyses with constructs containing 5'HS2 from the locus control region, modified gamma-globin genes, and the beta-globin gene were used. The -136 to +56 region of the gamma-globin promoter is necessary for competitive inhibition, as the beta-globin gene was inappropriately expressed in mouse embryos and in K562 and HEL cells containing constructs in which this region was deleted. Independently, the -140 to +56 region of gamma-globin gene was not sufficient to inhibit beta-globin transcription in mouse embryos or in cultured cells. Competitive inhibition of beta-globin gene expression was observed in K562 and HEL cells having a gamma-globin gene with a -161 promoter. The data suggest that the -161 gamma-globin promoter, which includes the CACCC box, two CCAAT boxes, the stage selector element (SSE), and TATA box, has a major role in suppressing beta-globin transcription early in development. Proteins binding to these or other gamma-globin promoter elements may interact with those binding to the locus control region, consequently precluding beta-globin transcription.

Animals↗
PubMed2002

Spectrum of beta-thalassemia mutations and their association with allelic sequence polymorphisms at the beta-globin gene cluster in an Eastern Indian population.

In this report, the spectrum of beta-thalassemia mutations and genotype-to-phenotype correlations were defined in large number of patients (beta-thalassemia carriers and major) with varying disease severity in an Eastern Indian population mainly from the state of West Bengal. The five most common beta-thalassemia mutations were detected, which included IVS1-5 (G-->C), codon 15 (G-->A), codon 26 (G-->A), codon 30 (G-->C), and codon 41/42 (-TCTT). These accounted for 85% in 80 beta-thalassemic alleles deciphered from 56 patients, including beta-thalassemia major and carriers, and 15% of alleles remained uncharacterized in these patients. Expression of the human beta-globin gene is regulated by an array of cis-acting DNA elements, including five DNase I hypersensitive sites (HSs) in the locus control region (LCR), promoters that incorporate certain silencer elements, and enhancers at 3' of the beta-globin gene. For detailed studies and to understand the molecular basis of beta-thalassemia, we studied two groups of subjects: a group of 12 patients from four families having beta-thalassemia major and carrier phenotype and a control group of 26 healthy individuals. In these two groups, we examined portions of the beta-globin gene locus control region HSs 1, 2, 3, and 4, which included the (CA)(x)(TA)(y) repeat motif, the (AT)(x)N(y)(AT)(z) repeat motif, the inverted repeat sequence TGGGGACCCCA, the promoter region of the (G)gamma-globin gene, an (AT)(x)(T)(y) repeat 5' of the silencer region, and the beta-globin gene and its 3' flanking region. We investigated the allelic sequence polymorphisms in these regions and their association with the beta-thalassemia mutations to know the possible genotype-phenotype relationship in beta-thalassemia patients. An analysis of cis-acting regulatory regions showed varied sequence haplotypes associated with some frequent beta-thalassemia mutations in this Eastern Indian population.

Adult↗
PubMed1995

Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains.

GATA-1, the founding member of a distinctive family of transcription factors, is expressed predominantly in erythroid cells and participates in the expression of numerous erythroid cell-expressed genes. GATA-binding sites are found in the promoters and enhancers of globin and nonglobin erythroid genes as well as in the alpha- and beta-globin locus control regions. To elucidate how GATA-1 may function in a variety of regulatory contexts, we have examined its protein-protein interactions. Here we show that GATA-1 self-associates in solution and in whole-cell extracts and that the zinc finger region of the molecule is sufficient to mediate this interaction. This physical interaction can influence transcription, as GATA-1 self-association is able to recruit a transcriptionally active but DNA-binding-defective derivative of GATA-1 to promoter-bound GATA-1 and result in superactivation. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, we have localized the minimal domain required for GATA-1 self-association to 40 amino acid residues within the C-terminal zinc finger region. Finally, we have detected physical interaction of GATA-1 with other GATA family members (GATA-2 and GATA-3) also mediated through the zinc finger domain. These findings have broad implications for the involvement of GATA factors in transcriptional control. In particular, the interaction of GATA-1 with itself and with other transcription factors may facilitate its function at diverse promoters in erythroid cells and also serve to bring together, or stabilize, loops between distant regulatory elements, such as the globin locus control regions and downstream globin promoters. We suggest that the zinc finger region of GATA-1, and related proteins, is multifunctional and mediates not only DNA binding but also important protein-protein interactions.

Amino Acid Sequence↗
PubMed2005

A general method to modify BACs to generate large recombinant DNA fragments.

Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA fragment containing the inverted human alpha-globin genes (theta, alpha1, alpha2, and zeta) from BAC191K2 and the locus control region (LCR) of human beta-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.

Chromosomes, Artificial, Bacterial↗
PubMed1998

Multiple control elements are required for expression of the human CD34 gene.

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.

Animals↗
PubMed1991

Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

Adult↗
PubMed1994

Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.

Antigens, CD↗
PubMed2005

HLA-G gene activation in tumor cells involves cis-acting epigenetic changes.

The tissue distribution of HLA-G molecules is broader than originally reported in trophoblastic cells. On the basis of numerous studies, HLA-G is also expressed in malignant tumors and involved in tumor immune escape. The mechanisms of HLA-G gene regulation differ from those of classical HLA class I genes and involve epigenetic processes. Here, we provide additional evidence on the influence of DNA demethylation on HLA-G activation. We also analyze the 5' regulatory region of HLA-G in 2 cellular models, melanoma (FON, M8) and choriocarcinoma (JEG-3, JAR), either expressing HLA-G transcripts or not. The data strongly suggest that HLA-G is silenced as a result of CpG site hypermethylation within a 5' regulatory region encompassing 450 bp upstream of the start codon, whereas it is activated upon demethylation. This result correlates with the acetylation status of histones within this region and the putative locus control region located at -1.2 kb. cis-acting epigenetic changes and the fact that demethylating agents activate HLA-G expression at least 5 days following treatment should be taken into account in epigenetic cancer therapies.

Burkitt Lymphoma↗
PubMed1995

DNase I-hypersensitive sites in the chromatin of rat growth hormone gene locus and enhancer activity of regions with these sites.

In this study, a determination was made of the chromatin structure of the rat growth hormone (GH) gene locus by DNase I sensitivity analysis using GC [GH+, prolactin (PRL)-], 235 (GH-, PRL+), GH3 (GH+, PRL+) and liver (GH-, PRL-) cells. From 7 kb upstream from the transcription start site to 19 kb downstream from the polyadenylation site, two major DNase I-hypersensitive sites (M-DHS; UIA, UIIA) and three M-DHS (DIA, DII, DIII) were found within 2 kb upstream and 7 kb downstream regions, respectively. Two minor DHS (m-DHS; UIB, UIIB) in the upstream region and one m-DHS (DIB) downstream were shown to be associated with M-DHS. Thus, a total of five M-DHS and three m-DHS were mapped on the rat GH gene locus. Among these, five (UIIB, UIA, UIB, DIB, DIA) including two (UIA, DIA) M-DHS were specific for GH-producing cells. UIIA and DIII were M-DHS only in PRL-producing 235 cells while the major hypersensitivity of DII was detected in GH-producing cells and liver cells. Assessment of the enhancing activity of the DHS regions indicated novel enhancers in one upstream and two downstream regions that function well with the GH promoter in GC cells. These enhancers, each appearing different, coincided with m-DHS but not M-DHS in GC cells, and were not activated by Pit-1. Based on these observations, the following functions of five M-DHS and three m-DHS regions were defined: enhancer; locus control region (LCR); switch region serving for conversion from GH/PRL-producing cells to PRL-producing cells; and a region having a structural function in chromatin.

Animals↗
PubMed1999

Nuclear matrix attachment regions antagonize methylation-dependent repression of long-range enhancer-promoter interactions.

The immunoglobulin intragenic mu enhancer region acts as a locus control region that mediates transcriptional activation over large distances in germ line transformation assays. In transgenic mice, but not in transfected tissue culture cells, the activation of a variable region (V(H)) promoter by the mu enhancer is dependent on flanking nuclear matrix attachment regions (MARs). Here, we examine the effects of DNA methylation, which occurs in early mouse development, on the function of the mu enhancer and the MARs. We find that methylation of rearranged mu genes in vitro, before transfection, represses the ability of the mu enhancer to activate the V(H) promoter over the distance of 1.2 kb. However, methylation does not affect enhancer-mediated promoter activation over a distance of 150 bp. In methylated DNA templates, the mu enhancer alone induces only local chromatin remodeling, whereas in combination with MARs, the mu enhancer generates an extended domain of histone acetylation. These observations provide evidence that DNA methylation impairs the distance independence of enhancer function and thereby imposes a requirement for additional regulatory elements, such as MARs, which facilitate long-range chromatin remodeling.

Acetylation↗