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Results for “Locus Control Region”

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At least 199 records · Page 11Linked to original sources

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↗

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↗

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↗

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↗

PubMed1998

The dynamics of globin gene expression and gene therapy vectors.

The most important level of the regulation of the beta-globin genes is by activation of all of the genes by the locus control region (LCR). Part of the developmental regulation of the locus is achieved by competition of the genes for the interaction with the LCR. Although this level of gene regulation is quantitatively of less importance than the direct repression mechanism for the early genes, it has important implications and has provided an excellent assay to probe the regulation of transcription at the single cell level. The results of these studies indicate that the LCR interacts with individual globin genes and that LCR/gene interactions are dynamic with complexes forming and dissociating continually. We conclude that transcription only appears to take place while the LCR and gene interact and that the level of transcription is determined by the frequency and duration of such interaction rather than by changes in the rate of transcription of the promoters. This mechanism has clear implications for the design of vectors for the purpose of gene therapy.

Animals↗

PubMed1997

Transcriptional behavior of LCR enhancer elements integrated at the same chromosomal locus by recombinase-mediated cassette exchange.

Efficient integration of transgenes at preselected chromosomal locations was achieved in mammalian cells by recombinase-mediated-cassette-exchange (RMCE), a novel procedure that makes use of the CRE recombinase together with Lox sites bearing different spacer regions. We have applied RMCE to the study of the human beta-globin gene Locus Control Region by integrating at the same genetic locus in MEL cells, a LacZ gene driven by the human beta-globin promoter linked to HS2 and HS3 alone or in combination with HS4. Expression studies at the cell population level and in individual cells before and after induction of differentiation with hemin or DMSO show that the presence of these enhancers is associated with variegated patterns of expression. We were able to show that the LCR fragments tested act by controlling both the probability of expression and the rate of transcription of the linked beta-globin promoter. Both of these factors were also dependent on the state of differentiation of the MELc and on the presence of a second transcription unit located in cis. The ability to manipulate by RMCE constructs integrated into chromosomes should help in the creation of complex, rationally designed, artificial genetic loci.

Animals↗

PubMed2000

A DNase I hypersensitive site near the murine gamma1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation.

Several cis-acting elements regulate the expression of germline transcripts of heavy chain constant region genes and their subsequent switch recombination. To study such elements in the murine gamma1 gene, we have utilized a transgenic approach. In this study we focused on a DNase I hypersensitive site (termed 'Site II') that lies about 2 kb 3' of the gamma1 promoter region and I exon, just 5' to the gamma1 switch region. We have reported that gamma1 transgenes with Site II display the characteristics of a locus control region (LCR) in that they are insertion site independent and copy number dependent. For the present study we prepared six lines of transgenic mice that have the promoter region and I exon, but lack Site II. Expression of RNA from gamma1 transgenes that lack Site II is not correlated with transgene copy number; expression is insertion site dependent. This result indicates that DNase hypersensitive Site II is an important part of the LCR-like elements in the murine gamma1 gene. RNA expression from the gamma1 transgenes that lack Site II is inducible by IL-4 and by CD40 ligation. However, the induction of transgenic RNA expression by CD40 ligation is greater than expected, suggesting that elements within Site II participate in negative regulation of the amount of germline transcripts after CD40 ligation.

Animals↗

PubMed1997

The human beta globin locus introduced by YAC transfer exhibits a specific and reproducible pattern of developmental regulation in transgenic mice.

The human beta globin locus spans an 80-kb chromosomal region encompassing both the five expressed globin genes and the cis-acting elements that direct their stage-specific expression during ontogeny. Sequences proximal to the genes and in the locus control region, 60 kb upstream of the adult beta globin gene, are required for developmental regulation. Transgenic studies have shown that altering the structural organization of the locus disrupts the normal pattern of globin gene regulation. Procedures for introducing yeast artificial chromosomes (YACs) containing large genetic loci now make it possible to define the sequences required for stage-restricted gene expression in constructs that preserve the integrity of the beta globin locus. We demonstrate that independent YAC transgenic lines exhibit remarkably similar patterns of globin gene expression during development. The switch from gamma to beta globin predominant expression occurs between day 11.5 and 12.5 of gestation, with no more than twofold differences in human beta globin mRNA levels between lines. Human beta globin mRNA levels were twofold to fourfold lower than that of mouse betamaj, revealing potentially significant differences in the regulatory sequences of the two loci. These findings provide an important basis for studying regulatory elements within the beta globin locus.

Adult↗

PubMed2000

Regulation of the promoter for human immunoglobulin gamma3 germ-line transcription and its interaction with the 3'alpha enhancer.

The mechanism underlying the differential regulation of switching to human IgG subclasses is still largely unknown. We demonstrate that the region upstream of the initiation sites for gamma3 germ-line (GL) transcripts contains a functional promoter which is synergistically induced by IL-4, antibody to CD40 and phorbol dibutyrate in transient transfection assays in the human DG75 cell line. Linker-scanning mutations identified multiple elements in the 3' half of the evolutionarily conserved sequence that are required for inducibility. Electrophoretic mobility shift assays showed that Stat6 and NF-kappaB p50 / p65 are induced after stimulation, and bind to specific sequence motifs within the promoter. Overexpression of Stat6, NF-kappaB p50 / p65 and C / EBPgamma synergistically induced the GL gamma3 promoter. Insertion of DNA segments from the human 3' IgH regions, which may function as a locus control region for switch recombination, greatly activated the promoter in an orientation-independent manner. Duplication of the enhancer fragments resulted in a further increase of promoter activity. The greater enhancement of the HS1,2 fragment from the 3' alpha1 rather than the alpha2 locus may suggest a mechanistic explanation for the differential expression of various isotypes.

B-Lymphocytes↗

PubMed1998

Insertion of the IgH locus 3' regulatory palindrome in expression vectors warrants sure and efficient expression in stable B cell transfectants.

We have explored the effect of inserting 3' immunoglobulin heavy chain (IgH) locus transcriptional regulatory elements in stable expression vectors driven by a heavy chain variable gene promoter (pVH). A cassette was constructed, associating three enhancer elements from the palindromic part of the 3' IgH regulatory region, namely Calpha3'/hs3 reverse, alpha3'E/hs1-2, and hs3. As regard to stable expression, this cassette carried some features of a locus control region (LCR) and conferred expression to an associated cat reporter gene in the majority of B cells having integrated the transgene. The palindromic cassette was inserted in an expression vector carrying Ig light chain coding sequences. In this construct, transcription driven by a pVH promoter/Emu cassette upstream of the transcription initiation site was boosted by the palindromic cassette located downstream of the coding sequence. This potent expression plasmid mimicking the architecture of endogenous Ig loci, definitely manifested a potent stimulatory activity for stable transcription, outscoring conventional ubiquitous or B-cell specific expression vectors.

Animals↗

PubMed1995

Genetic interactions in thalassemia intermedia: analysis of beta-mutations, alpha-genotype, gamma-promoters, and beta-LCR hypersensitive sites 2 and 4 in Italian patients.

In order to verify the genetic factors influencing the clinical expression of beta-thalassemia we have studied 292 Italian patients, 165 with thalassemia intermedia and 127 with thalassemia major. The beta-globin gene mutations were defined in all cases. The number of alpha-globin genes and the integrity of specific control regions of the beta-globin cluster--gamma promoters and beta-Locus Control Region (beta-LCR)--were studied in selected cases. Homozygosity for mild mutations (group I) accounts for 24% of the intermedia patients and it is not represented among major patients. Forty-four percent of intermedia patients had combinations of mild/severe (group II) mutations and 32% had homozygosity or double heterozygosity for severe mutations (group III). Seventy-six percent of patients with thalassemia major were classified in group III and 24% in group II. Deletion type-alpha3.7 thalassemia, assessed in a part of the cases, was found in 5% of thalassemia major and 19.5% of intermedia patients in groups II and III. Structural analysis of gamma promoters and beta-LCR HS2 and HS4 regions, carried out in order to look for alterations associated with Hb F increase, did not reveal new mutations. Only rare polymorphic changes were observed at the HS2 and HS4 level. The -158G gamma C T change was found with an increased incidence in intermedia patients in groups II and III. A subset of 10 beta-thalassemia heterozygotes with mild intermedia phenotype resulted from coinheritance of a triplicated alpha-locus. We have been unable to find a molecular basis for the benign clinical course in approximately 20% of patients with thalassemia intermedia. Other genetic or acquired factors must be hypothesized which ameliorate the clinical condition.

Adolescent↗

PubMed1993

Extraembryonic expression of the human MHC class I gene HLA-G in transgenic mice. Evidence for a positive regulatory region located 1 kilobase 5' to the start site of transcription.

Trophoblast, the only fetal tissue in direct contact with maternal cells, fails to express the polymorphic HLA class I molecules HLA-A and -B, but does express the nonpolymorphic class I molecule HLA-G. It is thought that HLA-G may provide some of the functions of a class I molecule without stimulating maternal immune rejection of the fetal semiallograft. As a first step in identifying the cis-acting DNA regulatory elements involved in the control of class I expression by extraembryonic tissue, several types of transgenic mice were produced. Two HLA-G genomic fragments were used, 5.7 and 6.0 kb in length. These included the entire HLA-G coding region, 1 kb of 3' flanking sequence, and 1.2 or 1.4 kb of 5' flanking sequence, respectively. A hybrid transgene, HLA-A2/G, was produced by replacing the 5' flanking sequence, first exon, and early first intron of HLA-G with the corresponding elements of HLA-A. Comparison of transgene mRNA expression patterns seen in HLA-A2/G and HLA-G transgenic mice suggests that 5' flanking sequences are largely responsible for the differing patterns of expression typical of the classical class I and HLA-G genes. Studies comparing the extraembryonic HLA-G expression levels of founder embryos transgenic for either the 5.7- or 6.0-kb HLA-G transgene showed that the 6.0-kb transgene directed HLA-G expression far more efficiently than did the 5.7-kb HLA-G transgene, producing extraembryonic HLA-G mRNA levels similar to those seen in human extraembryonic tissues. The results of these studies suggest that the 250-bp fragment present at the extreme 5' end of the 6.0-kb HLA-G transgene and absent from the 5.7-kb HLA-G transgene contains an important positive regulatory element. This 250-bp fragment lies further upstream than any of the previously documented class I regulatory regions and may function as a locus control region.

Animals↗