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Results for “Locus Control Region”

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PubMed1994

Cloning and functional characterization of LCR-F1: a bZIP transcription factor that activates erythroid-specific, human globin gene expression.

DNase I hypersensitive site 2 (HS 2) of the human beta-globin Locus Control Region (LCR) directs high level expression of the beta-globin gene located 50 kilobases downstream. Experiments in cultured cells and in transgenic mice demonstrate that duplicated AP1-like sites in HS 2 are required for this powerful enhancer activity. A cDNA clone encoding a basic, leucine-zipper protein that binds to these sites was isolated and designated Locus Control Region-Factor 1 (LCR-F1). This protein is a member of a new family of regulatory factors that contain a 63 amino acid 'CNC domain' overlapping the basic region. This domain is approximately 70% identical in the Drosophila Cap N Collar (CNC) protein, NF-E2 and LCR-F1. LCR-F1 transactivates an HS 2/gamma-globin reporter gene over 170-fold in transient transfection experiments specifically in erythroid cells. These results suggest that LCR-F1 may be a critical factor involved in LCR-mediated, human globin gene expression.

Animals↗

PubMed1998

Studying the recruitment of Sp1 to the beta-globin promoter with an in vivo method: protein position identification with nuclease tail (PIN*POINT).

Transcription is thought to be regulated by recruitment of transcription factors, adaptors, and certain enzymes to cis-acting elements through protein-DNA interactions and protein-protein interactions. To better understand transcription, a method with the capability to detect in vivo recruitment of these individual proteins will be essential. Toward this end, we use a previously undescribed in vivo method that we term protein position identification with nuclease tail (PIN*POINT). In this method, a fusion protein composed of a chosen protein linked to a nonsequence-specific nuclease is expressed in vivo, and the binding of the protein to DNA is made detectable by the nuclease-induced cleavage near the binding site. In this article, we used the technique protein position identification with nuclease tail to study the effect of the beta-globin locus control region (LCR) and promoter elements on the recruitment of transcription factor Sp1 to the beta-globin promoter. We present evidence that the hypersensitive sites of the LCR synergistically enhance the recruitment of a multimeric Sp1 complex to the beta-globin promoter and that this may be accomplished by protein-protein interactions with proteins bound to the LCR, the upstream activator region, and, possibly, general transcription factors bound near the "TATA" box.

Base Sequence↗

PubMed1996

Regulation of azurophil granule-associated serine protease genes during myelopoiesis.

During myelopoiesis, the primary or azurophil granules are synthesized at the promyelocyte stage of development. The biogenesis of these granules requires the coordinated regulation of a large number of genes that comprise the constituent proteins of the granules themselves. The genes encoding the granule-associated proteins appear to be regulated primarily at the level of gene transcription; that is, most of the granule genes appear to be transcriptionally activated at the beginning of the promyelocyte stage and are transcriptionally repressed at the transition to myelocytes. The cis-acting DNA elements and trans-regulatory factors that control the regulation of granule genes are just beginning to be defined. The granule-associated genes are dispersed throughout the genome; however, several of the serine protease genes expressed in these granules are tightly clustered and the tight clustering may play an important role for their regulation. Recent transgenic studies suggest that DNA elements required for myeloid-specific targeting may be located just upstream from or very near to the granule genes themselves. DNA elements similar to the globin locus control region will probably play a role in the expression of these genes, although locus control elements have not yet been defined for any azurophil granule genes. Future studies directed at defining these key regulatory elements will enhance our understanding of the molecular events that underlie myeloid development.

Azure Stains↗

PubMed1998

Chromatin interaction mechanism of transcriptional control in vivo.

We have used a kinetic analysis to distinguish possible mechanisms of activation of transcription of the different genes in the human beta globin locus. Based on in situ studies at the single-cell level we have previously suggested a dynamic mechanism of single genes alternately interacting with the locus control region (LCR) to activate transcription. However, those steady-state experiments did not allow a direct measurement of the dynamics of the mechanism and the presence of loci with in situ primary transcript signals from two beta-like genes in cis has left open the possibility that multiple genes in the locus could initiate transcription simultaneously. Kinetic assays involving removal of a block to transcription elongation in conjunction with RNA FISH show that multiple beta gene primary transcript signals in cis represent a transition between alternating transcriptional periods of single genes, supporting a dynamic interaction mechanism.

Animals↗

PubMed1998

Individual LCR hypersensitive sites cooperate to generate an open chromatin domain spanning the human beta-globin locus.

BACKGROUND: The human beta-globin locus control region (LCR) is composed of five DNase I hypersensitive (HS) sites located 5' to the multiple genes it regulates. The LCR has been shown to comprise, among other essential properties, an activity that is required for generating a chromatin structure which renders the entire beta-globin gene locus accessible to exogenous nucleases. This nuclease-sensitive state is generally believed to be reflective of the chromatin environment that is permissive for transcriptional activation of the globin genes. RESULTS: Here we show, in mice bearing intact YAC transgenes that encompass the whole human beta-globin locus, that the deletion of individual core LCR HS sites negatively affects the ability of the LCR to confer this open chromatin conformation throughout the locus, and when analysed in concert with the effect that these same mutations have on transcription, the data show that the chromatin opening activity is a necessary, but not sufficient, prerequisite for globin gene expression. The results also show that after deletion of individual hypersensitive sites, the mutated LCR is no longer able to provide an accessible chromatin environment that is independent from the site of YAC transgene integration. CONCLUSIONS: These experiments provide further evidence for the hypothesis that the HS sites must act cooperatively to fulfil the multiple functions that are attributable to the LCR.

Adult↗

PubMed1993

V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer.

We have assessed the importance of the immunoglobulin heavy chain (IgH) intron enhancer for recombination of variable gene segments (V, D and J) during B cell development. We generated chimeric mice with embryonic stem cells lacking the intron enhancer from one of their IgH loci. The IgH intron enhancer was substituted by a short oligonucleotide through homologous recombination using the 'Hit and Run' procedure. V(D)J recombination occurred less frequently on mutant alleles, but was not blocked completely. Quantitative polymerase chain reaction analyses demonstrated that 15-30% of the mutated loci in mature B cells were unrearranged, in striking contrast to the wild-type alleles. The remainder of the mutated loci underwent D-J (65-80%) as well as V-DJ rearrangements, although the latter were less frequent (3-6%). These results are in line with previous data which showed that the V(D)J recombination machinery is modulated through cis-regulatory elements within the intron enhancer. However, our data predict the existence of additional cis-regulatory element(s) which, together with the intron enhancer, are required to activate the V(D)J recombination machinery fully. Such cis-regulatory element(s) might function as an enhancer of recombination or as a locus control region regulating the accessibility of the IgH locus.

Animals↗

PubMed1992

Production of functional human hemoglobin in transgenic swine.

A construct containing the locus control region (LCR) from the human beta globin locus together with two copies of the human alpha 1 gene and a single copy of the human beta A gene was used to obtain three transgenic pigs. The transgenic pigs are healthy, not anemic, and grow at a rate comparable to non-transgenic littermates. All animals expressed the human genes. However, alpha globin was consistently expressed at higher levels than beta globin. Isolation of the human hemoglobin from both porcine hemoglobin and other non-hemoglobin proteins was accomplished by ion exchange chromatography. The purified porcine derived human hemoglobin exhibited an oxygen affinity similar to that of human derived human hemoglobin.

Animals↗

PubMed1991

Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.

Amino Acid Sequence↗

PubMed1993

Locating well-conserved regions within a pairwise alignment.

Within a single alignment of two DNA sequences or two protein sequences, some regions may be much better conserved than others. Such strong conservation may reveal a region that possesses an important function. When alignments are so long that it is infeasible, or at least undesirable, to inspect them in complete detail, it is helpful to have an automatic process that computes information about the varying degree of conservation along the alignment and displays the information in a graphical representation that is readily assimilated. This paper presents methods for computing several such 'robustness measures' at each position of a given alignment. These methods are all very space-efficient; they use only space proportional to the sum of the two sequence lengths. To illustrate their effectiveness, one of the methods is used to locate particularly well-conserved regions in the beta-globin gene locus control region and in the 5' flank of the gamma-globin gene.

Algorithms↗

PubMed1998

A case of non-beta-globin gene linked beta thalassaemia in a Dutch family with two additional alpha-gene defects: the common -alpha3.7 deletion and the rare IVS1-116 (A-->G) acceptor splice site mutation.

We describe a family with beta thalassaemia, apparently not linked to the beta-globin gene cluster, in combination with alpha thalassaemia. The propositus, an adult Dutch Caucasian male, and his son presented with microcytic hypochromic parameters. Their lysates displayed the normal adult pattern on electrophoresis. The HbA2 concentration, which is usually increased in beta thalassaemia, was normal. The in vitro biosynthetic rate of the globin chains was strongly unbalanced even in the presence of a coexisting alpha-thalassaemia defect. Routine analysis of the beta genes, including the promoter region, was performed repeatedly by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGCE) and direct sequencing. No molecular abnormalities were detected. Large beta deletions were excluded by haplotype determination, using seven polymorphic markers distributed over an area of 50 kb, from 1 kb 5' of the epsilon gene to 4 kb 3' of the beta gene. The haplotype analysis of the beta-gene cluster revealed that the unaffected daughter had received the same beta haplotype as her beta-thalassaemic brother from their beta-thalassaemic father. These data suggest that the beta-gene cluster shared by father and son was not directly associated with a reduced beta-globin chain expression. In order to exclude the remote possibility of a beta-locus-control region (LCR) rearrangement in the paternal haplotype of the daughter, the sequence of the HS2 element was examined in the nuclear family. We compared the haematological and clinical data of this family with the data reported in the limited number of similar cases. We discuss the possibility that the mutation of a trans-acting erythroid factor(s), not linked to the beta-genes cluster, may impair the beta-gene expression of both alleles.

Adolescent↗

PubMed1998

Genetic treatment of severe hemoglobinopathies: the combat against transgene variegation and transgene silencing.

Gene addition strategies are rational approaches to the treatment of sickle cell anemia and thalassemia. The goal of such genetic treatments is to introduce a functional globin transcription unit in hematopoietic stem cells and express the transgene in a manner that is erythroid-specific, elevated, relatively constant from one cell to another, and sustained over time. Gene transfer is mediated by an expanding array of viral and nonviral vectors. High-titer retroviral vectors harboring the human beta-globin gene and the core sequences of the human beta-globin locus control region yield erythroid-specific gene expression in erythroid cell lines and in short-term murine bone marrow chimeras. However, we show that expression remains subject to position effect variegation and often decreases over time in vivo. Rather than a progressive transcriptional silencing in all cells, we ascribe the waning expression to the gradual emergence in blood of erythroid progeny derived from more and more primitive precursor cells in the months after transplantation. In our model, transgene expression is therefore determined by the integration site and the differentiation stage of the transduced cell at the time of integration. Globin expression is thus different in the progeny of a transduced erythroid progenitor cell and in the erythroid progeny of a transduced hematopoietic stem cell, reflecting the effect of flanking chromatin in differentiated cells and of chromatin remodeling at the site of integration in the progeny of multipotential cells. This model predicts that insulators and matrix attachment regions could be highly valuable to gene therapy in combination with potent transcriptional activators. When efficient gene transfer in hematopoietic stem cells is achieved at last, the challenge will be to regulate gene expression in vivo and overcome transgene variegation and transgene silencing.

Animals↗

PubMed1998

Multiple epsilon-promoter elements participate in the developmental control of epsilon-globin genes in transgenic mice.

To delineate the regulation of the human epsilon-globin gene, we investigated epsilon-gene expression during the development of transgenic mice carrying constructs with epsilon-promoter truncations linked to a micro-locus control region (microLCR). Expression levels were compared with those of microLCR epsilon mice carrying a 2 kilobase epsilon-promoter and betaYAC controls. epsilon mRNA in the embryonic cells of microLCR (-179)epsilon mice were as high as in microLCR epsilon mice suggesting that the proximal epsilon-promoter contains most elements required for epsilon-gene activation. epsilon mRNA in adult microLCR (-179) epsilon mice was significantly lower than in the embryonic cells indicating that elements involved in epsilon-gene silencing are contained in the proximal epsilon-promoter. Extension of the promoter sequence to -463 epsilon decreased epsilon-gene expression in the definitive erythroid cells, supporting previous evidence that the -179 to -463epsilon region contains an epsilon-gene silencer. However, the epsilon-gene of the microLCR(-463)epsilon mice was not silenced in the definitive cells of fetal and adult erythropoiesis indicating that additional silencing elements are located upstream of position -463epsilon. These results provide in vivo evidence that multiple elements of the distal as well as the proximal promoter contribute to epsilon-gene silencing.

Animals↗

PubMed1998

Detection of protein-DNA interactions at beta-globin gene cluster in intact human cells utilizing hedamycin as DNA-damaging agent.

The DNA sequence specificity of hedamycin (HDM) damage was investigated in the single-copy human beta-globin gene cluster in an erythroid cell line, a nonerythroid cell line, and purified genomic DNA. The target DNA sequences for this study were the beta-globin gene locus control region (LCR) hypersensitive site 2 (HS-2) and the beta-globin gene promoter. The DNA fragments produced by HDM damage in these target sequences were selectively amplified by the ligation-mediated polymerase chain reaction (LMPCR) and analyzed at nucleotide resolution by DNA-sequencing gel electrophoresis. The DNA sequences damaged by HDM in the cellular environment were found to be similar to that observed in the purified genomic DNA. However, substantial differences did occur between the intensity of cellular and purified genomic DNA reaction products at discrete regions corresponding to transcription factor-binding motifs. This was most apparent in the LCR HS-2 at the tandem NF-E2/AP-1 motif, where the DNA damage activity of HDM was severely impaired. This motif has been shown to bind to the erythroid-specific nuclear factor-erythroid 2 (NF-E2) and the widely distributed activator protein-1 (AP-1). The HDM damage protection patterns or "genomic footprints" observed at this motif were probably caused by protein-DNA interactions with one or both of these transcription factors. This result indicates that the DNA damaging activity of HDM in cells is sensitive to bound nuclear factors. Because HDM can enter intact cells, where its DNA damaging activity is modulated by protein-DNA interactions, it may have application in genomic footprinting experiments.

Alkylating Agents↗

PubMed1998

Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase.

To identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse effects on B cell function, and essentially corrected the X-linked immunodeficiency (xid) phenotype in Btk- mice. In contrast, expression of a constitutively activated form of Btk carrying the E41K gain-of-function mutation resulted in a B cell defect that was more severe than xid. The mice showed a marked reduction of the B cell compartment in spleen, lymph nodes, peripheral blood and peritoneal cavity. The levels in the serum of most immunoglobulin subclasses decreased with age, and B cell responses to both T cell-independent type II and T cell-dependent antigens were essentially absent. Expression of the E41K Btk mutant enhanced blast formation of splenic B cells in vitro in response to anti-IgM stimulation. Furthermore, the mice manifested a disorganization of B cell areas and marginal zones in the spleen. Our findings demonstrate that expression of constitutively activated Btk blocks the development of follicular recirculating B cells.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1998

The dynamics of globin gene expression and gene therapy vectors.

The most important level of regulation of the beta-globin genes is by activation of all of the genes by the Locus Control Region (LCR) and repression of the early genes by an as yet unknown factor acting on sequences flanking the genes. Superimposed on this is a mechanism in which the early genes (epsilon and gamma) suppress the late genes (delta and beta) by competition for the interaction with the LCR. Although this extra level of gene regulation is quantitatively of less importance than the direct repression mechanism, it has important implications and has provided an excellent assay system to probe the regulation of transcription at the single cell level. These studies indicate that the LCR interacts with individual globin genes and that LCR/gene interactions are dynamic with complexes forming and dissociating continually. The levels of expression of each of the genes appear to depend on: 1) the frequency of interaction which is itself dependent on the distance of the gene to the LCR, 2) the affinity of the LCR for the gene and 3) the stability of the LCR/gene complex. The latter two are dependent on the balance of transcription factors. 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 promoters.

Animals↗

PubMed1998

Stochastic, stage-specific mechanisms account for the variegation of a human globin transgene.

The random insertion of transgenes into the genomic DNA of mice usually leads to widely variable levels of expression in individual founder lines. To study the mechanisms that cause variegation, we designed a transgene that we expected to variegate, which consisted of a beta-globin locus control region 5' HS-2 linked in tandem to a tagged human beta-globin gene (into which a Lac-Z cassette had been inserted). All tested founder lines exhibited red blood cell-specific expression, but levels of expression varied >1000-fold from the lowest to the highest expressing line. Most of the variation in levels of expression appeared to reflect differences in the percentage of cells in the peripheral blood that expressed the transgene, which ranged from 0.3% in the lowest expressing line to 88% in the highest; the level of transgene expression per cell varied no more than 10-fold from the lowest to the highest expressing line. These differences in expression levels could not be explained by the location of transgene integration, by an effect of beta-galactosidase on red blood cell survival, by the half life of the beta-galactosidase enzyme or by the age of the animals. The progeny of all early erythroid progenitors (BFU-E colony-forming cells) exhibited the same propensity to variegate in methylcellulose-based cultures, suggesting that the decision to variegate occurs after the BFU-E stage of erythroid differentiation. Collectively, these data suggest that variegation in levels of transgene expression are due to local, integration site-dependent phenomena that alter the probability that a transgene will be expressed in an appropriate cell; however, these local effects have a minimal impact on the transgene's activity in the cells that initiate transcription.

Age Factors↗

PubMed1997

Position effects in mice carrying a lacZ transgene in cis with the beta-globin LCR can be explained by a graded model.

We studied transgenic mice carrying the lacZ reporter gene linked to the erythroid-specific beta-globin promoter and beta-globin locus control region (LCR). Previously, we had demonstrated that the total level of expression of beta-galactosidase enzyme, which is the product of the lacZ gene, varies widely between different transgenic mice due to position effects at the sites of transgene integration. Here, using the X-gal based in situ assay for beta-galactosidase activity, we found that the percent erythroid cells that expressed the transgene also varied widely between the mice. Moreover, a kinetic analysis showed that the average beta-galactosidase content per expressing cell varied both between samples of different transgenic descent and between erythroid cells within each sample, demonstrating that the variable expression of this lacZ transgene was being controlled in a graded manner. These results suggest that the beta-globin LCR enhancers function through a graded model, which is described, rather than the binary mechanism that has been proposed previously for other enhancers.

Animals↗

PubMed1998

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice.

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.

Animals↗