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PubMed1992

Black beta-thalassemia homozygotes with specific sequence variations in the 5' hypersensitive site-2 of the locus control region have high levels of fetal hemoglobin.

We have sequenced the 5' hypersensitive-2 (5'HS-2) site of the locus control region (LCR) and the promoters of the two gamma-globin genes located on chromosome 11 of a black patient with mild beta-thalassemia (beta-thal) major due to a homozygosity for the C----T mutation at position -88 of the beta promoter and with a high Hb F level. Sequence variations in the 5'HS-2 were the same as observed for the beta s chromosome with haplotype number 3, while most of the G gamma promoter and the A gamma promoter had sequences similar to that of the beta S chromosome with haplotype number 19. This atypical haplotype (number 19A) is apparently associated with an increased gamma chain production which is particularly evident during periods of severe hematopoietic stress. Additional studies on relatives of the proband and on 10 unrelated black beta-thal homozygotes with either the C----T mutation at -88 or the A----G mutation at -29, confirm the possible importance of the sequence differences in the 5'HS-2, and also suggest that at least two additional factors, namely a C----T mutation at position -158 of the G gamma promoter and a relative deficiency in alpha chain synthesis play a (perhaps less important) role in the increased Hb F synthesis in these patients.

Adenine↗
PubMed1998

A long terminal repeat of the human endogenous retrovirus ERV-9 is located in the 5' boundary area of the human beta-globin locus control region.

Transcription of the human beta-like globin genes in erythroid cells is regulated by the far-upstream locus control region (LCR). In an attempt to define the 5' border of the LCR, we have cloned and sequenced 5 kb of new upstream DNA. We found an LTR retrotransposon belonging to the ERV-9 family of human endogenous retroviruses in the apparent 5' boundary area of the LCR. This ERV-9 LTR contains an unusual U3 enhancer region composed of 14 tandem repeats with recurrent GATA, CACCC, and CCAAT motifs. This LTR is conserved in human and gorilla, indicating its evolutionary stability in the genomes of the higher primates. In both recombinant constructs and the endogenous human genome, the LTR enhancer and promoter activate the transcription of cis-linked DNA preferentially in erythroid cells. Our findings suggest the possibility that this LTR retrotransposon may serve a relevant host function in regulating the transcription of the beta-globin LCR.

Animals↗
PubMed1994

Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.

Animals↗
PubMed1994

Developmental regulation of human gamma- and beta-globin genes in the absence of the locus control region.

Two lines of transgenic mice carrying a normal 40-kb Kpn I beta-globin cluster transgene lacking the locus control region (LCR) were analyzed for the expression of human gamma- and beta-globin genes during mouse development. After RNase protection assays, the ratios of human G gamma-, A gamma-, or beta-mRNAs relative to endogenous mouse zeta + alpha mRNAs were obtained for each stage of development. The two gamma transgenes were expressed in day-11.5 blood (embryonic stage) and day-13.5 blood (early fetal stage), but their expression was markedly decreased by day 16.5 of fetal life. Expression of the beta transgene was essentially absent at day 13.5, appeared at a low level by day 16.5, and was maximal by day 18.5, reaching a level similar to that observed in adult mice. Therefore, developmentally regulated expression of the human gamma- and beta-globin transgenes was obtained in the absence of the LCR. The relative expression of human gamma- and beta-globin genes was also examined in mice carrying 40-kb Kpn I beta-cluster transgenes with two different base substitutions associated with nondeletion forms of hereditary persistence of fetal hemoglobin (HPFH), -202 C-->G G gamma HPFH and -117 G-->A A gamma HPFH. The ratio of G gamma- to beta-globin transcripts was markedly increased in red blood cells of adult mice from three different lines carrying the transgene with the -202 G gamma HPFH mutation. This result confirms our previous preliminary results (Tanaka et al: Ann NY Acad Sci, 612:167, 1990) indicating that the -202 G gamma HPFH phenotype was reproduced in transgenic mice. The relatively low levels of G gamma-mRNA expression in adult mice carrying the non-HPFH transgene excludes a major influence of the 3' beta-globin enhancer, present upstream of the G gamma gene because of the tandem repeat insertion, as a factor in the persistent G gamma gene expression observed in blood of adult mice carrying the -202 G gamma HPFH transgene. This conclusion is also supported by the fact that, in mice carrying the -117 A gamma HPFH transgene, G gamma-globin mRNA was detected in blood of adult animals only at low levels similar to that observed in the non-HPFH lines. However, the A gamma-HPFH phenotype was not reproduced in the transgenic lines carrying the -117A gamma HPFH mice.

Animals↗
PubMed1997

A 150-base pair 5' region of the MHC class I HLA-B7 gene is sufficient to direct tissue-specific expression and locus control region activity: the alpha site determines efficient expression and in vivo occupancy at multiple cis-active sites throughout this region.

To characterize cis- and trans-acting mechanisms that regulate MHC class I transcription during development and in adult tissues, we have used transgenic mice to study a series of human MHC (HLA)-B7 class I gene constructs. Previous studies identified the 5' -0.66-kb to -0.075-kb region as sufficient to direct appropriate and efficient tissue-specific levels of HLA-B7 RNA relative to H-2 class I. Results here show that DNA 5' of -0.26 kb is not required for any aspect of expression. As the expression level correlated with the transgene copy number, was comparable to H-2 or a per-gene copy basis and was independent of integration site, the -0.075 to -0.26-kb segment also functions as a locus control region. With this region, sequences 3' of -0.075 kb, possibly at the promoter, appear to direct the appropriate tissue distribution. Of conserved sequences in the -0.075 to -0.26-kb region, enhancer B box is nonessential. In contrast, in vivo "footprinting" implicated region I/ enhancer A/NF-kappaB, IFN consensus/response sequence, and alpha in class I regulation as they are "occupied" in a tissue-specific pattern that correlates with expression. Mutation of alpha leads to decreased expression and loss of occupancy not only at alpha but also at region I/enhancer A/NF-kappaB and IFN consensus/response sequence. Thus, site alpha is an essential class I regulatory element, the dominant function of which is to mediate tissue-specific occupancy at multiple adjacent cis-active sites, possibly by facilitating stable synergistic interactions between factors at these distinct elements.

Animals↗
PubMed1998

A locus control region regulates yeast recombination.

The yeast Saccharomyces can switch its mating type by a highly choreographed recombination event in which 'a' or 'alpha' sequences at the mating-type (MAT) locus are replaced by opposite mating-type sequences copied from one of two donors, HML and HMR, located near the two ends of the same chromosome III. MAT alpha cells 'know' to choose HML, while MAT alpha cells preferentially recombine with HMR. Donor preference is regulated by a 250 bp recombination enhancer, that controls recombination of the entire left arm of chromosome III. Recent studies have shown how this locus-control region is turned on and off.

Enhancer Elements, Genetic↗
PubMed1998

DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression.

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between -14.6 kb and -32 kb 5' to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (-14.6 to -16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen in vivo. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.

3T3 Cells↗
PubMed1999

Bilateral macular atrophy in blue cone monochromacy (BCM) with loss of the locus control region (LCR) and part of the red pigment gene.

PURPOSE: To describe unusual macular abnormalities in a family with blue cone monochromacy (BCM, or X-linked incomplete achromatopsia) and deletion of about 9.5 kb comprising part of the red pigment gene and the region upstream of the red pigment gene. METHODS: The molecular structure of the red and green pigment genes and the locus control region (LCR) upstream of the red gene were studied for deletions, rearrangements and point mutations by Southern blot analysis and PCR. Four affected males (ages 33, 45, 51, and 59) and a carrier female (age 58) were examined by funduscopy and fluorescein angiography. Extensive color vision testing as well as rod and cone electroretinography (ERG) were performed on two of them. RESULTS: Analysis showed that the 6 kb proximal red gene region, exon 1 and about 3.1 kb of intron 1 of the red gene are deleted in this family. Exons 2-6 of the red gene, all the exons of the green gene and the Tex 28 gene were present. Four affected males had bilateral macular changes, including three with overt atrophy. All had visual acuity of 20/200 and their color vision was typical for BCM, with the absence of long- and middle-wavelength sensitive cone function. The ERG showed normal rod responses, whereas the photopic cone and 30-Hz flicker responses were >95% reduced. CONCLUSIONS: We report the unusual association between macular atrophy and BCM resulting from the loss of an approximately 9.5 kb region encompassing the LCR, proximal red gene promoter elements and exon 1 of the red gene. However, loss of the LCR and promoter is not sufficient to explain the phenotype since we have observed other BCM families with similar deletions who do not exhibit macular changes.

Adult↗
PubMed1992

In vivo protein-DNA interactions at hypersensitive site 3 of the human beta-globin locus control region.

The expression of beta-globin genes in developing erythroid cells is dependent on distant, upstream regulatory sequences, known as the locus control region (LCR), which are marked in chromatin by DNase I hypersensitive sites (HS-1 to HS-4). Linkage of the beta-globin gene complex LCR or fragments surrounding core regions of 200-300 base pairs to the human beta-globin gene permits consistent, high-level expression of the transgene in mice. To define the array of nuclear factors interacting with beta-LCR HS-3, we have performed in vivo dimethyl sulfate footprinting of the active HS-3 core in erythroid cells by a modified procedure that permits assessment of protein-DNA contacts at adenine, as well as guanine, residues. In vivo protein occupancy differs considerably from that predicted from previous in vitro binding analyses. In vivo footprinting detects protein binding at four sites recognized by the erythroid transcription factor GATA-1, at two CACC/GT motifs, and at a single AP-1/NF-E2 site. The regulatory elements occupied in vivo in HS-3 appear similar to those described previously in globin gene promoters and 3' enhancers. These findings suggest that the distinctive properties of the HS-3 region may be attributable to the organization of these occupied motifs and the consequent protein interactions, rather than to the binding of unique LCR regulatory factors.

Animals↗
PubMed1996

The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene.

The beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been described. Expression of the beta-like globin genes is absolutely dependent on the presence of the LCR. The developmental expression pattern of the genes in the cluster is achieved through competition of the promoters for the activating function of the LCR. Transgenic mice experiments suggest that subtle changes in the transcription factor environment lead to the successive silencing of the embryonic epsilon-globin and fetal gamma-globin promoters, resulting in the almost exclusive transcription of the beta-globin gene in adult erythropoiesis. In this paper, we have asked the question whether the LCR and its individual hypersensitive sites 5'HS1-4 can activate a basic promoter in the absence of any other globin sequences. We have employed a minimal promoter derived from the mouse Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The results show that the muLCR and 5'HS3 direct erythroid-specific, embryonic expression of this construct, while 5'HS1, 5'HS2 and 5'HS4 are inactive at any stage of development. Expression of the muLCR and 5'HS3 transgenes is repressed during fetal stages of development. The transgenes are in an inactive chromatin conformation and the lacZ gene is not transcribed, as shown by in situ hybridization. These data are compatible with the hypothesis that the LCR requires the presence of an active promoter to adopt an open chromatin conformation and with models proposing progressive heterochromatization during embryogenesis. The results suggest that the presence of a beta-globin gene is required for LCR function as conditions become more stringent during development.

Animals↗
PubMed1996

The beta-globin locus control region enhances transcription of but does not confer position-independent expression onto the lacZ gene in transgenic mice.

The beta-globin locus control region (LCR) confers high levels of position-independent, copy number-dependent expression onto globin transgenes. Here > 40 independent transgenic mouse lines and founders that carried the LCR in cis with the beta-globin gene promoter driving a lacZ reporter gene were studied. Expression of the lacZ transgene was assayed by measuring beta-galactosidase enzyme activity in fetal liver extracts, the levels of which correlated with the quantity of lacZ mRNA determined using RNase protection assays. Unexpectedly, expression of the lacZ transgene was found to show strong position effects, varying as much as 700-fold per transgene copy. These position effects occurred even if the whole beta-globin gene was incorporated as part of the lacZ reporter gene. Moreover, DNase I-hypersensitive sites appeared in the transgene LCR in high expressing but not in low expressing lines, suggesting that the LCR itself was position dependent. In contrast, MEL cell clones, in which transcriptionally active integration sites were selected for, gave < 13-fold variation in expression per copy of an LCR-lacZ construct. These results show that the lacZ reporter affects the ability of the LCR to activate chromatin in mice and that culture cells are not an adequate model for position-independent gene expression studies.

Animals↗
PubMed1998

The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes.

In chromosome translocations characteristic of Burkitt lymphomas (BL) and murine plasmacytomas, c-myc genes become juxtaposed to immunoglobulin heavy-chain (IgH) sequences, resulting in aberrant c-myc transcription. Translocated c-myc alleles that retain the first exon exhibit increased transcription from the normally minor c-myc promoter, P1, and increased transcriptional elongation through inherent pause sites proximal to the major c-myc promoter, P2. We recently demonstrated that a cassette derived from four DNase I-hypersensitive sites (HS1234) in the 3'Calpha region of the IgH locus functions as an enhancer-locus control region (LCR) and directs a similar pattern of deregulated expression of linked c-myc genes in BL and plasmacytoma cell lines. Here, we report that the HS1234 enhancer-LCR mediates a widespread increase in histone acetylation along linked c-myc genes in Raji BL cells. Significantly, the increase in acetylation was not restricted to nucleosomes within the promoter region but also was apparent upstream and downstream of the transcription start sites as well as along vector sequences. Histone hyperacetylation of control c-myc genes, which was induced by the deacetylase inhibitor trichostatin A, mimics the effect of the HS1234 enhancer on expression from the c-myc P2 promoter, but not that from the P1 promoter. These results suggest that the HS1234 enhancer stimulates transcription of c-myc by a combination of mechanisms. Whereas HS1234 activates expression from the P2 promoter through a mechanism that includes increased histone acetylation, a general increase in histone acetylation is not sufficient to explain the HS1234-mediated activation of transcription from P1.

Acetylation↗
PubMed1998

Development of a condensed locus control region cassette and testing in retrovirus vectors for A gamma-globin.

Retrovirus vectors for A gamma-globin are being developed for the treatment of beta chain hemoglobinopathies. Toward the goal of achieving therapeutic expression levels, core elements of the beta-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhancer activity in erythroid MEL and K562 cell lines using a drug-resistant colony assay. When used alone, core elements of HS1, HS3, and HS4 showed no activity and a fragment for HS2 showed only modest activity in the colony assay. However, a 1.1 kb combination of fragments for HS2, HS3, and HS4 (termed a nLCR) enhanced colony formation 17-fold in K562 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhanced nLCR activity only modestly in MEL cells. When linked to a beta-globin gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per copy of mouse alpha-globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a beta-globin promoter and various A gamma-globin gene expression cassettes resulted in extreme genetic instability and reduced titers. Specific deletions were abrogated by removing homologous sequences, but random recombinations were still observed at significant frequencies. In MEL cells containing intact provirus, A gamma-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse alpha-globin) compared to the same vector without the nLCR. These data suggest that vector elements detract from the ability of the nLCR to enhance expression of the beta pr.A gamma cassettes.

Animals↗
PubMed2003

Beta-globin locus control region HS2 and HS3 interact structurally and functionally.

The overall structure of the DNase I hypersensitive sites (HSs) that comprise the beta-globin locus control region (LCR) is highly conserved among mammals, implying that the HSs have conserved functions. However, it is not well understood how the LCR HSs, either individually or collectively, activate transcription. We analyzed the interactions of HS2, HS3 and HS4 with the human epsilon- and beta-globin genes in chromatinized episomes in fetal/embryonic K562 cells. Only HS2 activates transcription of the epsilon-globin gene, while all three HSs activate the beta-globin gene. HS3 stimulates the beta-globin gene constitutively, but HS2 and HS4 transactivation requires expression of the transcription factor EKLF, which is not present in K562 cells but is required for beta-globin expression in vivo. To begin addressing how the individual HSs may interact with one another in a complex, we linked the beta-globin gene to both the HS2 and HS3. HS2 and HS3 together resulted in synergistic stimulation of beta-globin transcription. Unexpectedly, mutated, inactive forms of HS2 impeded the activation of the beta-globin gene by HS3. Thus, there appear to be distinct interactions among the HSs and between the HSs and the globin genes. These preferential, non-exclusive interactions may underlie an important structural and functional cooperativity among the regulatory sequences of the beta-globin locus in vivo.

Binding Sites↗
PubMed2001

Distinct mechanisms control RNA polymerase II recruitment to a tissue-specific locus control region and a downstream promoter.

Histone acetylation precedes activation of many genes. However, the establishment and consequences of long-range acetylation patterns are poorly understood. To define molecular determinants of the developmentally dynamic histone acetylation pattern of the beta-globin locus, we compared acetylation of the locus in MEL and CB3 erythroleukemia cells. CB3 cells lack the beta-globin locus control region (LCR) binding protein p45/NF-E2. We found that p45/NF-E2 was required for histone hyperacetylation at adult beta-globin promoters approximately 50 kilobases downstream of the LCR, but not at the LCR. Surprisingly, RNA polymerase II associated with the LCR in a p45/NF-E2-independent manner, while its recruitment to the promoter required p45/NF-E2. We propose that polymerase accesses the LCR and p45/NF-E2 induces long-range transfer of polymerase to the promoter, resulting in transcriptional activation.

Acetylation↗
PubMed1999

Effects of altered gene order or orientation of the locus control region on human beta-globin gene expression in mice.

The five human beta-type-globin genes, epsilon, Ggamma, Agamma, delta and beta, are close together and are regulated by a locus control region (LCR) located at the 5' end of the locus. Here we investigate the functional consequences of this organization with respect to temporal regulation of the individual genes, by using recombination techniques to invert the order of either the genes or the LCR in vivo. Our analysis of transgenic mice bearing either normal or mutant transgenes leads to two new observations. First, the position of the epsilon-globin gene next to the LCR is mandatory for its expression during the yolk-sac stage of erythropoiesis. Second, LCR activity is orientation dependent, and so the LCR does not act as a simple enhancer to stimulate transcription of the globin genes. Thus, in the absence of any change in transgene integration position, transgene copy number, trans-acting factors or other resident genetic information, simple inversion of the human genes or the LCR fundamentally alters the transcription of beta-type globin genes.

Animals↗
PubMed2000

Control of organ-specific demethylation by an element of the T-cell receptor-alpha locus control region.

DNA methylation is important for mammalian development and the control of gene expression. Recent data suggest that DNA methylation causes chromatin closure and gene silencing. During development, tissue specifically expressed gene loci become selectively demethylated in the appropriate cell types by poorly understood processes. Locus control regions (LCRs), which are cis-acting elements providing stable, tissue-specific expression to linked transgenes in chromatin, may play a role in tissue-specific DNA demethylation. We studied the methylation status of the LCR for the mouse T-cell receptor alpha/delta locus using a novel assay for scanning large distances of DNA for methylation sites. Tissue-specific functions of this LCR depend largely on two DNase I-hypersensitive site clusters (HS), HS1 (T-cell receptor alpha enhancer) and HS1'. We report that these HS induce lymphoid organ-specific DNA demethylation in a region located 3.8 kilobases away with little effect on intervening, methylated DNA. This demethylation is impaired in mice with a germline deletion of the HS1/HS1' clusters. Using 5'-deletion mutants of a transgenic LCR reporter gene construct, we show that HS1' can act in the absence of HS1 to direct this tissue-specific DNA demethylation event. Thus, elements of an LCR can control tissue-specific DNA methylation patterns both in transgenes and inside its native locus.

Animals↗
PubMed2002

Interaction of NF-E2 in the human beta-globin locus control region before chromatin remodeling.

When transcription is initiated under repressive conditions, such as when chromatin are packed together, binding followed by the functioning of key components in the transcriptional apparatus should be appropriately facilitated in the chromatin architecture. We provide evidence that the erythroid-specific enhancer- binding protein NF-E2 interacts with the cognate motif at DNase I-hypersensitive site 2 of the human beta-globin locus control region in a repressive state. The nucleosome containing the NF-E2-binding site showed characteristic rotational and translational phases in vitro. The binding site had less affinity to the histone octamers than nearby regions while showing greater accessibility to DNase I and micrococcal nuclease. Furthermore, the motif was recognized by the exogenous NF-E2 protein expressed in HeLa cells, which have a repressive state of chromatin at the beta-globin locus, as shown by ligation-mediated PCR and chromatin immunoprecipitation assay. These lines of evidence indicate that NF-E2 interacts with the cognate motif on the nucleosome before chromatin is remodeled.

Base Sequence↗