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Results for “Locus Control Region”

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PubMed2001

Variegated expression and delayed retinal pigmentation during development in transgenic mice with a deletion in the locus control region of the tyrosinase gene.

Deletion of the tyrosinase locus control region (LCR) in transgenic mice results in variegated expression in the skin. Here we investigate the pigmentation pattern of other tissues that express tyrosinase: iris, choroid, and retina in the same animals. A mosaic distribution of pigmentation appears in the iris and choroid. Interestingly, a markedly different mosaic pattern is found in the retina, where central areas contain little or no melanin while pigmentation rises to normal levels towards periphery. Further, there is a temporal delay in the initiation and accumulation of pigment in retinal pigmented epithelium (RPE) cells during development, and patterns of adult retinal melanisation in these mice appear arrested at a stage found in early embryogenesis in wild-type mice. These results demonstrate that the tyrosinase LCR is needed for the correct establishment and maintenance of this expression domain throughout development, but particularly during the later stages of retinal melanisation.

Alleles↗
PubMed1994

Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator.

The human beta locus control region (LCR) consists of five DNAse I hypersensitive sites (HS), four of which are erythroid specific and one, the further upstream located 5'HS5, is constitutive. To characterize the function of 5'HS5 we analyzed globin gene expression of various constructs containing HS3 as an enhancer, HS5, and the beta gene as a reporter. Expression was analyzed in stably transfected MEL cells. We found that the enhancing effect of hypersensitive site 3 is blocked when the HS5 is interposed between HS3 and the beta globin gene. These data suggest that the human 5'HS5 has the properties of a chromatin insulator.

Animals↗
PubMed1998

The locus control region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells.

Studies in many systems have led to the model that the human beta-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the beta-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous beta-globin locus in an erythroid background, transcription of all beta-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the beta-globin locus are not affected by the deletion. In contrast, deletion of 5'HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on beta-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an "open" conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the beta-like globin genes.

Adult↗
PubMed2000

A large "footprint" at the boundary of the human beta-globin locus control region hypersensitive site-2.

The 5'-boundary region of the human beta-globin locus control region hypersensitive site-2 (HS-2) was examined for protein-DNA interactions. The HS-2 is an erythroid specific DNase I hypersensitive site that extends for approximately 600 bp. Erythroid K562 cells and non-erythroid HeLa cells were damaged by bleomycin and hedamycin--these agents are able to "footprint" nucleosome cores and proteins bound to DNA. The fragments generated by DNA damage were amplified by the ligation-mediated polymerase chain reaction with primers specific for the 5'-boundary region of HS-2 and examined at base pair resolution on DNA sequencing gels. The intensity of damage in intact cells was compared with that in purified DNA. The comparison between intact cells and purified DNA revealed a protected region of 226 bp with bleomycin and 182 bp with hedamycin in K562 cells. The length of the protected region was consistent with the presence of a nucleosome core. We postulate that an erythroid-specific protein binds next to the positioned nucleosome at the boundary of HS-2 to prevent sliding of the nucleosome into the hypersensitive site--this would also account for the large size of the protected region. HeLa cells (lacking a hypersensitive site in the beta-globin cluster) did not have an area of protection in this region.

Anthraquinones↗
PubMed1991

Primary structure of the goat beta-globin locus control region.

The goat beta-globin cluster is composed of a triplicated four-gene set. A locus control region (LCR) containing elements homologous to 5'DNase I hypersensitive sites (HS) 1, 2, and 3 of the human beta-globin LCR has been identified at the 5' end of this locus. We determined 10.2 kb of nucleotide sequence from the goat beta-globin locus control region. Self-comparison of this sequence by dot matrix analysis revealed the presence of six complete and three incomplete artiodactyl repeats. A novel repeated element, termed D repeat, was also identified. Southern blotting analysis demonstrated that these elements exist in the goat genome as a low to medium frequency interspersed repeat family. The absence of any other large region of self-homology (direct or inverted) in the goat LCR suggests that 5'HSs 1, 2, and 3 did not arise through duplication, but rather evolved independently. By comparing goat 5'HS 1 to those of human, rabbit, and mouse, we show a greater than 80% conservation in sequence between the four species. This level of evolutionary conservation suggests that 5'HS 1 plays an important role in the regulation of beta-globin loci.

Animals↗
PubMed2002

The human beta-globin locus control region.

The human beta-globin gene locus is the subject of intense study, and over the past two decades a wealth of information has accumulated on how tissue-specific and stage-specific expression of its genes is achieved. The data are extensive and it would be difficult, if not impossible, to formulate a comprehensive model integrating every aspect of what is currently known. In this review, we introduce the fundamental characteristics of globin locus regulation as well as questions on which much of the current research is predicated. We then outline a hypothesis that encompasses more recent results, focusing on the modification of higher-order chromatin structure and recruitment of transcription complexes to the globin locus. The essence of this hypothesis is that the locus control region (LCR) is a genetic entity highly accessible to and capable of recruiting, with great efficiency, chromatin-modifying, coactivator, and transcription complexes. These complexes are used to establish accessible chromatin domains, allowing basal factors to be loaded on to specific globin gene promoters in a developmental stage-specific manner. We conceptually divide this process into four steps: (a) generation of a highly accessible LCR holocomplex; (b) recruitment of transcription and chromatin-modifying complexes to the LCR; (c) establishment of chromatin domains permissive for transcription; (d) transfer of transcription complexes to globin gene promoters.

Chromatin↗
PubMed1999

Locus control regions: coming of age at a decade plus.

The beta-globin locus control region (LCR) is the founding member of a novel class of cis-acting regulatory elements that confer high level, tissue-specific, site-of-integration-independent, copy number-dependent expression on linked transgenes located in ectopic chromatin sites. Knowledge from beta-globin and other LCR studies has shed light on our understanding of the long-range interaction between enhancers and promoters, the relationship between chromatin conformation and transcriptional regulation, and the developmental regulation of multiple gene loci. After over a decade of investigation and discovery, we take a retrospective look at the beta-globin LCR and other LCRs, summarize their properties and review models of LCR function.

Animals↗
PubMed1993

Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes.

We have tested the effect of the individual DNase I hypersensitive site (HS) regions of the globin locus control region (LCR) on the developmental expression pattern of the human gamma and beta genes in transgenic mice. The results show that HS3 is the most active site during the embryonic period. It is also the only site capable of high level expression of the gamma genes during fetal hematopoiesis, in a population of cells that are capable of expressing both the gamma and beta genes. Region HS4 shows the highest activity during the adult stage and expresses the gamma genes only at low levels during the embryonic period. HS2 drives equivalent levels of gamma or beta transgene expression throughout development. HS1 has a similar pattern to HS2, although the activity of HS1 is very low. From these results we conclude that the HS regions have distinct developmental specificities and suggest that in the complete LCR they interact with each other to form a larger complex which, in turn, interacts with the globin genes.

Animals↗
PubMed1993

Homology of a 130-kb region enclosing the alpha-globin gene cluster, the alpha-locus controlling region, and two non-globin genes in human and mouse.

The human alpha-globin gene cluster (30 kb) is embedded in a GC-rich isochore very close to the telomere of Chromosome (Chr) 16p. The alpha-Locus Controlling Region (alpha-LCR) is located upstream of the adult alpha-globin genes and has been shown to be essential for their expression. In this study we have been looking for expressed genes in the region upstream of the alpha-globin cluster to understand the role of the LCR-like element in the expression and replication timing of flanking gene clusters. We show that the upstream alpha-globin region is conserved over a 75-kb range and includes at least two oppositely transcribed non-globin genes, here referred to as Mid1 and Dist1. Complementary DNA sequences of 250 bp and 2.5 kb from Mid1 (coordinate -68) and Dist1 (coordinate -90 to -99), respectively, were isolated from human and mouse. The deduced partial amino acid sequences of these cDNAs are 81% and 95% identical for the Mid1 and Dist1 gene respectively. We have cloned a mouse cosmid "contig" which includes Dist1, Mid1, and the entire murine alpha-globin cluster. The murine homolog of the alpha-LCR was mapped upstream of the mouse globin genes at approximately the same position as in the human locus. Our results indicate that, in mouse and human, the alpha-globin loci and their flanking sequences are homologous over a range of at least 130 kb. The structural homology of this region in both mammals suggests also a functional one and indicates the mouse as a potential model for studying the role of the alpha-LCR controlling element in the regulation of expression and replication timing of the flanking gene clusters.

Amino Acid Sequence↗
PubMed2000

Both locus control region and proximal regulatory elements direct the developmental regulation of beta-globin gene cluster.

Using ligation-mediated PCR and in vivo footprinting methods to study the status of DNA-protein interaction at hypersensitive site 2 of locus control region and beta(maj) promoter of erythroid cells of fetal liver and adult bone marrow, we found that during different developmental periods, the status of DNA-protein interaction at both hypersensitive site 2 and beta(maj) promoter changed significantly, and indicated that locus control region might function through a looping mechanism to regulate the expression of downstream genes, and that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of beta-globin gene cluster participate in the regulation of developmental specificity.

Animals↗
PubMed1995

NF-E2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human beta-globin locus control region.

The beta-like globin genes require the upstream locus control region (LCR) for proper expression. The active elements of the LCR coincide with strong erythroid-specific DNase I-hypersensitive sites (HSs). We have used 5' HS4 as a model to study the formation of these HSs. Previously, we identified a 101 bp element that is required for the formation of this HS. This element binds six proteins in vitro. We now report a mutational analysis of the HS4 HS-forming element (HSFE). This analysis indicates that binding sites for the hematopoietic transcription factors NF-E2 and GATA-1 are required for the formation of the characteristic chromatin structure of the HS following stable transfection into murine erythroleukemia cells. Similarly arranged NF-E2 and GATA binding sites are present in the other HSs of the human LCR, as well as in the homologous mouse and goat sequences and the chicken beta-globin enhancer. A combination of DNase I and micrococcal nuclease sensitivity assays indicates that the characteristic erythroid-specific hypersensitivity of HS4 to DNase I is the result of tissue-specific alterations in both nucleosome positioning and tertiary DNA structure.

Animals↗
PubMed1994

Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region.

Hypersensitive site 2 located in the beta-globin locus control region confers high levels of expression to the genes of the beta-globin cluster. A tandem repeat of the consensus sequence for the transcription factors AP1 and NF-E2 (activating protein 1 and nuclear factor erythroid 2, respectively) is present within hypersensitive site 2 and is absolutely required for strong enhancer activity. This sequence binds, in vitro and in vivo, to ubiquitous proteins of the AP1 family and to the recently cloned erythroid-specific transcription factor NF-E2. Using the tandem repeat as a recognition site probe to screen a lambda gt11 cDNA expression library from K562 cells, we isolated several DNA binding proteins. Here, we report the characterization of one of the clones isolated. The gene, which we named Nrf2 (NF-E2-related factor 2), is encoded within a 2.2-kb transcript and predicts a 66-kDa protein with a basic leucine zipper DNA binding domain highly homologous to that of NF-E2. Although Nrf2 is expressed ubiquitously, a role of this protein in mediating enhancer activity of hypersensitive site 2 in erythroid cells cannot be excluded. In this respect, Nrf2 contains a powerful acidic activation domain that may participate in the transcriptional stimulation of beta-globin genes.

Amino Acid Sequence↗
PubMed1996

NF-E2 disrupts chromatin structure at human beta-globin locus control region hypersensitive site 2 in vitro.

The human beta-globin locus control region (LCR) is responsible for forming an active chromatin structure extending over the 100-kb locus, allowing expression of the beta-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HS1 to -4). DNase I hypersensitive sites are thought to represent nucleosome-free regions of DNA which are bound by trans-acting factors. Of the four hypersensitive sites only HS2 acts as a transcriptional enhancer. In this study, we examine the binding of an erythroid protein to its site within HS2 in chromatin in vitro. NF-E2 is a transcriptional activator consisting of two subunits, the hematopoietic cell-specific p45 and the ubiquitous DNA-binding subunit, p18. NF-E2 binds two tandem AP1-like sites in HS2 which form the core of its enhancer activity. In this study, we show that when bound to in vitro-reconstituted chromatin, NF-E2 forms a DNase I hypersensitive site at HS2 similar to the site observed in vivo. Moreover, NF-E2 binding in vitro results in a disruption of nucleosome structure which can be detected 200 bp away. Although NF-E2 can disrupt nucleosomes when added to preformed chromatin, the disruption is more pronounced when NF-E2 is added to DNA prior to chromatin assembly. Interestingly, the hematopoietic cell-specific subunit, p45, is necessary for binding to chromatin but not to naked DNA. Interaction of NF-E2 with its site in chromatin-reconstituted HS2 allows a second erythroid factor, GATA-1, to bind its nearby sites. Lastly, nucleosome disruption by NF-E2 is an ATP-dependent process, suggesting the involvement of energy-dependent nucleosome remodeling factors.

Adenosine Triphosphate↗
PubMed2002

Specification of unique Pit-1 activity in the hGH locus control region.

The human GH (hGH) gene cluster is regulated by a remote 5' locus control region (LCR). HSI, an LCR component located 14.5 kb 5' to the hGH-N promoter, constitutes the primary determinant of high-level hGH-N activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity in vivo. Furthermore, these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted hGH-N transgene activation. In contrast, the hGH-N transgene is not activated by Pit-1 sites native to either the hGH-N or rat (r)GH gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of hGH in the developing pituitary.

Animals↗
PubMed2000

Beta-globin gene switching and DNase I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region.

We have generated mice with a targeted deletion of the beta-globin locus control region (LCR). Mice homozygous for the deletion die early in embryogenesis but can be rescued with a YAC containing the human beta-globin locus. After germline passage, deletion of the LCR leads to a severe reduction in expression of all mouse beta-like globin genes, but no alteration in the developmental specificity of expression. Furthermore, a DNase I-sensitive "open" chromatin conformation of the locus is established and maintained. Thus, the dominant role of the LCR in the native locus is to confer high-level transcription, and elements elsewhere in the locus are sufficient to establish and maintain an open conformation and to confer developmentally regulated globin gene expression.

Animals↗
PubMed1999

Activation by locus control regions?

On the basis of homologous recombination experiments to delete the murine beta-globin locus control region (LCR) in embryonic stem cells, it was recently suggested that the LCR is not required for the activation of the murine beta-globin locus. This conclusion is in direct contradiction to the findings and conclusions that have been obtained with the human beta-globin LCR; thus the murine and human LCR may functionally be different or there may be a different interpretation of the results.

Animals↗
PubMed1992

Activation of the beta-globin locus control region precedes commitment to the erythroid lineage.

The beta-globin locus control region (LCR) is characterized by erythroid-specific DNase I hypersensitive sites and is involved in the chromatin organization, transcriptional potentiation, developmental regulation, and replication timing of the entire beta-globin gene cluster. When and how the LCR is first activated during erythropoiesis is not known. Here we analyze the chromatin structure of the LCR during early hematopoietic differentiation using nontransformed, multipotential, growth factor-dependent, murine hematopoietic progenitor cells. We show that LCR hypersensitive sites characteristic of erythroid cells are present in three independent multilineage progenitors [FDCP (factor-dependent cell, Paterson)-mix A4, B6SUtA, and LyD9] under conditions of self-renewal. Induction of differentiation down a nonerythroid pathway causes a progressive loss of hypersensitivity in the LCR. These results show that the beta-globin LCR is in an active chromatin configuration prior to erythroid commitment and indicate a significant role for selective gene repression in lineage specification.

Animals↗
PubMed1996

Locus control region function and heterochromatin-induced position effect variegation.

Human CD2 locus control region (LCR) sequences are shown here to be essential for establishing an open chromatin configuration. Transgenic mice carrying an hCD2 mini-gene attached only to the 3' CD2 transcriptional enhancer exhibited variegated expression when the transgene integrated in the centromere. In contrast, mice carrying a transgene with additional 3' sequences showed no variegation even when the latter integrated in centromeric positions. This result suggests that LCRs operate by ensuring an open chromatin configuration and that a short region, with no enhancer activity, functions in the establishment, maintenance, or both of an open chromatin domain.

Animals↗