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PubMed1996

Dynamic changes in the locus control region of erythroid progenitor cells demonstrated by polymerase chain reaction.

The locus control region (LCR) far upstream of the human beta-like globin genes is defined by the preferential chromatin accessibility/DNase I hypersensitivity of four constituent DNA sites HS4, 3, 2, and 1. In an attempt to understand the mechanism of LCR function during early stages of erythropoiesis, a new polymerase chain reaction (PCR) method has been developed to examine the chromatin structure/DNase I hypersensitivity of the LCR in progenitor cells logistically available in limited cell numbers. In erythroid progenitors as well as in multipotent cells with erythroid potential, hypersensitive sites HS4, 3, 2, and 1 were present and the chromatin structure of the LCR was accessible. Moreover, the chromatin structure of the LCR underwent dynamic changes during erythropoiesis. In early erythroid progenitors, the HS2 site was more accessible than the HS3 site. In more mature erythroid progenitors, HS2 became less accessible than HS3 and the other sites. The results indicate that the transcriptional program of the globin genes is encoded, at least in part, in the chromatin accessibility of the LCR. This globin program was apparently initiated in multipotent cells and maintained in erythroid progenitors. Furthermore, the program could be modulated in response to cellular changes accompanying differentiation of the progenitor cells.

Adult↗
PubMed1997

Phylogenetic footprinting of hypersensitive site 3 of the beta-globin locus control region.

Hypersensitive site 3 (HS3) of the beta-like globin locus control region has been implicated as an important regulator of the beta-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two gamma genes (galago gamma is embryonic; human gamma is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of gamma in adult life).

Animals↗
PubMedDate not supplied

[Locus-controlling regions: description in the LCR-TRRD data base].

The structural and functional organization of locus control regions (LCR) was analyzed using data of the LCR-TRR Database. The role of several transcription factors in the LCR function was considered. A study was made of the possible nucleosomal packing of enhancer regions in LCR. The structure and the format of LCR-TRRD are described. The database has been constructed for SRS and is available at http://wwwmgs.bionet.nsc.ru/mgs/dbase/LCR/.

DNA↗
PubMed1997

Conserved E boxes function as part of the enhancer in hypersensitive site 2 of the beta-globin locus control region. Role of basic helix-loop-helix proteins.

The human beta-globin gene cluster is regulated in part by a distal locus control region that is required for opening a chromatin domain in erythroid cells and enhancing expression of the beta-like globin genes at the correct developmental stages. One part of the locus control region, called hypersensitive site 2 (HS2), functions as a strong enhancer. Matches to the consensus binding sites for basic helix-loop-helix (bHLH) proteins (E boxes) are well conserved within the HS2 core. We show that mutations of the HS2 core that alter an invariant E box cause a 3.5-fold reduction in enhancement of expression of an epsilon-globin reporter gene in transiently transfected K562 cells, both before and after induction. Mutations of the HS2 core that alter a less-highly conserved E box cause a more modest reduction in enhancement. Footprint analysis shows binding of erythroid nuclear proteins in vitro to the invariant E box as well as an adjacent CAC/GTG box. Probes containing the E box regions form sequence-specific complexes with proteins from both K562 and MEL nuclear extracts; these are disrupted by the same mutations that decrease enhancement. Some of these latter complexes contain known bHLH proteins, as revealed by specific loss of individual complexes when treated with antibodies against TAL1 and USF. Interaction between the E boxes and the bHLH proteins, as well as other binding proteins, could account for the role of these sites in enhancement by HS2.

Base Sequence↗
PubMed1998

Expression pattern of CD2 locus control region containing retroviral vectors in hemopoietic cells in vitro and in vivo.

We introduced the locus control region (LCR) of the human CD2 gene in eight different ways into retroviral constructs containing the human adenosine deaminase (hADA) gene. Two of these constructs were tested in transgenic mice. Neither expressed hADA in any examined tissue, nor did control vectors without the LCR insert. Amphotropic retrovirus vector producer cell clones were isolated and analyzed for provirus integrity and vector titer. Three constructs yielded recombined proviruses in 20-100% of transduced clones, whereas the other five constructs always rendered viruses that remained stable upon replication and gave vector titers comparable to the control lacking an LCR. Human ADA-deficient T cells and murine fibroblasts transduced with these recombinant viruses showed considerable hADA expression levels that were, however, not significantly different from those of cells transduced with the control vector lacking an LCR insert. Furthermore, no difference in hADA expression levels could be detected in spleen, thymus and bone marrow of long-term repopulated mice that had received bone marrow cells transduced with either the control vector or one of two different CD2-LCR containing vectors. In conclusion, the CD2-LCR does not alleviate the expression block for recombinant retroviruses in the germ line and does not enhance the LTR-driven expression in T cells.

Adenosine Deaminase↗
PubMed1992

Mechanism of DNase I hypersensitive site formation within the human globin locus control region.

The human beta-like globin gene locus contains embryonic, fetal, and adult globin genes that are regulated in a developmentally timed, as well as a tissue-specific, manner. The locus control region (LCR), located 5' of the globin genes, is characterized by four erythroid-specific nuclease-hypersensitive sites within native chromatin. These sites contain the active elements of the LCR. The LCR establishes an active chromatin conformation across the globin locus and enhances globin gene expression in transfected erythroleukemia cells and transgenic mice. We have used 5' DNase I hypersensitive site (HS) 4 as a model to define the minimum elements necessary for site formation. We have identified a 101-base-pair fragment within 5' HS4 that is the active site-forming element. DNase I footprint and gel-mobility shift assays have identified binding sites for transcription factors AP-1/NF-E2, Sp-1, and GATA-1 within the HS-forming element. We conclude that HS formation, the characteristic feature of the LCR in nuclear chromatin, requires interaction between erythroid-specific and ubiquitous nuclear proteins.

Animals↗
PubMed1998

Description and targeted deletion of 5' hypersensitive site 5 and 6 of the mouse beta-globin locus control region.

The most upstream hypersensitive site (HS) of the beta-globin locus control region (LCR) in humans (5' HS 5) and chickens (5' HS 4) can act as an insulating element in some gain of function assays and may demarcate a beta-globin domain. We have mapped the most upstream HSs of the mouse beta-globin LCR and sequenced this region. We find that mice have a region homologous to human 5' HS 5 that is associated with a minor HS. In addition we map a unique HS upstream of 5' HS 5 and refer to this novel site as mouse 5' HS 6. We have also generated mice containing a targeted deletion of the region containing 5' HS 5 and 6. We find that after excision of the selectable marker in vivo, deletion of 5' HS 5 and 6 has a minimal effect on transcription and does not prevent formation of the remaining LCR HSs. Taken together these findings suggest that the most upstream HSs of the mouse beta-globin LCR are not necessary for maintaining the beta-globin locus in an active configuration or to protect it from a surrounding repressive chromatin environment.

Animals↗
PubMed2000

Long-distance control of origin choice and replication timing in the human beta-globin locus are independent of the locus control region.

DNA replication in the human beta-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the beta-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5'HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5'HS2-5 in the normal chromosomal context of the human beta-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5'HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5'HS2-5 in the classically defined LCR do not control replication in the human beta-globin locus. Recent studies also show that targeted deletion of 5'HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

Animals↗
PubMed1996

Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.

Animals↗
PubMed2001

The murine beta-globin locus control region regulates the rate of transcription but not the hyperacetylation of histones at the active genes.

Locus control regions (LCRs) are defined by their ability to confer high-level tissue-specific expression to linked genes in transgenic assays. Previously, we reported that, at its native site, the murine beta-globin LCR is required for high-level beta-globin gene expression, but is not required to initiate an open chromatin conformation of the locus. To further investigate the mechanism of LCR-mediated transcriptional enhancement, we have analyzed allele-specific beta-globin expression and the pattern of histone acetylation in the presence and absence of the LCR. In single cells from mice heterozygous for a deletion of the LCR, beta-globin expression from the LCR-deleted allele is consistently low ( approximately 1-4% of wild type). Thus, the endogenous LCR enhances globin gene expression by increasing the rate of transcription from each linked allele rather than by increasing the probability of establishing transcription per se. Furthermore, in erythroid cells from mice homozygous for the highly expressing wild-type beta-globin locus, hyperacetylation of histones H3 and H4 is localized to the LCR and active genes. In mice homozygous for the LCR deletion reduced histone hyperacetylation is observed in LCR proximal sequences; however, deletion of the LCR has no effect on the localized hyperacetylation of the genes. Together, our results suggest that, in its native genomic context, the LCR follows the rate model of enhancer function, and that the developmentally specific hyperacetylation of the globin genes is independent of both the rate of transcription and the presence of the LCR.

Acetylation↗
PubMed2002

High-mobility group protein 2 may be involved in the locus control region regulation of the beta-globin gene cluster.

Expression regulation of the beta-globin gene cluster is a result of synergistic interactions between cis-elements and trans-acting factors. Previous studies usually concentrated on the core sequence of each hypersensitive site in the locus control region of the beta-globin gene cluster. But more and more evidence illustrates that the flanking regions are indispensable also. Using electrophoretic mobility shift assay and solid-phase DNase I footprinting methods, we identified a small nuclear protein from K562 cells that binds specifically to the first AT-rich region flanking the hypersensitive site 2 core sequence of the human beta-globin gene locus control region. N-terminal sequencing of the enriched protein proved that it is a member of the high-mobility group protein 2 family. This indicates that the AT-rich region in human hypersensitive site 2 may take part in the regulation of the beta-globin gene cluster by facilitating DNA bending, which is a prerequisite for the looping mechanism in this region.

Base Sequence↗
PubMed1997

Locus control regions of mammalian beta-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights.

Locus control regions (LCRs) are cis-acting DNA segments needed for activation of an entire locus or gene cluster. They are operationally defined as DNA sequences needed to achieve a high level of gene expression regardless of the position of integration in transgenic mice or stably transfected cells. This review brings together the large amount of DNA sequence data from the beta-globin LCR with the vast amount of functional data obtained through the use of biochemical, cellular and transgenic experimental systems. Alignment of orthologous LCR sequences from five mammalian species locates numerous conserved regions, including previously identified cis-acting elements within the cores of nuclease hypersensitive sites (HSs) as well as conserved regions located between the HS cores. The distribution of these conserved sequences, combined with the effects of LCR fragments utilized in expression studies, shows that important sites are more widely distributed in the LCR than previously anticipated, especially in and around HS2 and HS3. We propose that the HS cores plus HS flanking DNAs comprise a 'unit' to which proteins bind and form an optimally functional structure. Multiple HS units (at least three: HS2, HS3 and HS4 cores plus flanking DNAs) together establish a chromatin structure that allows the proper developmental regulation of genes within the cluster.

Animals↗
PubMed2001

Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement.

The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.

Binding Sites↗
PubMed2000

Independent formation of DnaseI hypersensitive sites in the murine beta-globin locus control region.

Mammalian beta-globin loci are composed of multiple orthologous genes whose expression is erythroid specific and developmentally regulated. The expression of these genes both from the endogenous locus and from transgenes is strongly influenced by a linked 15-kilobase region of clustered DNaseI hypersensitive sites (HSs) known as the locus control region (LCR). The LCR encompasses 5 major HSs, each of which is highly homologous among humans, mice, and other mammals. To analyze the function of individual HSs in the endogenous murine beta-globin LCR, we have used homologous recombination in embryonic stem cells to produce 5 mouse lines, each of which is deficient for 1 of these major HSs. In this report, we demonstrate that deletion of the conserved region of 5'HS 1, 2, 3, 4, or 5/6 abolishes HS formation at the deletion site but has no influence on the formation of the remaining HSs in the LCR. Therefore, in the endogenous murine locus, there is no dominant or initiating site whose formation must precede the formation of the other HSs. This is consistent with the idea that HSs form autonomously. We discuss the implications of these findings for current models of beta-globin regulation.

Animals↗
PubMed1993

Synthetic human beta-globin 5'HS2 constructs function as locus control regions only in multicopy transgene concatamers.

Transgenes linked to the beta-globin locus control region (LCR) are transcribed in a copy-dependent manner that is independent of the integration site. It has previously been shown that the LCR 5'HS2 region does not require its NF-E2 dimer binding site for LCR activity. In this paper we analyse synthetic 5'HS2 core constructs containing point mutations in the other factor binding sites 3' of the NF-E2 dimer site. The results show that 5'HS2 core is a partially active LCR that functions in a concatamer of at least two copies but not when present as a single copy in transgenic mice and that no single binding site within 5'HS2 is required for position-independent expression. In addition, the H-BP factor is identical to upstream stimulatory factor (USF) and full enhancement levels by 5'HS2 core in MEL cells require a combination of all the factor binding sites. We suggest that 5'HS2 cores in a concatamer interact with each other to establish an area of open chromatin and that this process may be the basis of LCR function.

Animals↗
PubMed1995

Autonomous, erythroid-specific DNase I hypersensitive site formed by human beta-globin locus control region (LCR) 5' HS 2 in transgenic mice.

The human beta-globin locus control region (LCR) is composed of four erythroid-specific, DNase I hypersensitive (HS) sites that are located 6 to 18 kb upstream of the epsilon-globin gene. The beta-globin LCR appears to have two major functions. First, the sequences "open" a chromosomal domain that includes the epsilon-, gamma-, and beta-globin genes and, second, the LCR directs high-level, erythroid-specific expression of each globin gene family member. An LCR subfragment containing only 5' HS 2 can confer these properties on a linked beta-globin gene. To determine whether 5' HS 2 can form an erythroid-specific, DNase I hypersensitive site in the absence of a linked globin gene, a 1.9-kb DNA fragment containing this site was injected into fertilized mouse eggs and DNase I hypersensitivity was analyzed in the animals that developed. In 9 of 10 transgenic mouse lines, the human 5' HS 2 fragment formed a DNase I hypersensitive site in fetal liver but not in fetal brain. These results suggest that human 5' HS 2 can function autonomously to organize an open chromatin domain specifically in erythroid cells.

Animals↗
PubMed2003

Chromatin remodeling and extragenic transcription at the MHC class II locus control region.

In vivo, a wild-type pattern of major histocompatibility complex (MHC) class II expression requires a locus control region (LCR). Whereas the role of promoter-proximal MHC class II regulatory sequences is well established, the function of the distal LCR remained obscure. We show here that this LCR is bound by the MHC class II-specific transactivators regulatory factor X (RFX) and class II transactivator (CIITA). Binding of these factors induces long-range histone acetylation, RNA polymerase II recruitment and the synthesis of extragenic transcripts within the LCR. The finding that RFX and CIITA regulate the function of the MHC class II LCR reveals an unexpected degree of complexity in the mechanisms controlling MHC class II gene expression.

Animals↗