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PubMed2001

Chromatin structure at the 3'-boundary of the human beta-globin locus control region hypersensitive site-2.

Chromatin structure was examined at the 3'-boundary region of the human beta-globin locus control region hypersensitive site-2 (LCR HS-2) using several footprinting agents. Erythroid K562 cells (possessing HS-2) were damaged by the footprinting agents: hedamycin, bleomycin and four nitrogen mustard analogues. Purified DNA and non-erythroid HeLa cells (lacking HS-2) were also damaged as controls for comparison with K562 cells. The comparison between intact cells and purified DNA showed several protected regions in K562 cells. A large erythroid-specific protected region of 135 bp was found at the boundary of HS-2. The length of this protected region (135 bp) was close to that of DNA contained in a nucleosome core (146 bp). Another two protected regions were found upstream of the protected region. A 16-bp erythroid-specific footprint co-localised with a GATA-1 motif-this indicated that the GATA-1 protein could be involved in positioning the nucleosome. Further upstream, a 100-bp footprint coincided with an AT-rich region. Thus our footprinting results suggest that the 3'-boundary of LCR HS-2 is flanked by a positioned nucleosome and that an erythroid-specific protein binds to the sequence adjacent to the nucleosome and acts to position the nucleosome at the boundary of the hypersensitive site.

Alkylating Agents↗
PubMed1996

Role of DNA sequences outside the cores of DNase hypersensitive sites (HSs) in functions of the beta-globin locus control region. Domain opening and synergism between HS2 and HS3.

The roles of each DNase hypersensitive site (HS), and the DNA sequences between them, in the activity of the locus control region of the mammalian beta-globin gene domain were examined by placing human and rabbit restriction fragments containing the cores of HS2, HS3, HS4, and HS5, along with varying amounts of flanking DNA, upstream of a hybrid epsilon-globin-luciferase reporter gene and testing for effects on expression both prior to and after integration into the chromosomes of K562 cells, a human erythroid cell line. Prior to integration, fragments containing HS2 enhanced expression to the greatest extent, and the modest enhancement by some fragments containing HS3 correlated with the presence of a well-conserved binding site for AP1/NFE2. The stronger effects of larger locus control region DNA fragments in clones of stably transfected cells indicates a role for sequences outside the HS cores after integration into the genome. The strong effect of a 1.9-kilobase HindIII fragment containing HS3 after, but not prior to, integration argues for the presence of a chromatin domain-opening activity. Use of a rabbit DNA fragment containing both HS2 and HS3 demonstrated a synergistic interaction between the two HSs when their natural context and spacing are preserved.

Animals↗
PubMed2001

Reconstitution of human beta-globin locus control region hypersensitive sites in the absence of chromatin assembly.

The human beta-globin genes are regulated by the locus control region (LCR), an element composed of multiple DNase I-hypersensitive sites (HS sites) located 5' to the genes. Various functional studies indicate that the LCR confers high-level, position-independent, and copy number-dependent expression to linked globin genes in transgenic mice. However, the structural basis for LCR function is unknown. Here we show that LCR HS sites can be reconstituted in an erythroid cell-specific manner on chromatin-assembled LCR templates in vitro. Surprisingly, HS2 and HS3 are also formed with erythroid proteins in the absence of chromatin assembly, indicating that sensitivity to nucleases is not simply a consequence of nucleosome reorganization. The generation of LCR HS sites in the absence of chromatin assembly leads to the formation of S1- and KMnO(4)-sensitive regions in HS2 and HS3. These sites are also sensitive to S1 nuclease in erythroid cells in vivo, suggesting a distorted DNA structure in the LCR core enhancer elements. Finally, we show that RNA polymerase II initiates transcription in the HS2 and HS3 core enhancer regions in vitro. Transcription in both HS2 and HS3 proceeds in a unidirectional manner. Taken together, the data suggest that erythroid proteins interact with the core enhancer elements, distort the DNA structure, and recruit polymerase II transcription complexes. These results further our understanding of the structural basis for LCR function and provide an explanation for why the LCR core regions are so extremely sensitive to nucleases in erythroid cells.

Animals↗
PubMed1996

A novel DNA-binding protein, HS2NF5, interacts with a functionally important sequence of the human beta-globin locus control region.

We have identified a previously unreported DNA-binding protein, HS2NF5, which interacts with a conserved sequence within hypersensitive site II (HS2) of the human beta-globin locus control region. A minimal DNA recognition sequence of TGTTCTCA was defined. The binding site for HS2NF5 overlaps an E box, which is a preferred recognition site in vitro for the erythroid-specific transcription factor TAL1 (SCL). No evidence for TAL1 (SCL) binding was found using nuclear extracts from K562 and MEL erythroleukemia cells. Mutations that prevent HS2NF5 binding reduce the enhancer activity of HS2 by 40 and 38% in transient and stable transfection assays, respectively. Analytical gel filtration and velocity centrifugation studies revealed a Stokes' radius of 23.0 A and an s20,w of 3.45 for HS2NF5. Based on these parameters, a native molecular mass of 34,679 Da was calculated. An ultraviolet light cross-linking assay was used to cross-link HS2NF5 to a minimal oligonucleotide. The cross-linking results are consistent with a protein of 33,396-38,309 Da. We propose that HS2NF5 is a novel DNA-binding protein that modulates the transcriptional activation property of the beta-globin locus control region.

Base Sequence↗
PubMed1998

Developmental specificity of the interaction between the locus control region and embryonic or fetal globin genes in transgenic mice with an HS3 core deletion.

The human beta-globin locus control region (LCR) consists of five erythroid-lineage-specific DNase I-hypersensitive sites (HSs) and is required for activation of the beta-globin locus chromatin domain and globin gene expression. Each DNase I-HS of the LCR consists of a highly conserved core element and flanking sequences. To analyze the functional role of the core elements of the HSs, we deleted a 234-bp fragment encompassing the core of HS3 (HS3c) from a beta-globin locus residing on a 248-kb beta-locus yeast artificial chromosome and analyzed its function in F2 progeny of transgenic mice. Human epsilon-globin gene expression was absent at day 10 and severely reduced in the day 12 embryonic erythropoiesis of mice lacking HS3c. In contrast, gamma-globin gene expression was normal in embryonic erythropoiesis but it was absent in definitive erythropoiesis in the fetal liver. These results indicate that the core element of HS3 is necessary for epsilon-globin gene transcription in embryonic cells and for gamma-globin gene transcription in definitive cells. Normal gamma-globin gene expression in embryonic cells and the absence of gamma-globin gene expression in definitive cells show that different HSs interact with gamma-globin gene promoters in these two stages of development. Such results provide direct evidence for developmental stage specificity of the interactions between the core elements of HSs and the promoters of the globin genes.

Animals↗
PubMed2001

Acetylation of a specific promoter nucleosome accompanies activation of the epsilon-globin gene by beta-globin locus control region HS2.

On stably replicating episomes, transcriptional activation of the epsilon-globin promoter by the beta-globin locus control region HS2 enhancer is correlated with an increase in nuclease sensitivity which is limited to the TATA-proximal nucleosome (N1). To elucidate what underlies this increase in nuclease sensitivity and the link between chromatin modification and gene expression, we examined the nucleoprotein composition and histone acetylation status of transcriptionally active and inactive promoters. Micrococcal nuclease digestion of active promoters in nuclei released few nucleosome-like nucleoprotein complexes containing N1 sequences in comparison to results with inactive promoters. We also observed that N1 DNA fragments from active promoters are of a subnucleosomal length. Nevertheless, chromatin immunoprecipitation experiments indicate that histones H3 and H4 are present on N1 sequences from active promoters, with H3 being dramatically hyperacetylated compared with that from inactive promoters and vector sequences. Strikingly, H3 in the adjacent upstream nucleosome (N2) does not appear to be differentially acetylated in active and inactive promoters, indicating that the nucleosome modification of the promoter that accompanies transactivation by HS2 is highly directed and specific. However, global acetylation of histones in vivo by trichostatin A did not activate transcription in the absence of HS2, suggesting that HS2 contributes additional activities necessary for transactivation. N1 sequences from active promoters also contain reduced levels of linker histone H1. The detection of a protected subnucleosomal sized N1 DNA fragment and the recovery of N1 DNA sequences in immunoprecipitations using anti-acetylated H3 and H4 antibodies argue that N1 is present, but in an altered conformation, in the active promoters.

Acetylation↗
PubMed1997

The complete sequences of the galago and rabbit beta-globin locus control regions: extended sequence and functional conservation outside the cores of DNase hypersensitive sites.

The locus control region (LCR) of mammalian beta-globin genes covers at least 17 kb at the 5' end of the gene cluster and has been implicated in chromatin domain opening, enhancement, and insulation from neighboring sequences. Functional dissection of the LCR has defined the minimal cores for four of the five major DNase hypersensitive sites (HSs) that mark this regulatory region. To examine fully the patterns of conserved sequences in the mammalian homologs to the beta-globin LCR, we determined the complete DNA sequence of the galago beta-globin LCR and completed previously unsequenced regions of the rabbit LCR. Simultaneous alignment of these sequences with the human, goat, and mouse LCRs revealed conserved sequences (phylogenetic footprints) detected using three largely independent methods. The most highly conserved segments are found both within the HS cores and in some but not all regions flanking the cores. These results argue for an extended pattern of well-conserved sequences, many of which lie outside the minimal cores, and we show that a key sequence required for domain opening by the region including HS3 maps about 1 kb 5' to the minimal core. Differential phylogenetic footprints, containing sequences conserved in nonhuman mammals but not in humans, are found primarily around HS3, consistent with some species-specific differences in function that may be important for differences in hemoglobin switching during development.

Animals↗
PubMed1993

Rhabdomyosarcoma arising in transgenic mice harboring the beta-globin locus control region fused with simian virus 40 large T antigen gene.

The beta-globin locus control region (LCR) confers a high level of erythroid-specific and copy-number-dependent expression to human globin genes in transgenic mice. Simian virus 40 T (tumor) antigen (Tag) with its own natural enhancer causes choroid plexus tumors in mice. We investigated the effect of the LCR on Tag gene expression, reasoning that mice harboring a LCR-Tag fusion gene might develop hematopoietic malignancies. To test this hypothesis we introduced an enhancerless Tag gene downstream of a LCR cassette into the germ lines of mice. The phenotypes of the transgenic mice depended on the copy number of the transgene. While mice with 1-2 copies matured normally, those with 3-7 copies developed rhabdomyosarcomas in different anatomic sites at high frequency and showed hyperplasia of the pancreatic islet cells which progressed to pancreatic islet tumors. In addition, the mice bearing 7 copies of the transgene had hypoglycemia and were stunted in growth. Mice with more than 10 copies were markedly stunted in growth and died within 2-4 weeks. Tag expression was detected at high levels in the mouse tumors but not in any other tissues, including the hematopoietic cells.

Animals↗
PubMed2000

A negative cis-element regulates the level of enhancement by hypersensitive site 2 of the beta-globin locus control region.

The core of DNase hypersensitive site (HS) 2 from the beta-globin locus control region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5' and 3' ends of the core. Analysis of constructs with and without the 5' negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5' end of the HS2 core map the negative element within a 20-base pair region containing two conserved sequences.

Adult↗
PubMed2001

The human growth hormone locus control region mediates long-distance transcriptional activation independent of nuclear matrix attachment regions.

Expression of the human growth hormone (hGH-N) transgene in the mouse pituitary is dependent on a multicomponent locus control region (LCR). The primary determinant of hGH LCR function maps to the pituitary-specific DNase I hypersensitive sites (HS) HSI,II, located 15 kb 5' to the hGH-N gene. The mechanism by which HSI,II mediates long-distance activation of the hGH locus remains undefined. Matrix attachment regions (MARs) comprise a set of AT-rich DNA elements postulated to interact with the nuclear scaffold and to mediate long-distance interactions between LCR elements and their target promoters. Consistent with this model, sequence analysis strongly predicted a MAR determinant in close proximity to HSI,II. Surprisingly, cell-based analysis of nuclear scaffolds failed to confirm a MAR at this site, and extensive mapping demonstrated that the entire 87 kb region encompassing the hGH LCR and contiguous hGH gene cluster was devoid of MAR activity. Homology searches revealed that the predicted MAR reflected the recent insertion of a LINE 3'-UTR segment adjacent to HSI,II. These data point out discordance between sequence-based MAR predictions and in vivo MAR function and predict a novel MAR-independent mechanism for long-distance activation of hGH-N gene expression.

3' Untranslated Regions↗
PubMed1998

Conserved elements containing NF-E2 and tandem GATA binding sites are required for erythroid-specific chromatin structure reorganization within the human beta-globin locus control region.

Proper expression of the genes of the human beta-globin gene locus requires the associated locus control region (LCR). Structurally, the LCR is defined by the presence of four domains of erythroid-specific chromatin structure. These domains, which have been characterized as DNase I hypersensitive sites (HSs), comprise the active elements of the LCR. The major focus of this research is to define the cis -acting elements which are required for the formation of these domains of unique chromatin structure. Our previous investigations on the formation of LCR HS4 demonstrated that NF-E2 and tandem, inverted GATA binding sites are required for the formation of the native HS. Similarly arranged NF-E2 and tandem GATA sites are present within the core regions of the other human LCR HSs and are evolutionarily conserved. Using site-directed mutagenesis of human HSs 2 and 3 we have tested the hypothesis that these NF-E2 and GATA sites are common requirements for the formation of all LCR HSs. We find that mutation of these elements, and particularly the GATA elements, results in a decrease or complete loss of DNase I hypersensitivity. These data imply the presence of common structural elements within the core of each LCR HS which are required for erythroid-specific chromatin structure reorganization.

Base Sequence↗
PubMed2002

Definition of transcriptional promoters in the human beta globin locus control region.

Our previous studies on the human beta globin gene cluster revealed the presence of intergenic transcripts throughout the locus, and demonstrated that transcription of the locus control region (LCR) initiates within an ERV9 endogenous retroviral long-terminal repeat (LTR) upstream of DNase I hypersensitive site 5. We show, using a combination of assays, that there are additional sites of transcription initiation within the LCR at hypersensitive sites 2 and 3. We have defined sites of transcription initiation, which occurs at discrete positions in a direction towards the globin genes. In addition, we show that mutation of specific transcription factor binding sites within HS2 leads to a reduction in transcription levels from within this site. We propose that these initiation events within the LCR can account for the observed orientation dependence of LCR function, and contribute to the open chromatin configuration of the beta globin locus. In addition, transcription from within the LCR hypersensitive sites could compensate for the absence of the ERV9 LTR in many transgenic mice lines, which nevertheless regulate their globin clusters correctly.

Binding Sites↗
PubMed2002

[A study of the effect of locus control region elements HS2 and HS3 on beta-globin gene expression].

HS2 and HS3 are important elements of human beta-globin locus control region (LCR). To study the effect of HS2, HS3, and HS2-HS3 on human beta-globin gene expression, a series of expression cassettes were constructed, in which the reporter gene encoding the enhanced green fluorescent protein (EGFP) was driven by the beta-globin promotor and under the control of HS2, HS3, and HS2-HS3. These constructs were transfected transiently into MEL and K562 cell lines mediated with liposome and their expression was measured with FACS as well as semi-quantitative RT-PCR method. The results showed that 3.2-kb HS2 could significantly enhance the activity of beta-globin promotor in MEL and K562 cells, while 3-kb HS3 could do only in MEL cells. There was no synergistic function in transient expression in the cassettes with the combination of HS2 and HS3 in MEL and K562 cells.

Gene Expression Regulation↗
PubMed1999

Correct function of the locus control region may require passage through a nonerythroid cellular environment.

The function of the beta-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb beta-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a beta-globin locus (beta-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb beta-YAC that encompasses the entire beta-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). The Ppo-155 beta-YAC was used to directly lipofect MEL 585 cells. In 7 beta-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, epsilon + gamma + beta) per copy of integrated beta-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the beta-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked beta-YAC DNA. To test whether passage of the beta-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(beta-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human beta-globin mRNA (ie, epsilon + gamma + beta) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

Animals↗
PubMed1999

A novel element upstream of the Vgamma2 gene in the murine T cell receptor gamma locus cooperates with the 3' enhancer to act as a locus control region.

Transgenic expression constructs were employed to identify a cis-acting transcription element in the T cell receptor (TCR)-gamma locus, called HsA, between the Vgamma5 and Vgamma2 genes. In constructs lacking the previously defined enhancer (3'E(Cgamma1)), HsA supports transcription in mature but not immature T cells in a largely position-independent fashion. 3'E(Cgamma1), without HsA, supports transcription in immature and mature T cells but is subject to severe position effects. Together, the two elements support expression in immature and mature T cells in a copy number-dependent, position-independent fashion. Furthermore, HsA was necessary for consistent rearrangement of transgenic recombination substrates. These data suggest that HsA provides chromatin-opening activity and, together with 3'E(Cgamma1), constitutes a T cell-specific locus control region for the TCR-gamma locus.

Animals↗
PubMed2000

Deletions within the mouse beta-globin locus control region preferentially reduce beta(min) globin gene expression.

The mouse beta-globin gene cluster is regulated, at least in part, by a locus control region (LCR) composed of several developmentally stable DNase I hypersensitive sites located upstream of the genes. In this report, we examine the level of expression of the beta(min) and beta(maj) genes in adult mice in which HS2, HS3, or HS5,6 has been either deleted or replaced by a selectable marker via homologous recombination in ES cells. Primer extension analysis of RNA extracted from circulating reticulocytes and HPLC analysis of globin chains from peripheral red blood cells revealed that all mutations that reduce the overall output of the locus preferentially decrease beta(min) expression over beta(maj). The implications of these findings for the mechanism by which the LCR controls expression of the beta(maj) and beta(min) promoters are discussed.

Animals↗
PubMed1992

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.

3T3 Cells↗
PubMed2002

Requirements for utilization of CREB binding protein by hypersensitive site two of the beta-globin locus control region.

Strong transactivation of the beta-globin genes is conferred by the beta-globin locus control region (LCR), which consists of four erythroid-specific DNase I hypersensitive sites (HS1-HS4). HS2 has a powerful enhancer activity dependent upon tandem binding sites for the erythroid cell- and megakaryocyte-specific transcription factor NF-E2. An important co-activator-mediating transactivation by HS2 is the histone acetyltransferase (HAT) CREB binding protein (CBP). We showed previously that recruitment of a GAL4-CBP fusion protein to HS2 largely bypassed the requirement of the NF-E2 sites for transactivation. To determine whether GAL4-CBP recruitment is sufficient for transactivation, we assessed the importance of cis-elements within HS2. Docking of GAL4-CBP upstream of an Agamma-globin promoter lacking HS2 only weakly activated the promoter, indicating that HS2 components are required for GAL4-CBP-mediated transactivation. Sequences upstream and downstream of the NF-E2 sites were required for maximal GAL4-CBP-mediated transactivation, and HAT catalytic activity of GAL4-CBP was critical. No single factor-binding site was required for GAL4-CBP-mediated transactivation. However, deletion of two sites, a CACC site and an E-box, abolished transactivation in transient and stable transfection assays. These results suggest that NF-E2 recruits CBP as a critical step in transactivation, but additional components of HS2 are required to achieve maximal enhancer activity.

Acetyltransferases↗