Abstract

Locus control regions (LCRs) are operationally defined by their ability to enhance the expression of linked genes to physiological levels in a tissue-specific and copy number–dependent manner at ectopic chromatin sites. Although their composition and locations relative to their cognate genes are different, LCRs have been described in a broad spectrum of mammalian gene systems, suggesting that they play an important role in the control of eukaryotic gene expression. The discovery of the LCR in the β-globin locus and the characterization of LCRs in other loci reinforces the concept that developmental and cell lineage–specific regulation of gene expression relies not on gene-proximal elements such as promoters, enhancers, and silencers exclusively, but also on long-range interactions of variouscis regulatory elements and dynamic chromatin alterations.

Introduction

Locus control regions (LCRs) are operationally defined by their ability to enhance the expression of linked genes to physiological levels in a tissue-specific and copy number–dependent manner at ectopic chromatin sites. The components of an LCR commonly colocalize to sites of DNAse I hypersensitivity (HS) in the chromatin of expressing cells. The core determinants at individual HSs are composed of arrays of multiple ubiquitous and lineage-specific transcription factor–binding sites.

The LCR was first identified in the human β-globin locus.1 (For a review, see Stamatoyannopoulos and Grosveld,2 Fraser and Grosveld,3 and Li et al.4) Early studies showed that a 5-kilobase (kb) β-globin gene segment, including a 1.5-kb promoter region, was expressed in erythroleukemia cell lines, implying that this fragment contains all the regulatory elements necessary for proper expression. However, this fragment did not uniformly promote gene expression in transgenic mice.5-7 The gene was expressed in only a small proportion of transgenic mice, but expression was far below physiologically significant levels and was variable between lines. These findings suggested that a major regulatory element required for reproducible, high-level expression in vivo was missing in this construct. Clues regarding the nature of the missing element came from several observations. For example, in some forms of β-thalassemia the genes of the β-globin locus are intact but not expressed.8,9 A defect common to the loci underlying these conditions was a large deletion upstream of the β-like globin genes. This deletion results in a closed chromatin conformation spanning the whole locus and leads to suppression of gene expression.8,10 Thus, these data suggested that the deleted DNA segment contained an indispensable cis-acting regulatory element required for β-globin expression in vivo. The existence of such a regulatory element was also implied by the presence of developmentally stable, erythroid-specific HSs 6 to 20 kb 5′ to the ε-globin gene.11,12 Definitive evidence for the presence of the LCR came from transgenic mouse studies.1 Linkage of this region to a β-globin gene resulted in expression of the gene at a level comparable to the endogenous mouse β-globin genes in a position-independent, copy number–dependent manner. LCRs have been described in a broad spectrum of mammalian gene systems, suggesting that they play an important role in the control of eukaryotic gene expression.

Properties of LCRs

Transcriptional enhancer activity

The most prominent property of the LCRs is their strong, transcription-enhancing activity. The β-globin LCR is located 6 to 22 kb 5′ to the first (embryonic) globin gene in the locus (Figure1). It consists of 5 DNAse I–hypersensitive sites, 5′HSs 1 to 5. HSs 1 to 4 are formed only in erythroid cells, while 5′HS5 is found in multiple lineages of cells, but it is not constitutive.13 When the LCR is absent, transcription of the human β-globin gene is usually less than 1% of the endogenous murine β-globin mRNA in transgenic mice, if it is expressed at all.5-7 Inclusion of the LCR increases β-globin gene expression to a level comparable to that of the mouse β-globin genes in all transgenic animals, indicating that the LCR has strong enhancer activity.1 LCR enhancer activity is also significant at its endogenous location, as demonstrated by LCR-deletion experiments.14-16 These deletions in the native chromosomes of mouse or human cell lines severely reduce the expression of globin genes.

Fig. 1.

The human and mouse β-globin loci.

The human locus consists of 5 functional genes, indicated as dark boxes, arrayed in their order of developmental expression, 5′-ε-Gγ-Aγ-δ-β-3′. There are 2 developmental switches in globin chain synthesis coincident with changes in site and type of erythropoiesis. During primitive erythropoiesis, the ε-globin gene is expressed in the embryonic yolk sac. The first switch occurs at approximately 8 weeks' gestation; the ε-globin gene is silenced and the Gγ- andAγ-globin genes are expressed during definitive erythropoiesis in the fetal liver. The second switch occurs shortly after birth; the γ-globin genes are silenced and the β-globin gene and, to a lesser extent, the δ-globin gene are activated in the bone marrow. The HSs 5′HS1 through 5′HS7 are located −6, −11, −15, −18, −22, −28, and −35 kb relative to the ε-globin gene, respectively, and are indicated by arrows. 5′HS1 through 5′HS4 are erythroid specific, but 5′HS5 through 5′HS 7 are not. Another HS (3′HS1) is located 20 kb downstream of the β-globin gene; 3′HS1 is found only in erythroid cells. Boxes represent globin genes and ovals represent olfactor receptor genes; filled ones represent the productive genes and shaded ones the pseudogenes. The lines below the diagram of the locus indicate deletions of the LCR discussed in “In vivo function of LCRs.” The Hispanic deletion, which causes (γδβ)0 thalassemia in humans, extends an additional 20 kb 5′ of the LCR 5′HS5.

Fig. 1.

The human and mouse β-globin loci.

The human locus consists of 5 functional genes, indicated as dark boxes, arrayed in their order of developmental expression, 5′-ε-Gγ-Aγ-δ-β-3′. There are 2 developmental switches in globin chain synthesis coincident with changes in site and type of erythropoiesis. During primitive erythropoiesis, the ε-globin gene is expressed in the embryonic yolk sac. The first switch occurs at approximately 8 weeks' gestation; the ε-globin gene is silenced and the Gγ- andAγ-globin genes are expressed during definitive erythropoiesis in the fetal liver. The second switch occurs shortly after birth; the γ-globin genes are silenced and the β-globin gene and, to a lesser extent, the δ-globin gene are activated in the bone marrow. The HSs 5′HS1 through 5′HS7 are located −6, −11, −15, −18, −22, −28, and −35 kb relative to the ε-globin gene, respectively, and are indicated by arrows. 5′HS1 through 5′HS4 are erythroid specific, but 5′HS5 through 5′HS 7 are not. Another HS (3′HS1) is located 20 kb downstream of the β-globin gene; 3′HS1 is found only in erythroid cells. Boxes represent globin genes and ovals represent olfactor receptor genes; filled ones represent the productive genes and shaded ones the pseudogenes. The lines below the diagram of the locus indicate deletions of the LCR discussed in “In vivo function of LCRs.” The Hispanic deletion, which causes (γδβ)0 thalassemia in humans, extends an additional 20 kb 5′ of the LCR 5′HS5.

The enhancer activity of the β-globin LCR resides in 5′HS2, 3, and 4, but not in 5′HS1 or 5 (for a review, see Stamatoyannopoulos and Grosveld,2 Fraser and Grosveld,3 Li et al,4 and Hardison et al17). 5′HS2 behaves as a classical enhancer; that is, its activity can be detected in transient transfection assays. Enhancer activity in 5′HS3 or 4 can be detected only when they are integrated into chromatin (for a review, see Hardison et al17 and references therein). A requirement for chromosomal integration suggests that alteration of chromatin structure may be involved in propagating the enhancer activity of these 2 HSs. 5′HS5 functions as a chromatin insulator.18-20 The function of 5′HS1 remains to be defined.

The enhancer activity of the β-globin LCR is tissue specific; that is, the expression of globin genes is confined to erythroid cells when linked to the β-globin LCR.1,21 In addition, the LCR is able to enhance expression of linked heterogeneous nonglobin gene promoters in erythrocytes. However, when a nonglobin gene was coupled to the LCR, ectopic expression was observed in some transgenic mice.22 In these instances, although the LCR conferred erythroid-specific gene expression on the heterogeneous gene, the natural function of the linked promoter allowed expression outside the erythroid compartment. Thus, tissue-specific control of basal transcription may reside in the promoter, as is the case for the globin genes, whereas tissue-specific enhancement of gene expression may be a property of the LCR. These data suggest that tissue specificity is not really an intrinsic property of the LCR but depends on both the LCR and the promoter that it interacts with.

Central to understanding the enhancer functions of β-globin LCR is the identification of the transcription factors mediating enhancer activity. Enhancer activity of 5′HSs 2-4 resides in a 200-bp to 300-bp core, which contains an array of binding sites for ubiquitous and erythroid-specific transfactors. A conserved sequence within 5′HS2, TGCTGA(C/G)TCA(T/C), is critical for strong enhancer activity.23,24 This Maf recognition element (MARE) is bound by multiple homodimeric and heterodimeric transcription factors in vitro.25 These factors include Maf homodimers, heterodimers containing a Maf subunit and another bZIP protein (NF-E2, Nrf1, Nrf2, Bach1, Bach2), and heterodimers lacking a Maf subunit (AP1).26-31 NF-E2 is the major protein found in nuclear extracts from murine erythroleukemia (MEL) cells that binds the tandem MAREs of 5′HS2, and globin gene expression closely parallels the level of NF-E2 binding activity.32 The MEL cell line CB3, which lacks p45, is severely impaired in globin gene expression, and transcription can be rescued by expression of NF-E2.32,33The erythroid-specific transactivator p45/NF-E2 binds directly and specifically to 5′HS2 in erythroleukemia cells and mouse fetal liver. Chromatin immunoprecipitation (ChIP) assay showed that specific recovery of the 5′HS2 sequences was dependent upon the presence of p45 and intact MARE sites within 5′HS2.34 Investigation of the binding of the p45/p18 (MafK) heterodimer or other small Maf proteins within the globin locus showed that prior to induction of MEL cell differentiation, the LCR was occupied by small Maf proteins, and that during erythroid maturation, the NF-E2 complex was recruited to the LCR and the active globin promoters, even though the promoters do not contain MAREs. This differentiation-coupled recruitment of the NF-E2 complex correlates with a more than 100-fold increase in β-major globin transcription, but is not associated with a significant change in locus-wide histone H3 acetylation. Thus, the β-globin gene locus may exist in a constitutively open chromatin conformation before terminal differentiation, and the recruitment of the NF-E2 complex to the LCR and active promoters may be a rate-limiting step in the activation of β-globin gene expression.35 While the in vivo association of NF-E2 and HS2 of the βLCR is confirmed by ChIP assay, a knockout of p45 gene does not inhibit globin gene expression.36 The absence of phenotype in the p45 knockout mice is not due to the result of compensation by Nrf-2, a factor closely related to p45, as demonstrated by the study of a double knockout of p45 and Nrf-2, which also fails to interfere with expression of the α- and β-globin genes.37 These observations suggest an interchangeable function between members of the cap'n collar (CNC) subfamily of bZIP transcription factors.

LCR functions may affect the basic transcription machinery directly. RNA polymerase II (pol II), one of the essential components of the eukaryotic transcription apparatus, was found to be associated with the β-globin LCR in a p45/NF-E2–independent manner, whereas its recruitment to the promoter required p45/NF-E2. These data suggest that pol II accesses the LCR and p45/NF-E2 induces long-range transfer of pol II to the promoter, resulting in transcriptional activation.38 

Copy number–dependent gene expression and chromatin domain-opening activity

Another property of the LCRs is their ability to confer position-independent, copy number–dependent expression on a linked gene.1 Copy number–dependent expression is widely considered to be indicative of open chromatin structure, that is, DNA that is accessible to transcription factors. Involvement of the β-globin LCR in creating open chromatin was suggested from analysis of β-thalassemia mutants with deletion of the LCR.8,10In the Hispanic form of β-thalassemia, an approximately 35-kb region upstream of 5′HS1 is deleted, but the remainder of the globin locus is intact. However, none of the globin genes is expressed. The deletion produces a closed chromatin conformation that spans the entire locus.10 Consistent with this, only the intact LCR (5′HS1-5) can provide position-independent chromatin-opening activity in single-copy transgenic mice carrying the entire β-globin locus.39 When one of the HSs was deleted from the LCR, expression of the β-globin genes appeared to be sensitive to the position of integration. In transgenic mice carrying single copies of small, recombinant 5′HS-globin gene constructs, only 5′HS3 is able to confer copy number–dependent gene expression. This observation led to the conclusion that 5′HS3 possesses the dominant chromatin-opening activity of the β-globin LCR.40 However, 5′HS3 chromatin-opening activity may not be dominant, since it appears to be dependent upon the constitution of the constructs.41Formation of hypersensitivity is a result of interaction of multiple ubiquitous and erythroid-specific transacting factors in the HS regions.42 

Recent studies have established that the human CD2 LCR achieves position-independent expression in the T cells of transgenic mice by overcoming heterochromatin-mediated position effect variegation (PEV).43 Fluorescence in situ hybridization (FISH) was used to identify the sites of transgene integration in individual mouse lines and allowed a correlation between the type of position effects induced by such chromosomal locations and the DNA sequences required to overcome them. Transgenic mice carrying a CD2 minigene attached only to the 3′ CD2 transcriptional enhancer (the CD2 HSs 1 and 2) exhibited variegated expression when the transgene integrated in the centromere. In contrast, mice carrying a transgene with additional 3′ sequences (the CD2 HS3) showed no variegation even when the latter integrated in centromeric positions. This indicates that the CD2 HS3 functions in the establishment and/or maintenance of an open chromatin domain and that human CD2 LCR is able to overcome the gene repression imposed by constitutive centromeric heterochromatin.

In conclusion, the ability to confer copy number–dependent expression of a transgene is used to distinguish a DNA fragment functioning as an LCR rather than a transcriptional enhancer. This criterion has been employed in identification of all LCR or LCR-like elements.

Timing and origin of DNA replication

The mammalian genome is made up of defined zones that undergo DNA replication in a programmed manner during the S phase of the cell cycle. Studies of individual genes have demonstrated that there is a correlation between replication timing and gene expression.44,45 The human β-globin locus replicates late in most cell types, but replicates early in erythroid cells.46,47 

Data generated from transgenic mice by FISH analysis mapped and characterized the replication zone surrounding the human globin locus on chromosome 11. These results showed that the β-globin LCR region (5′HSs 1-5) was sufficient for directing replication timing in a developmentally specific manner in vivo.48 The LCR (5′HSs 1-5) also plays a role in setting up regional erythroid-specific, open chromatin structure in transgenic mice, and this function is likely intertwined with the ability to direct early replication timing.49 Although early replication is generally correlated with gene expression, it has not been possible to decipher the cause-and-effect relationship between these 2 parameters.48 Other results using targeted deletion of the LCR (5′HSs 1-5) showed that early replication timing and an open chromatin structure do not, by themselves, guarantee high levels of globin transcription in erythroid cells.50 Therefore, an as yet undefined class of cis-acting elements may play a role in mediating control of replication timing, independent of transcription.

Many studies have emphasized the relationship between early replication and globin transcription in erythroid cells. However, these replication elements within the LCR also function in nonexpressing cell types. Thus, one of the major roles for replication timing control at the globin locus may be to set up late replication with its accompanying inactive chromatin structure in nonerythroid cells. In this manner, repression of background transcription may be achieved.48Perhaps this is accomplished by restricting the exposure of newly assembled nucleosomes to histone deacetylases, specifically during replication in late S phase.49 Recent evidence supporting this hypothesis suggests that HDAC2 is preferentially associated with late replication foci.51 Further data are required to determine whether the effects on replication are general features of LCRs and whether these effects influence transcription or are secondary to it.

Histone modification and heterochromatin

Despite numerous studies on the role of the LCR in controlling β-globin gene expression, the mechanism of long-range transactivation by the LCRs is poorly understood. Several models (including looping, tracking, linking, topologic alterations, and modification of proteins associated with chromatin) have been invoked to explain the functions of LCR.4,52-55 All the models, directly or indirectly, implicate the ability of LCRs to alter chromatin configuration and conformation.

The effects of LCRs on chromatin acetylation have been studied in different model systems. Function of the human growth hormone (hGH) LCR has been linked to specific patterns of core histone acetylation. The hGH locus consists of 5 genes expressed in either the pituitary or the placenta.56 This LCR consists of 5 HSs: 2 pituitary-specific (HSI, HSII), 1 placenta-specific (HSIV), and 2 shared (HSIII, HSV). In the pituitary, the LCR is encompassed in a somatotrope-specific domain of hyperacetylated chromatin that extends from the most 5′ LCR component to the hGH-N promoter. Further analysis shows that the hGH LCR, located 14.5 kb upstream from the hGH-N promoter, plays a critical, specific, and nonredundant role in facilitating promoter transacting factor binding and activation of hGH-N transcription. It also plays an essential role in establishing a 32-kb acetylated region that encompasses the entire hGH LCR contiguous with the hGH-N promoter. Separate positive elements in the LCR (HSI, HSII) for pituitary-expressed genes, or in gene-proximal sequences (P-elements) for placenta-expressed genes, activate their respective target genes by tissue-specific recruitment of different histone acetyltransferase activities, resulting in distinct patterns of acetylation across the locus.57 These data support a model for long-range gene activation via LCR-mediated targeting and extensive spreading of core histone acetylation.58 

The functions of the LCR in the β-globin locus appear to be different from those of the hGH gene cluster. Deletion of 5′HS2-5 of the human β-globin LCR did not affect the general pattern of histone H4 acetylation of a β-globin locus transgene.59 Other studies reported that although deletion of the murine β-globin LCR decreased the rate of β-globin transcription, it did not alter the acetylation status of histone H3 or H4 within the promoter region.60 Thus, histone H3 or H4 acetylation at the β-globin promoter may be independent of LCR function.

NF-E2 is required for histone hyperacetylation at the adult β-globin promoter, but not at the LCR.38,61 Other data demonstrated that the β-globin LCR and transcriptionally active promoters were enriched in acetylated histones in fetal liver relative to fetal brain, whereas the inactive promoters were hypoacetylated. In contrast, the LCR and both active and inactive promoters were hyperacetylated in yolk sac. 5′HS2 was also hyperacetylated in murine ES cells, whereas β-globin promoters were hypoacetylated. Thus, the acetylation pattern varied at different developmental stages. Histone deacetylase inhibition selectively increased acetylation at a hypoacetylated promoter in fetal liver, suggesting that active deacetylation contributes to silencing of promoters. Therefore, dynamic histone acetylation and deacetylation activities may play an important role in the developmental control of β-globin gene expression.61 

DNA methylation is important in mammalian development because it controls gene expression through chromatin closure and gene silencing. During development, gene loci expressed in a tissue-specific manner become selectively demethylated in the appropriate cell types by poorly understood processes. The LCRs may play a role in tissue-specific DNA demethylation. Studies of the methylation status of the LCR for the mouse T-cell receptor (TCR) α/δ locus support such a role. Tissue-specific functions of this LCR depend largely on 2 HSs, HS1 (T-cell receptor α enhancer) and HS1′. These HSs induce lymphoid organ–specific DNA demethylation in a region located 3.8 kb away, with little effect on intervening methylated DNA. Demethylation is impaired in mice with a germ line deletion of the HS1/HS1′ clusters. Using 5′-deletion mutants of a transgenic LCR reporter gene construct, HS1′ can act in the absence of HS1 to direct this tissue-specific DNA demethylation event. Therefore, elements of an LCR may control tissue-specific DNA methylation patterns both in transgenes and in native loci.62 

In vivo function of LCRs

As discussed above, LCRs possess all the properties necessary for opening a chromosome domain and preventing heterochromatinization at ectopic sites. This property of the LCR most prominently distinguishes it from enhancers. Thus, a broadly accepted model for the major role of the β-globin LCR in vivo is to open and/or maintain a permissive chromatin conformation within the β-globin locus in erythroid cells, although enhancement of transcription is also an essential function. Surprisingly, when the entire mouse β-globin LCR (5′HS1-6) was deleted by homologous recombination, the formation of the general DNAse I-sensitivity associated with the β-globin locus domain was not affected; however, transcription of all β-like globin genes was strikingly reduced.14,63 These observations raise several questions regarding the real in vivo function of the β-globin LCR. Is this LCR simply another enhancer in the β-globin locus? If the β-globin LCR functions only as an enhancer within endogenous β-globin loci, how can this fact be reconciled with observations from transgenic mouse studies in which chromatin-opening activity is characteristic?

Understanding the in vivo function of the LCRs is associated with our knowledge of the process of gene activation. A prevailing model for gene activation is that it is a stepwise process. The first step is chromatin opening. Opening allows transacting transcription factors and cofactors to access chromatin and assemble a functional transcription apparatus. Genes in open chromatin domains are poised for expression. When protein activators are present, transcription commences and high-level gene expression is achieved. Chromatin opening is manifested by an increase in its sensitivity to DNAse I or other nucleases. General DNAse I sensitivity represents a level of sensitivity on the order of one magnitude greater than bulk chromatin. General sensitivity may stretch over regions of several hundred kilobases. Within these regions of general sensitivity, small regions (< 300 bp) of DNAse I sensitivity may be 2 orders of magnitude more sensitive than bulk chromatin; these regions are termed DNAse I-hypersensitive sites. Although increased DNAse I sensitivity may be due to improved accessibility of DNA packed in chromatin, DNAse I sensitivity indeed is an ambiguous indicative of chromatin structure. The precise molecular nature of the alterations underlying accessibility has not been delineated. Questions persist as to whether the changes occur at the 30-nm fiber level or at the nucleosome level and whether all histone tails in the general DNAse I sensitive regions are modified (acetylated, methylated, or phosphorylated) in the same fashion. Although general DNAse I sensitivity was detected in both normal and β-globin LCR knockout mice in erythroid cells, previous data do not indicate whether the general DNAse I sensitivity detected in LCR knockout mice and that detected in normal mice represent identical or different chromatin configurations.

Regardless of what the chromatin configuration may be, chromatin of the globin locus is more sensitive than bulk DNA in LCR knockout mice. In the absence of the LCR, an alternate pathway for establishment and maintenance of open chromatin must exist. Chromatin is not a structurally inert entity. Most likely it undergoes many dynamic conformational transitions that may be important in facilitating interactions between transacting factors and DNA. DNA probably unwraps from the edge of the nucleosome, since sites within nucleosomal DNA are transiently separated from histones with a probability of 1 in 103 to 105 moving from the periphery of the nucleosome toward the center.64,65 Thus, given the dynamic nature of this system, factors present at sufficient concentrations and having high affinities for naked DNA may be able to compete efficiently with histone proteins for binding, thereby ensuring significant loading of these proteins at their cognate DNA elements in chromatin. Moreover, some transacting factors, such as GATA-4, are able to bind to compacted chromatin and open up a local chromatin.66 Other transacting factors then attain an opportunity to access enhancer or promoter elements and further remodel chromatin by recruiting and targeting chromatin modifying and remodeling machinery. Since a large number of factor-binding sites are scattered throughout the β-globin locus, particularly at promoters, they are able to recruit various proteins and cofactors in the erythroid environment. Accumulation of a large amount of small, qualitative changes may finally lead to a major change in chromatin structure. Such a synergistic mechanism could result in an open chromatin at low level in the absence of the LCR. Synergistic mechanisms have been postulated for transcription activation via cooperation of multiple transactivators67and for heterochromatin formation in a mass action model.68 Based on this model, the LCR does not necessarily possess a specified chromatin opening activity.

The LCR chromatin-opening activity manifested in transgenic studies indeed results from the unique feature of the LCR that numerous binding sites clustered in the region induce an exponent (synergistic) effect on chromatin structure. Closed chromatin is considered the default status and is found in the vast majority of the chromosome. Thus, most transgenes are integrated in sites of closed chromatin. When transgene expression is detected, the chromatin region surrounding the transgene is invariably open. Although it is easy to surmise that chromatin opening is a prerequisite of gene transcription, this conclusion is not definitive. The 2 processes may be separated during in vitro assays, but more likely these 2 events are mutually interdependent in vivo. Thus, the chromatin-opening activity of the LCR manifested in transgenic studies must also function at the endogenous locus in vivo in a similar manner. Experimental description of chromatin opening indeed includes multiple distinctive states in chromatin conformation and configuration. Chromatin-opening activity has to be considered as an integrated but not necessarily linear event in gene activation.

Mechanisms of globin gene activation by the LCR

Clearly, the LCR has a role in enhancement of globin gene expression, although some uncertainty exists regarding the direct effects of the LCR on chromatin conformation. Analysis of LCR function at its endogenous location in cell lines suggests that it is limited to globin gene transcription activation,14,15 whereas transgenic experiments suggest that it is also necessary for the establishment and maintenance of an open β-globin chromatin domain. Regardless of whether the LCR functions in one or both of these processes, it does so over a long distance. Studies using both native loci and constructs in transgenic mice offer insights as to how the β-globin LCR accomplishes transcriptional activation. Four models of LCR function have been proposed: looping, tracking, facilitated tracking, and linking (Figure 2). Available data neither strongly support nor preclude any of them.

Fig. 2.

Models of LCR function.

A globin gene is denoted by a green rectangular box with the promoter region indicated in a lighter green. Transcription factors are shown as colored ovals and circles. The 4 erythroid-specific hypersensitive site cores (HSs) are indicated by small red boxes. Blue boxes are the positions of 5′HS5 and 3′HS1, representing likely insulator elements. The flanking DNA sequences of the HSs are depicted as loops between the HS cores. Transcripts are denoted by wavy arrows. (A) Looping model. Transcription factors bind to the LCR HSs and the gene promoter. The LCR directly interacts with the gene promoter by looping out the intervening DNA, thus forming an active transcription complex at the gene promoter. (B) Tracking model. Sequence-specific transcription factors bind to the LCR, forming a complex that tracks down the DNA sequence, as depicted by the large black arrowhead, until encountering transcription factors bound to the appropriate gene promoter, initiating high-level gene expression. (C) Facilitated tracking model. Aspects of both looping and tracking models are combined. Sequence-specific transcription factors bind the LCR; looping then occurs to deliver the bound transcription factors proximal to the gene promoter, followed by tracking, until they encounter transcription factors bound to the appropriate gene promoter. (D) Linking model. Sequential binding of transcription factors along the DNA directs changes in chromatin conformation and defines the transcriptional domain. The transcription factors are linked to one another from the LCR to the gene promoter by non–DNA-binding proteins and chromatin modifiers (shown as small colored circles).

Fig. 2.

Models of LCR function.

A globin gene is denoted by a green rectangular box with the promoter region indicated in a lighter green. Transcription factors are shown as colored ovals and circles. The 4 erythroid-specific hypersensitive site cores (HSs) are indicated by small red boxes. Blue boxes are the positions of 5′HS5 and 3′HS1, representing likely insulator elements. The flanking DNA sequences of the HSs are depicted as loops between the HS cores. Transcripts are denoted by wavy arrows. (A) Looping model. Transcription factors bind to the LCR HSs and the gene promoter. The LCR directly interacts with the gene promoter by looping out the intervening DNA, thus forming an active transcription complex at the gene promoter. (B) Tracking model. Sequence-specific transcription factors bind to the LCR, forming a complex that tracks down the DNA sequence, as depicted by the large black arrowhead, until encountering transcription factors bound to the appropriate gene promoter, initiating high-level gene expression. (C) Facilitated tracking model. Aspects of both looping and tracking models are combined. Sequence-specific transcription factors bind the LCR; looping then occurs to deliver the bound transcription factors proximal to the gene promoter, followed by tracking, until they encounter transcription factors bound to the appropriate gene promoter. (D) Linking model. Sequential binding of transcription factors along the DNA directs changes in chromatin conformation and defines the transcriptional domain. The transcription factors are linked to one another from the LCR to the gene promoter by non–DNA-binding proteins and chromatin modifiers (shown as small colored circles).

The looping model suggests that the 5′HSs of the β-globin LCR fold to form a holocomplex, with the HS core elements forming an active site that binds transcription factors and the core-flanking sequences constraining the holocomplex in the proper conformation (Figure 2A). This structure physically “loops,” so that the LCR comes in close proximity to the appropriate promoter. Close association with gene-proximal promoter and enhancer elements allows the delivery of LCR-bound transcription proteins and other coactivators that interact with the basal transcription apparatus, already bound at the promoter to form a stable transcription complex, thus enhancing globin gene expression.39,52,69,70 A variation of this model suggests that the LCR initially serves as a multiple element receptor that acts as a hub for factor binding to direct chromatin remodeling.71 Once chromatin-remodeling activity has been completed, the LCR directly interacts with downstream genes to facilitate their expression.

Several data support the looping model. Deletion of the 5′HS2 core abolished expression of the ε-, γ-, and β-globin genes.70 However, when the entire 5′HS2 region of conserved sequence similarity (core and flanking sequences) was removed, the ε-, γ- and β-globin genes were expressed in the correct temporal order, although the levels of each were decreased severalfold.72 These data suggested that, in the case of the core deletion, the 5′HS2 flanking regions were able to interact with the flanking sequences of the remaining intact 5′HSs to form the normal holocomplex conformation. Removal of only the 5′HS2 core, in effect, destroyed the active site of the holocomplex, resulting in a dominant-negative mutation that crippled LCR function. In contrast, when the entire 5′HS2 region was deleted, the remaining 5′HS sites were able to adapt an alternate holocomplex conformation with a slightly less effective active site consisting of the remaining 5′HS cores and constrained in form by the remaining 5′HS flanking sequences.72 Similar results were found with 5′HS3 core deletions versus complete deletion of 5′HS3.72-74 

Further evidence supporting the notion of a holocomplex suggests that the LCR interacts with only 1 globin gene promoter at a time and that it may “flip-flop” between 2 or more promoters, depending on the stage of development.39,52 In this model, the LCR holocomplex is free to move from gene to gene. A parameter relevant to holocomplex function is the distance between the LCR and its target gene, which has been shown to affect the probability that these 2 elements will interact.52,75-77 This probability is constant for a gene at a specific stage of development. As development proceeds, the LCR has increasingly stable interactions with more distant globin genes, which is largely a function of the changing transcription factor milieu. Thus, it is mainly the availability of specific transcription factors and distance of a gene from the LCR that constrain the frequency of LCR-gene interactions during development.75-77 

In the tracking, or scanning, model, erythroid-specific and ubiquitous transcription factors and cofactors bind recognition sequences in the LCR sequences, forming an activation complex that migrates, or tracks, linearly along the DNA helix of the locus (Figure2B).53,78 When this transcription complex encounters the basal transcription machinery located at the correct (according to the developmental stage) promoter, the complete transcriptional apparatus is assembled and transcription of that gene is initiated. If this model is valid, the expectation would be that some aberrant transcripts would arise from cryptic start sites along the locus. In fact, transcripts were detected across the LCR and intergenic regions in erythroid cells, but not in nonerythroid cells.53,79 However, these transcripts were nuclear-specific; they were not found in the cytoplasm, suggesting that they were not processed into mature messenger RNAs. The function of such intergenic transcription may be to deliver transcription complex proteins to the globin gene promoters via the tracking mechanism. Alternatively, it is possible that the function of these transcripts is to establish and maintain an open chromatin conformation permissive for gene transcription, although the persistence of DNAse I sensitivity following deletion of the LCR in cell lines argues against this role.14,15 Deacetylases and methylases within the complex may reorganize chromatin after the transcription complex activates transcription, possibly to limit activation to a particular developmental stage.

The facilitated-tracking model incorporates aspects of both the looping and tracking models (Figure 2C).78 An LCR bound-transcription factor and coactivator complex loops to contact downstream DNA in promoter-distal regions, where the transcription factor complex is released. This complex then tracks in small steps along the chromatin until it encounters the appropriate promoter with its associated bound proteins. A stable loop structure is established and gene expression proceeds.

The linking model proposes that chromatin facilitator proteins bound throughout the locus define the domain to be transcribed and mediate the sequential stage-specific binding of transcription factors (Figure2D).55 Non–DNA-binding facilitator proteins form a continuous protein chain from the LCR to the globin gene to be transcribed, linking proteins bound at a transcriptionally primed gene to one another.54 Support for this model comes from theDrosophila Chip protein complex.80 Chip protein complexes interact with transcription factors bound at a promoter region at a specific developmental time point. The Chip-tagged promoter is targeted for transcriptional activation. It was speculated that a homologous mammalian protein complex may act as the facilitating guide for transcription initiation, associating with transcription factors in the globin gene promoter regions and aiding gene activation.55 This Chip-like protein complex may allow transcriptional activation of one gene at a time, while simultaneously blocking transcription outside of the region, accounting for the developmental stage-specific expression of the β-like globin genes. The β-globin locus may have several transcription factor–bound promoter regions linked in a chainlike fashion. Chip-like proteins then dissociate and move to another promoter link to target that promoter for LCR interaction. Thus, globin gene switching proceeds.

LCRs in other systems

Several elements have been characterized in mammals (including humans, mice, rats, chickens, rabbits, sheep, and goats) that meet the criteria for LCR function (Table1). Several other elements have been identified that likely will be confirmed as LCRs, including one in the medaka fish tyrosinase gene.81-84 Structurally, these LCRs are composed of varying numbers of tissue-specific DNAse I–hypersensitive sites. The HSs of nonglobin LCRs have been extensively characterized and consist of a 150- to 300-bp central core containing a high density of transcription factor binding sites.85-87 Although the β-globin LCR consists of 5 HSs clustered on one contiguous piece of DNA, the sequences that embody a complete LCR do not have to be located together, whether upstream of, downstream of, or within the genes they control. Other LCRs are a collection of elements with different numbers of HSs spread over large distances. The relative simplicity of the β-globin LCR with regard to its single group of HSs may have contributed to its early discovery. Identification of LCRs in complex multigene loci, where the elements are interspersed among the genes, is a difficult task. Functionally, they all exhibit some or all of the properties associated with the β-globin LCR, most commonly the hallmark of copy number–dependent, site-of-integration–independent expression of their cognate loci or linked transgenes.

Table 1.

Mammalian LCR and LCR-like elements

Human: 
  1. β-globin locus1 
  2. Adenosine deaminase gene, human86 
  3. Apolipoprotein E/C-1 gene locus, human105 
  4. T cell receptor α/δ locus, human96 
  5. CD2 gene, human95 
  6. S100 β gene, human106 
  7. Growth hormone gene, human107 
  8. Apolipoprotein B gene, human108 
  9. β myosin heavy chain gene, human109 
 10. MHC class I HLA-B7 gene, human87 
 11. Immunoglobulin heavy chain locus, human110 
 12. Immunoglobulin C alpha 1 & 2 genes, human111 
 13. Keratin 18 gene, human112 
 14. MHC class I HLA G gene, human113 
 15. Complement component C4A & B genes, human114 
 16. Red and green visual pigment genes, human115 
 17. CD4 gene, human116 
 18. α-lactalbumin, human117 
 19. Desmin gene, human118 
 20. CYP19 (aromatase) gene, human119 
 21. c-fesproto-oncogene, human120 
Mouse: 
  1. β-globin locus121 
  2. MHC Class II Ea gene, mouse122 
  3. Tyrosinase gene, mouse123 
  4. α-fetoprotein gene, mouse124 
  5. Immunoglobulin μ heavy chain, mouse125 
  6. CD34 gene, mouse126 
  7. Metallothionein II gene, mouse127 
  8. Kallikrein genes, mouse and rat128 
  9. Glycophorin gene, mouse129 
 10. γ1 heavy chain gene, mouse130 
 11. λ5-VpreB1 locus, mouse131 
 12. granzyme B gene, mouse132 
 13. T cell receptor γ locus, murine102 
 14. Interleukin-2 gene, mouse84 
Rat: 
  1. Aldolase C gene, rat133 
  2. Whey acidic protein (WAP) gene, rat134 
  3. Kallikrein genes, mouse and rat128 
  4. LAP (C/EBP β) gene, rat135 
Other: 
  1. β-globin locus, goat, rabbit136,137  
  2. β-lactoglobulin gene, ovine138 
Human: 
  1. β-globin locus1 
  2. Adenosine deaminase gene, human86 
  3. Apolipoprotein E/C-1 gene locus, human105 
  4. T cell receptor α/δ locus, human96 
  5. CD2 gene, human95 
  6. S100 β gene, human106 
  7. Growth hormone gene, human107 
  8. Apolipoprotein B gene, human108 
  9. β myosin heavy chain gene, human109 
 10. MHC class I HLA-B7 gene, human87 
 11. Immunoglobulin heavy chain locus, human110 
 12. Immunoglobulin C alpha 1 & 2 genes, human111 
 13. Keratin 18 gene, human112 
 14. MHC class I HLA G gene, human113 
 15. Complement component C4A & B genes, human114 
 16. Red and green visual pigment genes, human115 
 17. CD4 gene, human116 
 18. α-lactalbumin, human117 
 19. Desmin gene, human118 
 20. CYP19 (aromatase) gene, human119 
 21. c-fesproto-oncogene, human120 
Mouse: 
  1. β-globin locus121 
  2. MHC Class II Ea gene, mouse122 
  3. Tyrosinase gene, mouse123 
  4. α-fetoprotein gene, mouse124 
  5. Immunoglobulin μ heavy chain, mouse125 
  6. CD34 gene, mouse126 
  7. Metallothionein II gene, mouse127 
  8. Kallikrein genes, mouse and rat128 
  9. Glycophorin gene, mouse129 
 10. γ1 heavy chain gene, mouse130 
 11. λ5-VpreB1 locus, mouse131 
 12. granzyme B gene, mouse132 
 13. T cell receptor γ locus, murine102 
 14. Interleukin-2 gene, mouse84 
Rat: 
  1. Aldolase C gene, rat133 
  2. Whey acidic protein (WAP) gene, rat134 
  3. Kallikrein genes, mouse and rat128 
  4. LAP (C/EBP β) gene, rat135 
Other: 
  1. β-globin locus, goat, rabbit136,137  
  2. β-lactoglobulin gene, ovine138 

Most of the data regarding LCR function have come from studies of the human and murine β-globin LCRs. Several insights have been gained with studies of the chicken LCR. Organization of the chicken LCR is similar to its human counterpart except that one of the LCR elements is located between the adult βA and embryonic ɛ genes (the βA/ɛ enhancer).88 This enhancer is able to confer site-independent expression to the chicken βA-globin gene in transgenic mice.89Chromatin unfolding of the chicken β-locus requires the presence of both the LCR and the promoter.90 Chicken HS4 demarcates the 5′ border of the locus, which functions as a powerful chromatin insulator.91 The insulating function of chicken HS4 is manifested by enhancer blocking activity and position-effect protection. These two activities are separable:92 the former is mediated by a transacting factor CTCF93 and the latter function may be achieved by highly efficient recruitment of histone acetyltransferase by the HS4 element.94 

Some novel data regarding LCR function have come from studies of nonglobin LCRs. As previously discussed, the human CD2 LCR was shown to be essential for establishing an open chromatin configuration, even in the absence of enhancer activity.43,95 Thus, LCRs appear to operate by ensuring an open chromatin configuration. As discussed earlier, the T cell–specific TCR α/δ (TCRα) LCR has been implicated in tissue-specific DNA demethylation, an important role for LCRs, since DNA methylation may cause chromatin closure and gene silencing. Additional information regarding LCR function was obtained from studies of this LCR. The TCRα LCR consists of 8 HSs located downstream of the T-cell receptor (TCR) gene.96 It is a bifunctional element, regulating both the TCR gene and the adjacent, ubiquitously expressed Dad1 antiapoptosis gene. Two subregions of the TCRα LCR were identified: one that constituted a novel non–tissue-restricted chromatin-opening element and one an immediate upstream sequence comprising the 4 proximal HSs that restored tissue specificity to the downstream chromatin-opening element.97 The HSs of this tissue-specificity region map near 2 transcriptional silencers, the TCRα enhancer (HS1) and a region of unknown function; the region between the enhancer and the unknown HS appears to be responsible for the tissue specificity.98 The proximal tissue-specific element may insulate the TCR gene from the LCR in other tissues, without affecting the TCRα LCR-Dad1 interaction. The occurrence of activators and insulators in LCRs appears to be a common theme, suggesting that the interaction of these elements may modulate LCR function. In fact, the tissue-unrestricted HS element suppressed PEV of a linked transgene in a wide variety of tissues and was bound by several ubiquitously expressed transcription factors.99However, when the full-length LCR was present, tissue-specific binding of tissue-restricted proteins was observed, demonstrating that a widely active LCR element can interact synergistically with other LCR elements to produce tissue-specific LCR activity via differential protein binding.

Other results suggest that LCRs may, in some instances, activate gene expression through a mechanism that includes increased histone acetylation. A cassette derived from 4 HSs of the 3′ murine immunoglobulin heavy chain (IgH) locus LCR was linked to the c-myc gene.100 This LCR mediated a widespread increase in acetylation, not only within the promoter region of the c-myc gene, but also over substantial distances upstream and downstream of the transcription site. Studies of the hGH LCR described earlier suggest that LCRs may increase histone acetylation by targeted recruitment and subsequent spreading of histone acetyltransferase activity to encompass and activate remote target genes.101 

Two elements of the T-cell receptor γ (TCRγ) locus, a 3′ enhancer (3′E (Cγ1)) and a region called HsA located between the Vγ5 and Vγ2 genes, constitute an LCR.102 HsA alone supported position-independent transcription in mature, but not immature, T cells; 3′E (Cγ1) alone supported position-dependent expression in both immature and mature cells. Copy number–dependent, position-independent expression was obtained at both stages of development, suggesting that HsA provides chromatin-opening activity. In addition, HsA was required for rearrangement of transgenic recombination substrates, an essential component of TCR loci.

Another interesting variant of the LCR theme is the human keratin-18 (K18) gene, which contains a 323-bp fragment that confers position-independent, copy number–dependent expression upon heterologous transgenes.103 This fragment is composed primarily of an Alu repetitive element that was partly responsible for the protective effects of sequences flanking K18, perhaps through its pol III transcriptional potential and inhibition of transcriptional interference from neighboring genes. Thus, Alu elements may function as regulatory domains within LCRs.

A pair of DNAse I hypersensitive sites (site II) of the murine γ1 heavy-chain gene lie approximately 2 kb 3′ of the γ1 promoter and exon 1 and just 5′ of the γ1 switch region. Site II functions as an LCR, conferring insertion site independence and copy number dependence on linked transgene expression, the hallmark of LCRs.104Messenger RNA is induced from γ1 transgenes lacking site II by interleukin 4 (IL-4) and by CD40 ligation (CD40 ligand–CD40 interaction). However, in the absence of site II, the induction of transgenic RNA expression by CD40 ligation was greater than expected, suggesting that the elements within site II also participate in negative regulation of the number of germ line transcripts after CD40 ligation, an effect opposite to the enhancement of transcription observed with most LCRs.

Conclusions

LCRs have been identified in various loci of vertebrates. While their composition and location relative to their cognate genes are different, they share the common property of maintaining physiological levels of gene expression, either at their natural position or at ectopic sites. This feature highlights the complexity of gene regulation. Developmental and cell lineage–specific regulation of gene expression relies not upon gene-proximal elements such as promoters, enhancers, and silencers exclusively, but also upon long-range interactions of various cis regulatory elements and dynamic chromatin alterations. The discovery of the LCR in the β-globin locus and the characterization of LCRs in other loci reinforces the need to study in vivo transcriptional regulation in the context of whole loci, so that essential regulatory elements are not excluded or overlooked.

Prepublished online as Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-04-1104.

Supported by National Institutes of Health grants DK53510, HL67336, HL20899, DK61805, and DK61804 and a Faculty Scholar Award from the Madison and Lila Self Graduate Fellowship awarded to K.R.P.

References

References
1
Grosveld
F
van Assendelft
GB
Greaves
DR
Kollias
G
Position-independent, high-level expression of the human beta-globin gene in transgenic mice.
Cell.
51
1987
975
985
2
Stamatoyannopoulos
G
Grosveld
F
Hemoglobin switching.
Molecular Basis of Blood Diseases.
3rd ed.
Stamatoyannopoulos
G
Majerus
PW
Perlmutter
RM
Varmus
H
2001
135
182
WB Saunders
Philadelphia, PA
3
Fraser
P
Grosveld
F
Locus control regions, chromatin activation and transcription.
Curr Opin Cell Biol.
10
1998
361
365
4
Li
Q
Harju
S
Peterson
KR
Locus control regions: coming of age at a decade plus.
Trends Genet.
15
1999
403
408
5
Magram
J
Chada
K
Costantini
F
Developmental regulation of a cloned adult beta-globin gene in transgenic mice.
Nature.
315
1985
338
340
6
Townes
TM
Lingrel
JB
Chen
HY
Brinster
RL
Palmiter
RD
Erythroid-specific expression of human beta-globin genes in transgenic mice.
EMBO J.
4
1985
1715
1723
7
Kollias
G
Wrighton
N
Hurst
J
Grosveld
F
Regulated expression of human A gamma-, beta-, and hybrid gamma beta-globin genes in transgenic mice: manipulation of the developmental expression patterns.
Cell.
46
1986
89
94
8
Kioussis
D
Vanin
E
deLange
T
Flavell
RA
Grosveld
FG
Beta-globin gene inactivation by DNA translocation in gamma beta-thalassaemia.
Nature.
306
1983
662
666
9
Driscoll
MC
Dobkin
CS
Alter
BP
Gamma delta beta-thalassemia due to a de novo mutation deleting the 5′ beta-globin gene activation-region hypersensitive sites.
Proc Natl Acad Sci U S A.
86
1989
7470
7474
10
Forrester
WC
Epner
E
Driscoll
MC
et al. 
A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.
Genes Dev.
4
1990
1637
1649
11
Tuan
D
Solomon
W
Li
Q
London
IM
The “beta-like-globin” gene domain in human erythroid cells.
Proc Natl Acad Sci U S A.
82
1985
6384
6388
12
Forrester
WC
Thompson
C
Elder
JT
Groudine
M
A developmentally stable chromatin structure in the human beta-globin gene cluster.
Proc Natl Acad Sci U S A.
83
1986
1359
1363
13
Li
Q
Zhang
M
Duan
Z
Stamatoyannopoulos
G
Structural analysis and mapping of DNAse I hypersensitivity of HS5 of the beta-globin locus control region.
Genomics.
61
1999
183
193
14
Epner
E
Reik
A
Cimbora
D
et al. 
The beta-globin LCR is not necessary for an open chromatin structure or developmentally regulated transcription of the native mouse beta-globin locus.
Mol Cell.
2
1998
447
455
15
Reik
A
Telling
A
Zitnik
G
Cimbora
D
Epner
E
Groudine
M
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.
Mol Cell Biol.
18
1998
5992
6000
16
Bender
MA
Bulger
M
Close
J
Groudine
M
Beta-globin gene switching and DNAse I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region.
Mol Cell.
5
2000
387
393
17
Hardison
R
Slightom
JL
Gumucio
DL
Goodman
M
Stojanovic
N
Miller
W
Locus control regions of mammalian beta-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights.
Gene.
205
1997
73
94
18
Li
Q
Stamatoyannopoulos
G
Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator.
Blood.
84
1994
1399
1401
19
Li
Q
Zhang
M
Han
H
Rohde
A
Stamatoyannopoulos
G
Evidence that DNAse I hypersensitive site 5 of the human beta-globin LCR functions as a chromosomal insulator in transgenic mice.
Nucleic Acids Res.
30
2002
2484
2491
20
Farrell
CM
West
AG
Felsenfeld
G
Conserved CTCF insulator elements flank the mouse and human beta-globin loci.
Mol Cell Biol.
22
2002
3820
3831
21
Blom van Assendelft
G
Hanscombe
O
Grosveld
F
Greaves
DR
The beta-globin dominant control region activates homologous and heterologous promoters in a tissue-specific manner.
Cell.
56
1989
969
977
22
Tewari
R
Gillemans
N
Harper
A
et al. 
The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene.
Development.
122
1996
3991
3999
23
Ney
PA
Sorrentino
BP
McDonagh
KT
Nienhuis
AW
Tandem AP-1-binding sites within the human beta-globin dominant control region function as an inducible enhancer in erythroid cells.
Genes Dev.
4
1990
993
1006
24
Talbot
D
Grosveld
F
The 5′HS2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites.
EMBO J.
10
1991
1391
1398
25
Motohashi
H
Shavit
JA
Igarashi
K
Yamamoto
M
Engel
JD
The world according to Maf.
Nucleic Acids Res.
25
1997
2953
2959
26
Mignotte
V
Wall
L
deBoer
E
Grosveld
F
Romeo
PH
Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene.
Nucleic Acids Res.
17
1989
37
54
27
Andrews
NC
Erdjument-Bromage
H
Davidson
MB
Tempst
P
Orkin
SH
Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein.
Nature.
362
1993
722
728
28
Caterina
JJ
Donze
D
Sun
CW
Ciavatta
DJ
Townes
TM
Cloning and functional characterization of LCR-F1: a bZIP transcription factor that activates erythroid-specific, human globin gene expression.
Nucleic Acids Res.
22
1994
2383
2391
29
Chan
JY
Han
XL
Kan
YW
Cloning of Nrf1, an NF-E2-related transcription factor, by genetic selection in yeast.
Proc Natl Acad Sci U S A.
90
1993
11371
11375
30
Moi
P
Chan
K
Asunis
I
Cao
A
Kan
YW
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.
Proc Natl Acad Sci U S A.
91
1994
9926
9930
31
Oyake
T
Itoh
K
Motohashi
H
et al. 
Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.
Mol Cell Biol.
16
1996
6083
6095
32
Kotkow
KJ
Orkin
SH
Dependence of globin gene expression in mouse erythroleukemia cells on the NF-E2 heterodimer.
Mol Cell Biol.
15
1995
4640
4647
33
Lu
SJ
Rowan
S
Bani
MR
Ben-David
Y
Retroviral integration within the Fli-2 locus results in inactivation of the erythroid transcription factor NF-E2 in Friend erythroleukemias: evidence that NF-E2 is essential for globin expression.
Proc Natl Acad Sci U S A.
91
1994
8398
8402
34
Forsberg
EC
Downs
KM
Bresnick
EH
Direct interaction of NF-E2 with hypersensitive site 2 of the beta-globin locus control region in living cells.
Blood.
96
2000
334
339
35
Sawado
T
Igarashi
K
Groudine
M
Activation of beta-major globin gene transcription is associated with recruitment of NF-E2 to the beta-globin LCR and gene promoter.
Proc Natl Acad Sci U S A.
98
2001
10226
10231
36
Shivdasani
RA
Orkin
SH
Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2.
Proc Natl Acad Sci U S A.
92
1995
8690
8694
37
Martin
F
van Deursen
JM
Shivdasani
RA
Jackson
CW
Troutman
AG
Ney
PA
Erythroid maturation and globin gene expression in mice with combined deficiency of NF-E2 and nrf-2.
Blood.
91
1998
3459
3466
38
Johnson
KD
Christensen
HM
Zhao
B
Bresnick
EH
Distinct mechanisms control RNA polymerase II recruitment to a tissue-specific locus control region and a downstream promoter.
Mol Cell.
8
2001
465
471
39
Milot
E
Strouboulis
J
Trimborn
T
et al. 
Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription.
Cell.
87
1996
105
114
40
Ellis
J
Tan-Un
KC
Harper
A
et al. 
A dominant chromatin-opening activity in 5′ hypersensitive site 3 of the human beta-globin locus control region.
EMBO J.
15
1996
562
568
41
Li
Q
Stamatoyannopoulos
JA
Position independence and proper developmental control of gamma-globin gene expression require both a 5′ locus control region and a downstream sequence element.
Mol Cell Biol.
14
1994
6087
6096
42
Goodwin
AJ
McInerney
JM
Glander
MA
Pomerantz
O
Lowrey
CH
In vivo formation of a human beta-globin locus control region core element requires binding sites for multiple factors including GATA-1, NF-E2, erythroid Kruppel-like factor, and Sp1.
J Biol Chem.
276
2001
26883
26892
43
Festenstein
R
Tolaini
M
Corbella
P
et al. 
Locus control region function and heterochromatin-induced position effect variegation.
Science.
271
1996
1123
1125
44
Holmquist
GP
Role of replication time in the control of tissue-specific gene expression.
Am J Hum Genet.
40
1987
151
173
45
Selig
S
Okumura
K
Ward
DC
Cedar
H
Delineation of DNA replication time zones by fluorescence in situ hybridization.
EMBO J.
11
1992
1217
1225
46
Epner
E
Forrester
WC
Groudine
M
Asynchronous DNA replication within the human beta-globin gene locus.
Proc Natl Acad Sci U S A.
85
1988
8081
8085
47
Dhar
V
Skoultchi
AI
Schildkraut
CL
Activation and repression of a beta-globin gene in cell hybrids is accompanied by a shift in its temporal replication.
Mol Cell Biol.
9
1989
3524
3532
48
Simon
I
Tenzen
T
Mostoslavsky
R
Fibach
E
Lande
L
Milot
E
Gribnau
J
Grosveld
F
Fraser
P
Cedar
H
Developmental regulation of DNA replication timing at the human beta globin locus.
EMBO J.
20
2001
6150
6157
49
Allshire
R
Bickmore
W
Pausing for thought on the boundaries of imprinting.
Cell.
102
2000
705
708
50
Simon
I
Cedar
H
Temporal order of DNA replication.
DNA replication in eukaryotic cells.
De Pamphilis
ML
1996
387
408
Cold Spring Harbor Laboratory Press
Cold Spring Harbor, NY
51
Rountree
MR
Bachman
KE
Baylin
SB
DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci.
Nat Genet.
25
2000
269
277
52
Wijgerde
M
Grosveld
F
Fraser
P
Transcription complex stability and chromatin dynamics in vivo.
Nature.
377
1995
209
213
53
Tuan
D
Kong
S
Hu
K
Transcription of the hypersensitive site HS2 enhancer in erythroid cells.
Proc Natl Acad Sci U S A.
89
1992
11219
11223
54
Engel
JD
Tanimoto
K
Looping, linking, and chromatin activity: new insights into beta-globin locus regulation.
Cell.
100
2000
499
502
55
Bulger
M
Groudine
M
Looping versus linking: toward a model for long-distance gene activation.
Genes Dev.
13
1999
2465
2477
56
Su
Y
Liebhaber
SA
Cooke
NE
The human growth hormone gene cluster locus control region supports position-independent pituitary- and placenta-specific expression in the transgenic mouse.
J Biol Chem.
275
2000
7902
7909
57
Elefant
F
Su
Y
Liebhaber
SA
Cooke
NE
Patterns of histone acetylation suggest dual pathways for gene activation by a bifunctional locus control region.
EMBO J.
19
2000
6814
6822
58
Ho
Y
Elefant
F
Cooke
N
Liebhaber
S
A defined locus control region determinant links chromatin domain acetylation with long-range gene activation.
Mol Cell.
9
2002
291
302
59
Schubeler
D
Francastel
C
Cimbora
DM
Reik
A
Martin
DI
Groudine
M
Nuclear localization and histone acetylation: a pathway for chromatin opening and transcriptional activation of the human beta-globin locus.
Genes Dev.
14
2000
940
950
60
Schubeler
D
Groudine
M
Bender
MA
The murine beta-globin locus control region regulates the rate of transcription but not the hyperacetylation of histones at the active genes.
Proc Natl Acad Sci U S A.
98
2001
11432
11437
61
Forsberg
EC
Downs
KM
Christensen
HM
Im
H
Nuzzi
PA
Bresnick
EH
Developmentally dynamic histone acetylation pattern of a tissue-specific chromatin domain.
Proc Natl Acad Sci U S A.
97
2000
14494
14499
62
Santoso
B
Ortiz
BD
Winoto
A
Control of organ-specific demethylation by an element of the T-cell receptor-alpha locus control region.
J Biol Chem.
275
2000
1952
1958
63
Bender
MA
Mehaffey
MG
Telling
A
et al. 
Independent formation of DNAseI hypersensitive sites in the murine beta-globin locus control region.
Blood.
95
2000
3600
3604
64
Anderson
JD
Widom
J
Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites.
J Mol Biol.
296
2000
979
987
65
Polach
KJ
Lowary
PT
Widom
J
Effects of core histone tail domains on the equilibrium constants for dynamic DNA site accessibility in nucleosomes.
J Mol Biol.
298
2000
211
223
66
Cirillo
LA
Lin
FR
Cuesta
I
Friedman
D
Jarnik
M
Zaret
KS
Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4.
Mol Cell.
9
2002
279
289
67
Carey
M
The enhanceosome and transcriptional synergy.
Cell.
92
1998
5
8
68
Locke
J
Kotarski
MA
Tartof
KD
Dosage-dependent modifiers of position effect variegation in Drosophila and a mass action model that explains their effect.
Genetics.
120
1988
181
198
69
Gribnau
J
de Boer
E
Trimborn
T
et al. 
Chromatin interaction mechanism of transcriptional control in vivo.
EMBO J.
17
1998
6020
6027
70
Bungert
J
Tanimoto
K
Patel
S
Liu
Q
Fear
M
Engel
JD
Hypersensitive site 2 specifies a unique function within the human beta-globin locus control region to stimulate globin gene transcription.
Mol Cell Biol.
19
1999
3062
3072
71
Grosveld
F
Activation by locus control regions?
Curr Opin Genet Dev.
9
1999
152
157
72
Peterson
KR
Clegg
CH
Navas
PA
Norton
EJ
Kimbrough
TG
Stamatoyannopoulos
G
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.
Proc Natl Acad Sci U S A.
93
1996
6605
6609
73
Navas
PA
Peterson
KR
Li
Q
et al. 
Developmental specificity of the interaction between the locus control region and embryonic or fetal globin genes in transgenic mice with an HS3 core deletion.
Mol Cell Biol.
18
1998
4188
4196
74
Bungert
J
Dave
U
Lim
KC
et al. 
Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.
Genes Dev.
9
1995
3083
3096
75
Hanscombe
O
Whyatt
D
Fraser
P
et al. 
Importance of globin gene order for correct developmental expression.
Genes Dev.
5
1991
1387
1394
76
Peterson
KR
Stamatoyannopoulos
G
Role of gene order in developmental control of human gamma- and beta-globin gene expression.
Mol Cell Biol.
13
1993
4836
4843
77
Dillon
N
Trimborn
T
Strouboulis
J
Fraser
P
Grosveld
F
The effect of distance on long-range chromatin interactions.
Mol Cell.
1
1997
131
139
78
Blackwood
EM
Kadonaga
JT
Going the distance: a current view of enhancer action.
Science.
281
1998
61
63
79
Ashe
HL
Monks
J
Wijgerde
M
Fraser
P
Proudfoot
NJ
Intergenic transcription and transinduction of the human beta-globin locus.
Genes Dev.
11
1997
2494
2509
80
Dorsett
D
Distant liaisons: long-range enhancer-promoter interactions in Drosophila.
Curr Opin Genet Dev.
9
1999
505
514
81
Fu
L
Mambrini
M
Perrot
E
Chourrout
D
Stable and full rescue of the pigmentation in a medaka albino mutant by transfer of a 17 kb genomic clone containing the medaka tyrosinase gene.
Gene.
241
2000
205
211
82
Mathieu
N
Hempel
WM
Spicuglia
S
Verthuy
C
Ferrier
P
Chromatin remodeling by the T cell receptor (TCR)-beta gene enhancer during early T cell development: implications for the control of TCR-beta locus recombination.
J Exp Med.
192
2000
625
636
83
Agarwal
S
Rao
A
Long-range transcriptional regulation of cytokine gene expression.
Curr Opin Immunol.
10
1998
345
352
84
Yui
MA
Hernandez-Hoyos
G
Rothenberg
EV
A new regulatory region of the IL-2 locus that confers position-independent transgene expression.
J Immunol.
166
2001
1730
1739
85
Bonifer
C
Vidal
M
Grosveld
F
Sippel
AE
Tissue specific and position independent expression of the complete gene domain for chicken lysozyme in transgenic mice.
EMBO J.
9
1990
2843
2848
86
Aronow
BJ
Silbiger
RN
Dusing
MR
et al. 
Functional analysis of the human adenosine deaminase gene thymic regulatory region and its ability to generate position-independent transgene expression.
Mol Cell Biol.
12
1992
4170
4185
87
Kushida
MM
Dey
A
Zhang
XL
et al. 
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.
J Immunol.
159
1997
4913
4929
88
Felsenfeld
G
Chromatin structure and the expression of globin-encoding genes.
Gene.
135
1993
119
124
89
Reitman
M
Lee
E
Westphal
H
Felsenfeld
G
Site-independent expression of the chicken beta A-globin gene in transgenic mice.
Nature.
348
1990
749
752
90
Reitman
M
Lee
E
Westphal
H
Felsenfeld
G
An enhancer/locus control region is not sufficient to open chromatin.
Mol Cell Biol.
13
1993
3990
3998
91
Chung
JH
Whiteley
M
Felsenfeld
G
A 5′ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila.
Cell.
74
1993
505
514
92
Recillas-Targa
F
Pikaart
MJ
Burgess-Beusse
B
et al. 
Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities.
Proc Natl Acad Sci U S A.
99
2002
6883
6888
93
Bell
AC
West
AG
Felsenfeld
G
The protein CTCF is required for the enhancer blocking activity of vertebrate insulators.
Cell.
98
1999
387
396
94
Litt
MD
Simpson
M
Gaszner
M
Allis
CD
Felsenfeld
G
Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus.
Science.
293
2001
2453
2455
95
Greaves
DR
Wilson
FD
Lang
G
Kioussis
D
Human CD2 3′-flanking sequences confer high-level, T cell-specific, position-independent gene expression in transgenic mice.
Cell.
56
1989
979
986
96
Diaz
P
Cado
D
Winoto
A
A locus control region in the T cell receptor alpha/delta locus.
Immunity.
1
1994
207
217
97
Ortiz
BD
Cado
D
Chen
V
Diaz
PW
Winoto
A
Adjacent DNA elements dominantly restrict the ubiquitous activity of a novel chromatin-opening region to specific tissues.
EMBO J.
16
1997
5037
5045
98
Ortiz
BD
Cado
D
Winoto
A
A new element within the T-cell receptor alpha locus required for tissue-specific locus control region activity.
Mol Cell Biol.
19
1999
1901
1909
99
Ortiz
BD
Harrow
F
Cado
D
Santoso
B
Winoto
A
Function and factor interactions of a locus control region element in the mouse T cell receptor-alpha/Dad1 gene locus.
J Immunol.
167
2001
3836
3845
100
Madisen
L
Krumm
A
Hebbes
TR
Groudine
M
The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes.
Mol Cell Biol.
18
1998
6281
6292
101
Elefant
F
Cooke
NE
Liebhaber
SA
Targeted recruitment of histone acetyltransferase activity to a locus control region.
J Biol Chem.
275
2000
13827
13834
102
Baker
JE
Kang
J
Xiong
N
Chen
T
Cado
D
Raulet
DH
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.
J Exp Med.
190
1999
669
679
103
Willoughby
DA
Vilalta
A
Oshima
RG
An Alu element from the K18 gene confers position-independent expression in transgenic mice.
J Biol Chem.
275
2000
759
768
104
Adams
K
Ackerly
H
Cunningham
K
Dunnick
W
A DNAse I hypersensitive site near the murine gamma1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation.
Int Immunol.
12
2000
1705
1713
105
Dang
Q
Walker
D
Taylor
S
et al. 
Structure of the hepatic control region of the human apolipoprotein E/C-I gene locus.
J Biol Chem.
270
1995
22577
22585
106
Friend
WC
Clapoff
S
Landry
C
et al. 
Cell-specific expression of high levels of human S100 beta in transgenic mouse brain is dependent on gene dosage.
J Neurosci.
12
1992
4337
4346
107
Jones
BK
Monks
BR
Liebhaber
SA
Cooke
NE
The human growth hormone gene is regulated by a multicomponent locus control region.
Mol Cell Biol.
15
1995
7010
7021
108
Kalos
M
Fournier
RE
Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain.
Mol Cell Biol.
15
1995
198
207
109
Knotts
S
Rindt
H
Robbins
J
Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.
Nucleic Acids Res.
23
1995
3301
3309
110
Madisen
L
Groudine
M
Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt's lymphoma cells.
Genes Dev.
8
1994
2212
2226
111
Mills
FC
Harindranath
N
Mitchell
M
Max
EE
Enhancer complexes located downstream of both human immunoglobulin Calpha genes.
J Exp Med.
186
1997
845
858
112
Neznanov
N
Thorey
IS
Cecena
G
Oshima
RG
Transcriptional insulation of the human keratin 18 gene in transgenic mice.
Mol Cell Biol.
13
1993
2214
2223
113
Schmidt
CM
Ehlenfeldt
RG
Athanasiou
MC
et al. 
Extraembryonic expression of the human MHC class I gene HLA-G in transgenic mice. Evidence for a positive regulatory region located 1 kilobase 5' to the start site of transcription.
J Immunol.
151
1993
2633
2645
114
Tee
MK
Babalola
GO
Aza-Blanc
P
Speek
M
Gitelman
SE
Miller
WL
A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA: location of a cryptic CYP21 promoter element?
Hum Mol Genet.
4
1995
2109
2116
115
Wang
Y
Macke
JP
Merbs
SL
et al. 
A locus control region adjacent to the human red and green visual pigment genes.
Neuron.
9
1992
429
440
116
Wurster
AL
Siu
G
Leiden
JM
Hedrick
SM
Elf-1 binds to a critical element in a second CD4 enhancer.
Mol Cell Biol.
14
1994
6452
6463
117
Fujiwara
Y
Miwa
M
Takahashi
R
Hirabayashi
M
Suzuki
T
Ueda
M
Position-independent and high-level expression of human alpha-lactalbumin in the milk of transgenic rats carrying a 210-kb YAC DNA.
Mol Reprod Dev.
47
1997
157
163
118
Raguz
S
Hobbs
C
Yague
E
Ioannou
PA
Walsh
FS
Antoniou
M
Muscle-specific locus control region activity associated with the human desmin gene.
Dev Biol.
201
1998
26
42
119
Kamat
A
Graves
KH
Smith
ME
Richardson
JA
Mendelson
CR
A 500-bp region, approximately 40 kb upstream of the human CYP19 (aromatase) gene, mediates placenta-specific expression in transgenic mice.
Proc Natl Acad Sci U S A.
96
1999
4575
4580
120
Heydemann
A
Warming
S
Clendenin
C
Sigrist
K
Hjorth
JP
Simon
MC
A minimal c-fes cassette directs myeloid-specific expression in transgenic mice.
Blood.
96
2000
3040
3048
121
Moon
AM
Ley
TJ
Conservation of the primary structure, organization, and function of the human and mouse beta-globin locus-activating regions.
Proc Natl Acad Sci U S A.
87
1990
7693
7697
122
Carson
S
Wiles
MV
Far upstream regions of class II MHC Ea are necessary for position-independent, copy-dependent expression of Ea transgene.
Nucleic Acids Res.
21
1993
2065
2072
123
Ganss
R
Montoliu
L
Monaghan
AP
Schutz
G
A cell-specific enhancer far upstream of the mouse tyrosinase gene confers high level and copy number-related expression in transgenic mice.
Embo J.
13
1994
3083
3093
124
Hammer
RE
Krumlauf
R
Camper
SA
Brinster
RL
Tilghman
SM
Diversity of alpha-fetoprotein gene expression in mice is generated by a combination of separate enhancer elements.
Science.
235
1987
53
58
125
Jenuwein
T
Forrester
WC
Qiu
RG
Grosschedl
R
The immunoglobulin mu enhancer core establishes local factor access in nuclear chromatin independent of transcriptional stimulation.
Genes Dev.
7
1993
2016
2032
126
May
G
Enver
T
Targeting gene expression to haemopoietic stem cells: a chromatin-dependent upstream element mediates cell type-specific expression of the stem cell antigen CD34.
Embo J.
14
1995
564
574
127
Palmiter
RD
Sandgren
EP
Koeller
DM
Brinster
RL
Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice.
Mol Cell Biol.
13
1993
5266
5275
128
Smith
MS
Lechago
J
Wines
DR
MacDonald
RJ
Hammer
RE
Tissue-specific expression of kallikrein family transgenes in mice and rats.
DNA Cell Biol.
11
1992
345
358
129
Terajima
M
Nemoto
Y
Obinata
M
Inducible expression of erythroid-specific mouse glycophorin gene is regulated by proximal elements and locus control region-like sequence.
J Biochem (Tokyo).
118
1995
593
600
130
Elenich
LA
Ford
CS
Dunnick
WA
The gamma 1 heavy chain gene includes all of the cis-acting elements necessary for expression of properly regulated germ-line transcripts.
J Immunol.
157
1996
176
182
131
Sabbattini
P
Georgiou
A
Sinclair
C
Dillon
N
Analysis of mice with single and multiple copies of transgenes reveals a novel arrangement for the lambda5-VpreB1 locus control region.
Mol Cell Biol.
19
1999
671
679
132
Johnson
BA
John
VA
Henschler
R
et al. 
Upstream elements bestow T-cell and haemopoietic progenitor-specific activity on the granzyme B promoter.
Gene.
234
1999
101
107
133
Arai
Y
Kajihara
S
Masuda
J
et al. 
Position-independent, high-level, and correct regional expression of the rat aldolase C gene in the central nervous system of transgenic mice.
Eur J Biochem.
221
1994
253
260
134
Dale
TC
Krnacik
MJ
Schmidhauser
C
Yang
CL
Bissell
MJ
Rosen
JM
High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3' untranslated region.
Mol Cell Biol.
12
1992
905
914
135
Talbot
D
Descombes
P
Schibler
U
The 5' flanking region of the rat LAP (C/EBP beta) gene can direct high-level, position-independent, copy number-dependent expression in multiple tissues in transgenic mice.
Nucleic Acids Res.
22
1994
756
766
136
Li
QL
Zhou
B
Powers
P
Enver
T
Stamatoyannopoulos
G
Beta-globin locus activation regions: conservation of organization, structure, and function.
Proc Natl Acad Sci U S A.
87
1990
8207
8211
137
Slightom
JL
Bock
JH
Tagle
DA
et al. 
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.
Genomics.
39
1997
90
94
138
Whitelaw
CB
Harris
S
McClenaghan
M
Simons
JP
Clark
AJ
Position-independent expression of the ovine beta-lactoglobulin gene in transgenic mice.
Biochem J.
286
Pt 1
1992
31
39

Author notes

George Stamatoyannopoulos, Department of Genome Sciences, University of Washington, Box 357730,1705 NE Pacific St, Health Sciences K-357, Seattle WA 98195; e-mail:gstam@u.washington.edu.