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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-04-1104.
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Blood, 1 November 2002, Vol. 100, No. 9, pp. 3077-3086
REVIEW ARTICLE
Locus control regions
Qiliang Li,
Kenneth R. Peterson,
Xiangdong Fang, and
George Stamatoyannopoulos
From the Division of Medical Genetics and Department of
Genome Sciences, University of Washington, Seattle, and the Department
of Biochemistry and Molecular Biology and the Department of Anatomy and
Cell Biology, University of Kansas Medical Center, Kansas City.
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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  -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 various
cis regulatory elements and dynamic chromatin alterations.
(Blood. 2002;100:3077-3086)
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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 -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)
-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 -thalassemia the
genes of the -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 -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 -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  -globin gene.11,12 Definitive evidence
for the presence of the LCR came from transgenic mouse
studies.1 Linkage of this region to a -globin gene
resulted in expression of the gene at a level comparable to the
endogenous mouse -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.
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Properties of LCRs |
Transcriptional enhancer activity
The most prominent property of the LCRs is their strong,
transcription-enhancing activity. The -globin LCR is located 6 to 22 kb 5' to the first (embryonic) globin gene in the locus (Figure 1). 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 -globin gene is usually less than 1% of
the endogenous murine -globin mRNA in transgenic mice, if it is
expressed at all.5-7 Inclusion of the LCR increases
-globin gene expression to a level comparable to that of the mouse
-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.
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| Figure 1.
The human and mouse -globin loci.
The human locus consists of 5 functional genes, indicated as dark
boxes, arrayed in their order of developmental expression,
5'--G -A- --3'. There are 2 developmental switches in globin chain synthesis coincident with
changes in site and type of erythropoiesis. During primitive
erythropoiesis, the -globin gene is expressed in the embryonic yolk
sac. The first switch occurs at approximately 8 weeks' gestation; the
-globin gene is silenced and the G- and
A-globin genes are expressed during definitive
erythropoiesis in the fetal liver. The second switch occurs shortly
after birth; the -globin genes are silenced and the -globin gene
and, to a lesser extent, the -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 -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 -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
()0 thalassemia in humans, extends an additional
20 kb 5' of the LCR 5'HS5.
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The enhancer activity of the -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 -globin LCR is tissue specific; that
is, the expression of globin genes is confined to erythroid cells when
linked to the -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 -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-E2.32,33
The 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 -major
globin transcription, but is not associated with a significant change in locus-wide histone H3 acetylation. Thus, the -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 -globin gene expression.35 While the in
vivo association of NF-E2 and HS2 of the LCR 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  - and -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
-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
-globin LCR in creating open chromatin was suggested from analysis
of -thalassemia mutants with deletion of the LCR.8,10
In the Hispanic form of -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 -globin
locus.39 When one of the HSs was deleted from the LCR,
expression of the -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 -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.41
Formation 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 -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 -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.48
Perhaps 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
-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 -globin locus appear to be different
from those of the hGH gene cluster. Deletion of 5'HS2-5 of the
human -globin LCR did not affect the general pattern of histone H4
acetylation of a -globin locus transgene.59 Other studies reported that although deletion of the murine -globin LCR
decreased the rate of -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
-globin promoter may be independent of LCR function.
NF-E2 is required for histone hyperacetylation at the adult -globin
promoter, but not at the LCR.38,61 Other data demonstrated that the -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
-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 -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) / locus support such
a role. Tissue-specific functions of this LCR depend largely on 2 HSs,
HS1 (T-cell receptor 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
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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 -globin LCR in vivo is to open and/or maintain a permissive
chromatin conformation within the -globin locus in erythroid cells,
although enhancement of transcription is also an essential function.
Surprisingly, when the entire mouse -globin LCR (5'HS1-6) was
deleted by homologous recombination, the formation of the general DNAse
I-sensitivity associated with the -globin locus domain was not
affected; however, transcription of all -like globin genes was
strikingly reduced.14,63 These observations raise several
questions regarding the real in vivo function of the -globin LCR. Is
this LCR simply another enhancer in the -globin locus? If the
-globin LCR functions only as an enhancer within endogenous
-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
-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 -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 transactivators67
and 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.
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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 -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
-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.
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| Figure 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).
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The looping model suggests that the 5'HSs of the -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 -, -, and -globin
genes.70 However, when the entire 5'HS2 region of
conserved sequence similarity (core and flanking sequences) was
removed, the -, - and -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 (Figure
2B).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 (Figure
2D).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 the
Drosophila 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 -like globin genes.
The -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.
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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 (Table
1). 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 -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 -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 -globin LCR, most commonly the hallmark of copy
number-dependent, site-of-integration-independent expression of their
cognate loci or linked transgenes.
Most of the data regarding LCR function have come from studies of the
human and murine -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 A and embryonic  genes (the
A/ enhancer).88 This enhancer is able to
confer site-independent expression to the chicken
A-globin gene in transgenic mice.89
Chromatin unfolding of the chicken -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 / (TCR) 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 TCR 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 TCR 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 TCR 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 TCR 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.99
However, 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 (TCR) locus, a 3' enhancer
(3'E (C1)) and a region called HsA located between the V5 and
V2 genes, constitute an LCR.102 HsA alone supported position-independent transcription in mature, but not immature, T
cells; 3'E (C1) 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 1
heavy-chain gene lie approximately 2 kb 3' of the 1 promoter and
exon 1 and just 5' of the 1 switch region. Site II functions as an
LCR, conferring insertion site independence and copy number dependence
on linked transgene expression, the hallmark of LCRs.104 Messenger RNA is induced from 1 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.
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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 -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.
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Footnotes |
Submitted April 11, 2002; accepted June 3, 2002.
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.
Reprints: 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{at}u.washington.edu.
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References |
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Top
Abstract
Introduction