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Variability and Exclusion in Host and
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J Immunol 2006; 177:5767-5774; ;
doi: 10.4049/jimmunol.177.9.5767
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References
Shira Fraenkel and Yehudit Bergman
OF
THE
JOURNAL IMMUNOLOGY
BRIEF REVIEWS
Variability and Exclusion in Host and Parasite:
Epigenetic Regulation of Ig and var Expression1
Shira Fraenkel and Yehudit Bergman2
T
he immune system is made up of a large spectrum of
individual B and T cells, each of which express one specific AgR molecule. The DNA sequences that confer
Ag specificity to the BCR and the TCR are assembled at seven
different loci/clusters (three for the BCR: Ig H chain, ␬, or ␭ L
chains; four for the TCR: ␣, ␤, ␥, and ␦). Thus, each B or T cell
has initially to choose one locus/cluster for recombination. In
each cluster, recombination occurs between V (variable), J
(joining), and, in some cases, D (diversity) region gene segments. Thus, once a cluster is chosen, the cell must select one of
the V, J, and D gene segments for rearrangement. Rearrangement at each AgR locus is strictly regulated with respect to developmental timing (e.g., IgH before IgL) and lineage specificity (e.g., VH-to-DJH rearrangement in B but not in T cells).
Since every cell has two alleles for each of the seven receptor loci,
the ultimate choice of one receptor type (Ig or TCR) per cell
involves an extra process of selection of one allele, termed allelic
exclusion. Allelic exclusion is controlled at the V(D)J rearrangement level by initially restricting recombination to only one allele in each cell. The above-described choices seem to be
achieved through monoallelic epigenetic changes (1– 4).
Epigenetics is also involved in selecting single members of
gene families for expression in several other biological systems.
Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel
Received for publication June 20, 2006. Accepted for publication July 17, 2006.
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
In this review, we will analyze the role of epigenetics in choosing
one member of the var family of genes that encodes the malaria
parasite Plasmodium falciparum erythrocyte membrane protein
1 (PfEMP1)3 (5). During the proliferation phase of the parasite
infection, expression of PfEMP1 switches from one var gene to
another, giving rise to parasite antigenic variation and thus facilitating host immune evasion (6). The var switching process is
regulated at the level of transcription and follows the rule that
only one gene is expressed at a time in a single parasite (6). Similar to the Ig and TCR gene clusters, the var genes in P. falciparum genome also appear in several clusters. In parallel to the
series of molecular decisions in developing B and T cells, a parasite cell would initially select a certain gene cluster, after which
selection of a single var gene would have to take place. Unlike B
and T lineage cells, allelic selection is irrelevant to the process
since P. falciparum carries only a haploid genome in its
human host.
The molecular mechanisms that control mutually exclusive
expression are not completely understood in any eukaryotic system. In this review, we will discuss the transcriptional and epigenetic mechanisms used by the immune system to generate a
repertoire of Abs, as well as those used by P. falciparum to evade
attacks by host Abs. Expectantly, insight could be gained by
comparing the unifying features of these single-gene expressing
systems and by identifying the unique characteristics displayed
by each.
The Ig system
Ordered Ig rearrangements during B cell differentiation. Both the unrearranged H and L chain loci (Fig. 1) are
silenced in non-B lymphoid cells, as well as in early B cell precursors. In these cell types, the IgH and IgL chain loci are maintained in an extended conformation at the nuclear periphery,
which is regarded as a repressive environment (7).
All genes are packaged in a basic nucleosome-repeat structure, but alterations in histone-tail modifications can greatly influence both local and higher-order chromatin conformation
2
Address correspondence and reprint requests to Dr. Yehudit Bergman, Hubert H. Humphrey Center for Experimental Medicine and Cancer Research, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel. E-mail address: [email protected]
3
Abbreviations used in this paper: PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; TARE, telomere-associated repeat element.
1
This work was supported by research grants from the Israel Academy of Sciences, the
National Institutes of Health Program, and Philip Morris USA and Philip Morris
International.
Copyright © 2006 by The American Association of Immunologists, Inc.
0022-1767/06/$02.00
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The immune system generates highly diverse AgRs of different specificities from a pool of designated genomic loci,
each containing large arrays of genes. Ultimately, each B
or T cell expresses a receptor of a single type on its surface.
Immune evasion by the malaria parasite Plasmodium falciparum is mediated by the mutually exclusive expression
of a single member of the var family of genes, which encodes variant surface Ags. In this review, we discuss the
similarities as well as the unique characteristics of the epigenetic mechanisms involved in the establishment of mutually exclusive expression in the immune and parasite
systems. The Journal of Immunology, 2006, 177:
5767–5774.
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BRIEF REVIEWS: EPIGENETIC REGULATION OF Ig AND var EXPRESSION
(8). Repressive modifications such as histone H3 lysine 9 methylation (H3K9) can prevent histone acetylation (9) and can recruit chromodomain-containing factors such as HP1 that are
probably involved in stabilizing a silenced state (9). The Ig loci
are hypoacetylated and H3K9 methylated in non-B lymphoid
progenitor cells and in hemopoietic cells outside the B lineage
(reviewed in Ref. 3). Indeed, a recent study suggests that H3K9
methylation inhibits V(D)J recombination when directed to
the promoter of an artificial, stably integrated recombination
substrate (10).
One of the main mechanisms for mediating gene repression is
DNA methylation (11), which is established at the time of embryo implantation by de novo methylation of all DNA except
for CpG islands, and this state is preserved through all cell diFIGURE 2. IgH and Ig␬ configurations in B cell development. At the pro-B cell stage, both H chain alleles undergo D-to-J gene recombination, whereas the Ig␬ alleles
are in their germline configuration. Following transition to
the pre-B cell stage, one of the IgH alleles has recombined,
and the productive rearrangement results in the IgH-protein pairing with the surrogate light chains ␭ 5 and VpreB,
which, together with other subunits, form the pre-BCR
complex. The cell surface expression of the pre-BCR leads
to epigenetic changes occurring at the Ig␬ locus that culminate in Ig␬ rearrangement on one of the two ␬ alleles,
after which, transition to the immature B cell stage takes
place. A productive ␬ rearrangement enables surface expression of the BCR. See text for discussion.
visions. Consequently, immune receptor loci are initially configured in a repressed methylated state, and they remain this way until
they are about to rearrange during B cell development.
Ig rearrangement in pro-B cells. During pro-B cell development, only the H chain genes undergo rearrangement (Fig.
2). These cells are characterized by relocation of the IgH alleles
to central nuclear positions (7), changes in histone modification
(12–16), antisense transcription along the entire VH gene cluster (17), and long-range contraction of the IgH locus (18, 19),
which ultimately results in VH-DJH recombination. Two different silencing processes are involved in directing an ordered
monoallelic IgH rearrangement. One regulates the sequence of
the rearrangement in the IgH locus, i.e., DJH rearrangement
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FIGURE 1. Genomic organization of the mouse IgH and Ig␬ chains and the P. falciparum var family of genes. Distances are not drawn to scale. A, IgH and Ig␬
loci. The IgH locus consists of ⬃150 functional VH gene segments, 12–13 DH gene segments, 4 JH gene segments, and a few constant region genes starting with the
5⬘ C␮ exon. The entire locus spans ⬃3 Mb and resides near the mouse chromosome 12 telomere. The Ig␬ locus consists of ⬃100 functional V␬ gene segments, 5
J␬ gene segments, of which 4 are functional, and a single constant region gene. The entire locus spans ⬃3 Mb and resides in the mouse chromosome 6. B, The var
gene family. The P. falciparum genome contains ⬃60 var genes found in clusters at either subtelomeric or chromosome central regions. The chromosome end
structure is conserved, comprising TAREs 1– 6. The subtelomeric var genes are the most telomere-proximal genes located 3⬘ to TARE6 (Rep20). Also shown is the
conserved structure of the var genes, with a long 5⬘ exon followed by a short 3⬘ exon.
The Journal of Immunology
tone deacetylation probably occurs. Reduction in IL7-R signaling at the pre-B stage most likely underlies histone deacetylation and centromeric recruitment as treatment of B cells with
IL-7 interferes with centromeric recruitment of the IgH allele,
while simultaneously inducing histone acetylation of the distal
VH genes (12). Thus, several mechanisms may preclude the DJrearranged allele from further rearranging following the re-expression of the RAG proteins in small pre-B cells.
Ig␬ recombination that specifically initiates in pre-B cells is
regulated through targeted changes in chromatin accessibility
(28, 29). For example, recombination signal sequences in the J␬
region are only subject to cleavage in the nuclei of pre-B cells
(30). To achieve this level of accessibility, the ␬ locus seems to
undergo a programmed process that releases multiple layers of
repression in a stepwise manner. The Ig␬ gene becomes preferentially packaged into an active chromatin structure characterized by histone acetylation and methylation of histone H3K4
(13, 14, 31). Furthermore, the locus undergoes DNA looping
and locus contraction, which most probably facilitates VJ recombination as well (18).
It is pertinent to ask how the Ig␬ allele choice is dictated in
pre-B cells. There are several epigenetic molecular mechanisms
that enforce this phenomenon, resulting in allelic exclusion
(Fig. 3). It seems that allelic exclusion in pre-B cells is mediated
by a predetermined “instructive” mechanism that begins early
in development when the Ig␬ alleles first become asynchronously replicated (32). Once established, this differential state is
then maintained in a clonal manner in somatic cells, but only
during pre-B cell development are the consequences of this
marking established. It seems that the asynchronous replication
directs one of the ␬ alleles to heterochromatin, while causing
the other to become packaged with highly acetylated histones.
Experimental data indicate that the late replicating allele is almost always (80%) repositioned to heterochromatin, whereas
the early replicating allele remains detached from heterochromatin and is preferentially DNA-demethylated. Relocalization
to heterochromatin involves association with the heterochromatin protein HP1 (14) and Ikaros, which forms multimers
that colocalize with foci of pericentromeric heterochromatin in
pre-B cells (33, 34). It is thought that in this way it may be
involved in the transcriptional inactivation of specific genes in
the B lymphoid lineage (35).
Interestingly, the aforementioned changes in histone modifications occur preferentially on the nonheterochromatic allele,
but it is still not clear whether heterochromatinization occurs
before or as a consequence of these changes. It seems that the
Ig␬ gene remains fully methylated almost throughout pre-B cell
development (14). However, the active chromatin structure
and most probably Ig␬ nuclear repositioning are involved in
directing the demethylation machinery to one allele in each cell,
shortly after the proper demethylation trans-acting factors become available during the transition to B cells (36). Thus, by
late stages of pre-B cell development, the two ␬ alleles adopt
completely different chromatin structures, and in keeping
with these data, germline Ig␬ expression, which is biallelic in
pro-B cells (27), actually becomes monoallelic at about this
stage of differentiation (37). This monoallelic germline transcription could be due to a limiting concentration of a transcription factor that binds preferentially to the nonheterochromatinized allele, thus reinforcing the previously
instructed epigenetic marks.
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before VH-to-DJH, and the other ensures that the second rearrangement step occurs on one allele only.
Initially, the DH-C␮ region becomes hyperacetylated while
the VH gene segments remain hypoacetylated (20). This apparently accounts for DH to JH recombination occurring first during B cell development. To activate VH genes for rearrangement, the region requires the removal of repressive methylation
marks such as methylation of histone H3 on lysine 9. Evidently,
Pax5, a transcription factor required for B cell commitment, is
necessary and sufficient for the removal of H3K9 methylation
in the VH locus (21). Moreover, VH genes have to become packaged with nucleosomes enriched with histone H3 methylated
on lysine 4 and lysine 27 (for a subset of VH), as well as enriched
with acetylated histones. Both methylation of H3K27 and acetylation of H3 and H4 are dependent on IL-7R signaling (reviewed in Ref. 22). Furthermore, histone acetylation was specifically attributed to the IL-7 responsive transcription factor
STAT5 functioning as a signal-dependent coactivator (23).
Regulation of VH-to-DJH rearrangement varies between
DJH-proximal and DJH-distal region, with the proximal VH being a preferable target for rearrangement and a more promiscuous one (12, 13, 15, 19, 20, 24, 25). Following DJ rearrangement, genic and intergenic antisense transcripts are generated
throughout the IgH V region on both alleles (17). These transcripts appear to be long, extending across several genes, and are
rapidly lost from both alleles following VDJ recombination. It
still has not been determined whether these antisense transcripts participate in opening up a large chromatin domain for
possible rearrangement, or vice versa, they participate in RNAdependent silencing.
There is little mechanistic data for allelic epigenetic differences that will help in discriminating between the two IgH alleles. One very interesting process that occurs in pro-B cells is
the long-range contraction or compaction of the IgH locus,
which draws the VH region near the IgH DJ cluster and thus
facilitates the V-to-DJ recombination (18, 19). Interestingly,
one of the studies reported that these loops occur monoallelically and thus could account for choosing one allele to undergo
V-to-DJ recombination (26).
As for the ␬ chain gene, both alleles become centrally located
and disassociated from heterochromatic regions (7), produce
germline transcripts (27), and are packaged with acetylated histones (14), even though the DNA is still methylated (1), and
thus refractory to the recombination machinery that targets the
H chain locus.
Ig rearrangement in pre-B cells. Successful rearrangement
of the IgH locus leads to cell surface expression of the ␮ protein
as part of the pre-BCR, which functions as an important checkpoint to signal proliferative expansion of populations of large
pre-B cells to induce subsequent differentiation to small-pre-B
cells, in which the ␬ chain gene rearranges (Fig. 2). In small
pre-B cells, RAG proteins are re-expressed and thus silencing
mechanisms must be involved in restricting IgH rearrangement. Inhibition of VH-to-DJH recombination involves several
mechanisms, including the decontraction of both IgH loci, reduced accessibility and lower levels of histone acetylation at VH
segments (mostly of distal VH), and allelic recruitment to repressive centromeric domains (reviewed in Ref. 4). Based on the
observation that germline VHJ558 gene segments can revert to
a hypoacetylated state in the absence of replication, active his-
5769
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BRIEF REVIEWS: EPIGENETIC REGULATION OF Ig AND var EXPRESSION
Several trans-acting factors can influence the epigenetic status
of the locus, as well as actively participate in facilitating the recombination process itself. Pax5 is known to perform such dual
functions. It was recently shown that in addition to its ability to
induce proper chromosomal configuration and chromatin
structure, it is able to facilitate RAG-mediated recombination
through direct binding to individual VH coding regions and to
the RAG1-RAG2 complex (38). Specific cis-regulatory elements involved in demethylation or histone acetylation have
been identified (reviewed in Refs. 39 and 40). Recently, a novel
cis-acting element that negatively regulates rearrangement in
the Ig␬ locus has been identified (41). The element, termed Sis,
specifies the targeting of Ig␬ in pre-B and B cells to centromeric
heterochromatin, associates with Ikaros, and thus probably participates in Ig␬ allelic exclusion.
Editing in B cells. Immature B cells are screened for autoreactivity in the bone marrow environment so that the mature B
cell population is generally devoid of cells reactive with self-Ags.
These cells can undergo receptor editing, which involves secondary Ig rearrangement(s) that generate a new AgR with an
innocuous specificity. This process uses the same recombination machinery that mediates primary V(D)J joining and occurs
mainly at the Ig␬ locus through V␬-to-J␬ secondary rearrangement. In addition, receptor editing also occurs, at a low frequency, at the IgH locus through VH-to-VDJH rearrangement.
Thus, epigenetic silencing of the Ig loci must be reversed to allow for RAG-mediated recombination. Indeed, when B cells are
treated with IL-7, centromeric recruitment is prevented and histone hyperacetylation of VH genes is induced (12, 18). This
mechanism enables reversal of the initial choice and exemplifies
the built-in plasticity of this system.
The var system
As stated above, the variant Ags in P. falciparum are the
PfEMP1 family, which is encoded by the var family of genes.
Each genome contains ⬃60 members of this family, interspersed in groups at subtelomeric regions and in several groups
at chromosome central loci (42) (Fig. 1). The overall structure
of P. falciparum chromosome ends is conserved, with blocks of
repeated DNA defined as telomere-associated repeat elements
(TAREs/Rep20) found internal to the telomere. Subtelomeric
var genes are the most telomere-proximal genes and are located
downstream of TARE6/Rep20 (5). The repetitive element
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FIGURE 3. Silenced vs active states of Ig␬ and var genes. The upper and lower panels depict changes in the nuclei of B lineage and P. falciparum cells. Upper panel,
In progenitor B cells, the two ␬ alleles— one of which replicates early (red) and the other later—are localized to the periphery of the nucleus, and their DNA is
methylated. They are also not associated with hyperacetylated histones. A late pre-B cell exhibits notable epigenetic differences between its two ␬ alleles. The allele
selected for recombination has moved to the center of the nucleus and is associated with hyperacetylated histones. Also, shortly before recombination occurs, the
selected allele goes through the process of DNA demethylation. Conversely, the other allele is recruited to heterochromatin, is not associated with hyperacetylated
histones, and remains hypermethylated at the DNA level. Lower panel, When silenced, varn (red) is bound to PfSir2 proteins at the perinucleus and is not associated
with hyperacetylated histones. Transcriptional activation of varn correlates with its localization to a presumed privileged zone in the nucleus periphery that is
competent for transcription. Active vars are associated with hyperacetylated histones and are devoid of PfSir2 proteins.
The Journal of Immunology
Rep20 appears to be associated with var gene control since truncated chromosomes, in which the entire subtelomeric region including Rep20 is deleted, do not silence adjacent genes (43).
Also of interest for the analysis is the typical var gene organization. All var genes share a two-exon structure, with the 5⬘ exon
encoding the variable extracellular segment of the PfEMP1 protein and the 3⬘ exon encoding the cytoplasmic tail. The var intron present between the two exons has a highly conserved
structure and is implicated in var gene regulation (see below).
Cis-regulatory elements that control var expression
Epigenetic regulation of var expression
How is a single var gene elected and kept active while the rest are
maintained in a silenced state? It has been demonstrated that
epigenetic regulation plays an important role in setting up this
process. In the reported experimental system, a transgene inserted in the subtelomeric region of a P. falciparum chromosome can be reversibly silenced independent of the promoter
sequences driving its expression. Sites in the promoter that are
sensitive to micrococcal nuclease digestion in the active state are
protected against digestion when the promoter is silenced, indicating that chromatin packaging at the repressed transgene
locus is more compact than at the actively transcribing promoter (51).
Differential subnuclear localization is probably critical to var
gene regulation. Both subtelomeric and internal var genes were
found to be localized to the nuclear periphery (52). Fluorescence in situ hybridization analysis demonstrated that a reporter
gene occupied different positions in the perinucleus, depending
on its state of transcriptional activation, and that two different
selectable markers had significantly higher chances of colocalization when both were actively transcribed (51). Thus, the data
indicate that transcription at the periphery of the nucleus may
be confined to privileged zones and movements of var loci
within the periphery could either enable or disable their access to the spatially restricted transcriptional machinery.
One possible model to explain var mutually exclusive expression is the existence of a unique perinuclear compartment
that is responsible for transcription of a single var locus. It is
tempting to suggest that this compartment contains a cisregulatory element such as an enhancer localized to the privileged zone, which is able to trans enhance transcription
from a single promoter. Switching in var transcription
would then occur by competition of silenced var promoters
for occupancy of this compartment.
Several chromatin modifications which lead to the silencing
of the var genes have been discovered (45). One of these is heterochromatinization involving the P. falciparum homologue
of the yeast Silent Information Regulator 2 protein (PfSir2).
PfSir2 colocalizes with telomeric clusters, whereas it is absent
from the electron-sparse nuclear interior, and from central var
clusters. Moreover, it was shown that PfSir2 propagates from
telomers ⬎50 kb into the chromosome coding region. Rep20
sequences and subtelomeric var promoters are bound by PfSir2.
In contrast, no PfSir2 was associated with two expressed bloodstage genes, which are located ⬃100 kb from the end of the
telomer (51).
Reversible covalent modifications of histones were shown to
play a role in var gene transcriptional regulation as well. It was
demonstrated that the promoter of an active var gene was packaged with hyperacetylated histones H3 and H4, while the histones associated with the silenced var genes were hypoacetylated
(45). Furthermore, an inverse correlation between the presence
of PfSir and enrichment of nucleosomes with acetylated histones was observed. Since the yeast Sir2 is known to be a histone
deacetylase, it was reasonably suggested that PfSir2 deacetylates
histones packaging the nontranscribed var genes. This is probably followed by recruitment of additional silencing factors and
the establishment of a heterochromatin-like environment preventing transcriptional activation. Indeed, disruption of PfSir2
leads to increased transcription of members of the var gene family located in the subtelomeric regions, a result that is consistent
with the association of PfSir2 to the telomeric regions of P. falciparum chromosomes (45).
Previous studies described an AT-rich region inside the intron of var genes which harbors a promoter activity (49, 53).
These sterile promoters apparently give rise to noncoding RNA
that may take part in repressing var expression, in a manner
similar to Xist RNA-dependent X chromosome inactivation.
Yet, a recent study did not find a role for antisense RNA in the
transcriptional regulation of var genes (54). In addition, it is
possible that competition between the 5⬘ and the intronic var
promoters for either limiting amounts of transcription factors, or
entrance into a few privileged perinuclear location(s), could serve as
an auxiliary epigenetic repressing mechanism, contributing to the
expression of one var gene of 60 possible candidates.
The Ig loci and the var family
This review describes two essentially different systems that share
a common mode of gene regulation in which mutually exclusive
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Var expression has been shown to be controlled at the level of
transcriptional initiation, and DNA rearrangement, which is
crucial for Ig expression, is not involved (5, 6). As for cis-regulatory elements, var promoters were shown to play a cardinal
role in regulating mutually exclusive gene expression. Var genes
are flanked by one of several upstream sequences, upsA, upsB,
upsC, and upsE, where upsA- and upsB-type promoters are used
by the subtelomeric var genes, the upsC-type sequences are used
by the chromosome-central var genes, and the upsE uniquely
controls the var2CSA gene (5). Different mechanisms responsible for regulating var promoters in internal as opposed to subtelomeric regions were found (44, 45).
It is not clear whether the var intron participates in regulating
var mutually exclusive expression (46, 47). Yet, the intron sequences apparently do harbor an insulator activity that antagonizes spreading of chromatin activation into neighboring loci
(48). This agrees with previous results, suggesting that the upsCpromoter-intron interaction has a boundary function to protect
var genes in chromosome-central clusters from activation
through nearby euchromatic regions and neighboring var genes
(49).
Promoters from active or silenced var genes are capable of
driving expression of episome-based reporter genes, suggesting
that all var promoters are competent for transcription at one
time or another and that the majority of them are silenced by
epigenetic factors. Importantly, it was shown that a single active
var promoter is sufficient to silence endogenous var genes and
to ensure mutually exclusive expression of a single var gene (48,
50). Together, it is reasonable to suggest that interplay between
the promoter and intronic cis-regulatory elements generates a
stably inherited silenced chromatin environment.
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BRIEF REVIEWS: EPIGENETIC REGULATION OF Ig AND var EXPRESSION
Table I. Regulatory mechanisms of Ig and var choice
Differential histone acetylation
Differential DNA methylation
Noncoding RNA
Nuclear repositioning associated with
gene activity
Heterochromatinization of
nonselected genes
Feedback inhibition by protein
product
Reversible silencing
Concept of limiting resources
Ig␬ (Pre-B)
Var
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
Furthermore, once a selection is made, each system uses distinct mechanisms to disable subsequent selections from being
made. In large pre-B cells, for instance, RAG1/2 expression is
down-regulated, the VH region becomes hypoacetylated, and
the locus decontracts (4, 18). In parallel, at subtelomeric locations, a var gene that is no longer expressed becomes occupied
by PfSir2, which participates in heterochromatin assembly and
probably harbors a histone-deacetylase activity of its own, ensuring that a stably silenced state is maintained (45). However,
while termination of the rearrangement process depends on
negative feedback generated by signaling of pre- and B cell
membrane receptors, the mutually exclusive manner of expression at the var gene family relies entirely on noncoding elements
of the P. falciparum genome. Moreover, while DNA-demethylation precedes Ig␬ rearrangement and is essential for the rearrangement to take place (1, 2, 14), methylation patterns
are largely indistinguishable between active and inactive var
genes, although subtle differences may still exist (6). This is
in agreement with the highly reversible nature of the var decision, which precludes long-term modifications such as
DNA demethylation.
Numerous studies indicate that expression in both systems is
directed by cis-acting sequences that play both positive and negative roles. Another common feature of the mutually exclusive
expression involves timely transcription of sterile transcripts. At
the Ig loci, noncoding RNA is transcribed from both DNA
strands, giving rise to germline transcripts and to antisense
RNA (17, 27). This sterile transcription is restricted to a time
window just before rearrangement takes place (17, 28). In the
var system, the assumed noncoding transcripts are generated
from the so-called intronic promoters. In both systems, the
physiological role of these transcripts is not known. They could
simply be byproducts of a permissive chromatin configuration
or could play a more active role in directing mutually exclusive
expression (52).
Finally, and most intriguingly, gene choice in both systems
operates through regulated movements of the target genes
within the nucleus. Change in the position of the Ig loci is
known to precede rearrangement events. At the pro-B cell stage
the H and ␬ chain loci dissociate from the nuclear periphery,
which is regarded as a repressive environment, to become centrally located (7). Furthermore, during pre-B cell differentiation, one Ig␬ allele is recruited to heterochromatin while the
other remains centrally located and away from heterochromatin, apparently making it a better target for the recombination machinery (14). Activation of a single var gene also
involves its movement in the nucleus, and more specifically,
the activated gene occupies privileged zones at the nucleus
periphery, which are transcription permissive. Colocalization of active markers in the perinucleus is in agreement with
the limiting transcription zone hypothesis, which states that
competition over these specialized zones governs P. falciparum mutual exclusive expression.
Conclusions
We have examined, in some detail, the mechanisms used in two
biologically remote systems to achieve a similar goal, namely the
expression of one single copy of a gene in any given cell. In the
vertebrate immune system, Ig expression from one single allele
is used to allow for the generation of an infinitely diverse yet
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expression takes place (Fig. 3 and Table I). In both systems, the
cell has to choose one cluster of genes for either recombination
(Ig) or transcription (var), and from each cluster one gene segment has to be further selected. Choosing one var gene of 60 for
expression is reminiscent of choosing 1 of the 150 or 100 VH or
V␬ segments, respectively, for rearrangement. A related common feature is that gene usage is not completely random. For
example, certain families of VH were found to be preferentially
expressed in neonatal B cells (55) and, accordingly, switching to
some var genes occurs more frequently than to others (56).
Notably, both the Ig and the var systems are quite plastic in
nature. The parasite cells of P. falciparum keep changing their
choice of which PfEMP1 to express, making their decision
rather temporary. Yet, editing at the immature B cell stage (57),
as well as back-differentiation of mature B cells accompanied by
novel rearrangements (58), suggests a high plasticity of the immune system as well.
Both Ig allelic exclusion and var mutual exclusion can be explained by a stochastic mechanism. At the Ig loci, several lines of
evidence suggest that the process is likely to comply with a stochastic decision that instructs later choices that take place in
committed B lineage cells (14, 31). According to this model,
one of the two alleles is selected for recombination at an early
developmental stage, a choice that is manifested through asynchronous DNA replication (31). Subsequently, numerous
known (described above) and possibly unknown epigenetic
mechanisms are involved in maintaining the allelic asymmetry
before rearrangement in B cells. The ever-changing var gene
choice is likely to follow the stochastic model, which assumes
that most genes are equal candidates for each choice.
Both processes involve activation of the otherwise silenced
target gene, which becomes distinguished from the silenced
counter allele or member genes. The Ig and the var genes differentially associate with euchromatin and with heterochromatin according to their state of activation. For both systems, studies have shown that silenced genes are present in a more
compact packaging than their activated counterparts, and in the
two systems, histone H3 and H4 hyperacetylation characterizes
activity, whereas hypoacetylation is linked with a state of inactivity (13, 20, 45). One note of caution for both systems should
be considered. It is not clear whether hyperacetylation of one
var or one Ig in a cell is the cause or the result of choosing them.
It could be the case that histone acetylases are recruited to a
specific target either randomly or by a previous instructive
mark. However, it is equally probable that histone acetylation
occurs as a consequence of repositioning to a privileged active
nuclear zone.
The Journal of Immunology
highly specific array of active Ig genes. In the cells of P. falciparum, only one cell surface var gene is expressed at a time to avoid
detection and targeting by the immune system’s ever evolving
Ag specificity. In both systems, mutually exclusive expression is
apparently achieved by differential chromatin modifications
and unequal nuclear localization. In the future, privileged nuclear zones should be studied more at the molecular level to determine whether they harbor a cis-regulatory master element
that enables transcription and rearrangement. In addition, a
new understanding of interchromosomal pairing, as well as involvement of noncoding RNA, has added a new dimension to
the understanding of other mutually exclusive choices (59, 60).
It will be of great interest to discover whether these themes also
apply to Ig and var allelic exclusion.
Acknowledgments
We thank C. Rosenbluh and Dr. Howard Cedar for stimulating discussions and
constructive reading of the manuscript.
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