Choreography of Ig allelic exclusion

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Choreography of Ig allelic exclusion Howard Cedar1 and Yehudit Bergman2 Allelic exclusion guarantees that each B or T cell only produces a single antigen receptor, and in this way contributes to immune diversity. This process is actually initiated in the early embryo when the immune receptor loci become asynchronously replicating in a stochastic manner with one early and one late allele in each cell. This distinct differential replication timing feature then serves an instructive mark that directs a series of allele-specific epigenetic events in the immune system, including programmed histone modification, nuclear localization and DNA demethylation that ultimately bring about preferred rearrangement on a single allele, and this decision is temporally stabilized by feedback mechanisms that inhibit recombination on the second allele. In principle, these same molecular components are also used for controlling monoallelic expression at other genomic loci, such as those carrying interleukins and olfactory receptor genes that require the choice of one gene out of a large array. Thus, allelic exclusion appears to represent a general epigenetic phenomenon that is modeled on the same basis as X chromosome inactivation. Addresses 1 Department of Cellular Biochemistry and Human Genetics, Hebrew University Medical School, Jerusalem 91120, Israel 2 Department of Experimental Medicine and Cancer Research, Hebrew University Medical School, Jerusalem 91120, Israel Corresponding author: Cedar, Howard ([email protected]) and Bergman, Yehudit ([email protected])

Current Opinion in Immunology 2008, 20:308–317 This review comes from a themed issue on Lymphocyte Activation Edited by Anjana Rao and Kathryn Calame Available online 9th April 2008 0952-7915/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2008.02.002

Introduction Monoallelic expression can be either imprinted or random. Imprinted genes are expressed exclusively from either the maternally or paternally inherited chromosome, with the identity of the expressed allele being predetermined, usually by the establishment of differential DNA methylation in the male or female gametes [1]. Random monoallelic genes, on the other hand, are expressed from the maternal chromosome in some cells and the paternal chromosome in others. The best-known example of this phenomenon is X chromosome inactivation in female cells [2]. In this case, the choice of which Current Opinion in Immunology 2008, 20:308–317

X chromosome undergoes silencing is made early in embryonic development and is random. Once established, however, this decision is clonally inherited, resulting in adult females with mosaic expression of X-linked genes from maternally and paternally inherited X chromosomes. In addition to the X chromosome, an increasing number of random monoallelic regions are being identified on autosomes, and these usually contain multi-gene sequence arrays such as olfactory [3] or natural killer cell [4] receptors, interleukins [5–8], T-cell receptors (TCR) and immunoglobulins (Ig) [9,10]. In this review, we will discuss the molecular mechanisms that play a role in regulating monoallelic expression of immunoglobulin genes.

Developmental-specific regulation of antigen receptor gene rearrangement The generation of B or T cell antigen receptors takes place through a multi-step ordered process that involves developmentally regulated rearrangement events. In the B lineage, for example, this begins at the pro-B cell stage with rearrangement of the IgH locus, and light chain genes only rearrange after progression to the pre-B cell stage. Within the heavy chain locus itself DH segments are first joined to JH segments and this is then followed by VH-to-DJH recombination (reviewed in [11]). Similarly, in the T-lineage, rearrangement of TCRb occurs early in the double-negative (DN) cell compartment, and only after the production of this receptor does the TCRa region undergo recombination in a process that takes place in double-positive (DP) cells. In parallel to the B cell receptor system, rearrangement of Db to Jb segments within the TCRb locus always occurs before Vb-toDJb recombination (reviewed in [12,13]). Although this ordered process is probably directed by stage-specific trans acting factors, it is well established that the choice of which locus undergoes rearrangement is mainly dependent on lineage-specific and developmental-specific modulation of local chromatin structure [14– 16]. Thus, while RAG1 and RAG2 are highly expressed in pro-B cells, only the IgH locus appears to be accessible for recombination, while the light chain locus remains in a relatively closed conformation and thus retains its germ line configuration. Recent studies have shed a great deal of light on the molecular mechanisms that regulate this process.

Chromatin modification One of the major ways for controlling accessibility to the recombination machinery is through histone modification (reviewed in [12,13,17–19]). In B cells, for example, both www.sciencedirect.com

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the heavy and light chain loci undergo a series of chromatin changes in which they become hyperacetylated in histones H3 and H4, hypomethylated at H3K9 and enriched for histone H3K4me2,3, and all of these events take place before V(D)J rearrangement [18–22]. Much like recombination itself, these changes appear to be subject to strict temporal control. In the heavy chain locus, for example, the DHJH cluster initially becomes hyperacetylated in pre–pro-B cells, while acetylation of VH only occurs once the DHJH joints have actually been generated [23]. Histone modification is thought to affect chromatin structure by providing a molecular code that controls local protein-binding, but in most cases, the precise mechanism of how this influences DNA function is unknown. Three recent papers have now gone a long way towards deciphering how this may work on recombination in the immune system. According to these studies, the RAG2 protein contains a plant homeodomain (PHD) finger capable of recognizing H3K4me2 or me3 (preferred), and mutations that abrogate this binding site severely impair V(D)J recombination in vivo [24–26]. Furthermore, by reducing the level of H3K4me3 either by knocking down expression of the common histone H3K4 methyltransferase component WDR5, or by overexpressing the H3K4me3 demethylase, SMCX, also leads to a sharp decrease in V(D)J recombination [24]. This clearly indicates that H3K4 methylation plays a major role in marking specific sites as ‘accessible’ targets for the recombination machinery. This recognition system is obviously important during normal development in vivo, as well, since mutation of a conserved tryptophan residue (W453) within the PHD domainbinding site actually leads to a form of inherited immunodeficiency syndrome. Insight into how this molecular mechanism works at the structural level comes from experiments showing that non-core RAG2 (lacking the entire PHD domain) is more effective as a recombinase than RAG2 mutated in the PHD domain, possibly indicating that the non-core region contains an inhibitory domain whose function is decreased by binding of the PHD finger to H3K4me3. Indeed, crystallographic analysis shows that in the absence of methyl groups on H3K4, a peptide N-terminal to the RAG2-PHD domain occupies the substratebinding site, thus providing an alternate structure that may serve to inhibit enzymatic activation [24,26]. Thus, H3K4me3 represents both a docking site for RAG proteins and, at the same time plays an active role as an allosteric effector of recombinase function. Recent findings demonstrate that binding of the RAG2-PHD domain is not only dependent on H3K4me3, but is also affected by methylation of arginine 2 (R2) on the same H3 molecule, strongly suggesting that the ability to direct recombination depends on a highly defined code www.sciencedirect.com

that uses a combination of multiple histone modifications [26]. Taken together, these studies provide a molecular basis for chromatin-dependent gene recombination that, in cooperation with RSS recognition by the RAG heterodimer, could ensure specificity of the rearrangement machinery almost exclusively to antigen receptor genes.

Non-coding RNA Over 20 years ago it was observed that initiation of V(D)J recombination often coincides with non-coding (germline) transcription at antigen receptor loci [14], and since this usually appeared before rearrangement, it was suggested that it may be an essential precursor for efficient recombination. The most compelling evidence for this model has been obtained from studies on the TCRa locus where it has been shown that specific blockage of transcriptional elongation suppresses Va-to-Ja recombination and chromatin remodeling of Ja segments. This transcript, which initiates from the upstream TEA (T early a) promoter, appears to activate promoters associated with proximal Ja segments while inhibiting distal promoters, most probably through transcription interference, and this results in increased recombination of proximal Ja segments [27,28]. In the IgH locus, antisense intergenic transcription has been observed throughout the DHJH and VH regions and is clearly present before D-to-J or V-to-DJ recombination [29,30,31]. In light of the fact that the intronic enhancer, Em, regulates both DH antisense transcription, as well as DH-to-JH recombination [32,33], it has been suggested that this cis-acting element works by first activating germ line transcription, which then influences chromatin accessibility. While this appears to suggest a clear-cut role for local RNA, the actual relationship between transcription and chromatin structure is probably far more complex. Thus, while the entire DH region is marked by low level tissue-specific antisense transcription, there is a significant difference in chromatin structure between the outlying DH elements which have a relatively open structure and the more centrally placed elements which are associated with heterochromatic marks such as H3K9me2. It has been proposed that the multiple DH gene segments located centrally form a tandem array that is suppressed by repeat-induced antisense transcription and heterochromatinization, and this may explain their infrequent representation as compared to the flanking DH segments [31]. When taken together, these studies imply that germline transcription can function both as a positive and negative regulator of accessibility for V(D)J recombination. It should be noted that germline transcripts have also been shown to be required in targeting classswitch recombination but this is carried out by a completely different mechanism involving the formation of single-strand DNA substrates for activation-induced cytosine deaminase [34]. Current Opinion in Immunology 2008, 20:308–317

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Stochastic allelic exclusion can be fortuitous or instructive Allelic exclusion of the immunoglobulin genes is regulated at the level of V(D)J recombination, and two possible mechanisms have been proposed to account for this phenomenon. The first model suggests that monoallelic rearrangement comes about in a fortuitous manner because of low frequency activation and stochastic usage of the two equivalent alleles in pre-B cells (Figure 1a) [35,36]. Alternatively, the initial decision that differentiates between the two alleles may be carried out in a stochastic manner, but once made, it then becomes stably instructive, thereby resulting in two alleles that are differentially accessible to the rearrangement machinery (Figure 1b) [37]. According to this second model the initial decision occurs during early stages of embryogenesis when the Ig loci become marked by asynchronous DNA replication. Later on, during B cell development, these loci are then subject to additional monoallelic changes (see below) that are apparently directed by the initial mark, and this culminates in monoallelic rearrangement. Evidence supporting this model of allelic exclusion for B cell immunoglobulin gene receptors will be discussed below.

Initiation of allelic exclusion The entire genome is organized into chromosomal bands programmed to replicate at different points in S-phase. In a truly striking manner, expressed genes replicate in early S, while repressed domains replicate late [38]. In keeping with this pattern, genes that are expressed monoallelically at some stage in development have an intermediate pattern, with one allele undergoing replication relatively early in S-phase, while the second allele replicates late. Indeed, this behavior is the hallmark of monoallelic expression. In the case of imprinted regions, this replication pattern is initially set up in the gametes and then maintained throughout development [39], while random asynchronous replication is established during early embryogenesis [37]. This decision represents one of the earliest steps in the X chromosome inactivation process. Although the mechanism for this binary choice has not been fully elucidated, it now appears that it may be on the basis of the ability of the two X chromosomes to pair-up during the cell cycle, thus allowing the cis-based choice of one allele through exclusion of the other [40,41]. It should be noted that asynchronous replication of both B cell and TCR regions on autosomes is also established at the same time in early development, and it is very likely that this choice is on the basis of a similar binary mechanism. Once established by this stochastic decision making process, the choice of allele appears to become stably engraved, and is then maintained in a clonal manner throughout development [37,42].

Epigenetic changes associated with allelic exclusion Even though rearrangement of each receptor antigen appears to occur at a specific stage in B or T cell deCurrent Opinion in Immunology 2008, 20:308–317

velopment, this event is often preceded by distinct epigenetic changes that, in some instances, have been shown to occur monoallelically in each cell. An excellent example of this is the Igk light chain locus. During the pro-B cell stage, both alleles of this locus are found at centrally located, indistinguishable positions within the nucleus, but as cells progress to the pre-B cell stage, one allele is relocated to the pericentric heterochromatin region (Figure 2) [22,43–45]. This process is mediated by a newly identified rearrangement silencer element (Sis) that is located 50 to Jk1 and works by binding Ikaros, a protein known to colocalize with heterochromatin [46]. These results have also been confirmed by biochemical studies showing that some alleles are packaged with acetylated histones, while others are associated with heterochromatin protein 1 (HP1) in pre-B cells [44]. In situ studies indicate that the late replicating k allele in each cell is specifically located near heterochromatin, strongly suggesting that this monoallelic chromatin pattern may be directed by pre-existing epigenetic marks, consistent with the instructive model for allelic exclusion. In vivo experiments are still required to prove that this pathway actually occurs during normal development. Another epigenetic event associated with rearrangement is locus contraction, a process by which outlying V regions are brought into a position that makes them more available for recombination. Here too, this change occurs in a developmentally regulated manner with contraction always preceding rearrangement itself (Figure 2). This intra-molecular conformation has been observed at all four antigen receptor loci, IgH, Igk, TCRa and TCRb [47–49,50], but has been best studied at the heavy chain locus where genetic experiments have demonstrated that it is mediated by two key transcriptional regulators, Pax5 and YY1 [48,51]. In situ analysis of this locus in pro-B cells indicated that looping occurs monoallelically, clearly indicating that this event may also be directed by underlying epigenetic mechanisms [49]. Finally, it is well documented that DNA methylation represses V(D)J recombination in vivo during normal B cell maturation, and demethylation appears to be the last step required before rearrangement can take place [44]. At the k locus, it has been demonstrated that demethylation in the Jk region actually takes place monoallelically on the same exact allele that is destined for recombination [52,53]. Early models to explain the kinetics of rearrangement in B and T cells were on the basis of the assumption that both alleles are equally accessible and recombination is dependent on the presence of limiting cooperative factors that work stochastically at low frequency on only one allele at a time. Although reasonable, this model is not completely consistent, mainly because it would predict that most lymphocytes would only assemble a complete IgH, IgL or TCRb at one allele, and this is not the case www.sciencedirect.com

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Figure 1

Fortuitous versus instructive decisions in the regulation of Igk allelic exclusion. (a) During early stages of embryogenesis (pre- and post-implantation) the two k alleles (red and blue) are equivalent. The initial stochastic decision occurs in pre-B cells, where rearrangement comes about in a fortuitous manner either on the maternal or the paternal allele, perhaps as a result of limiting factor interactions that allow time for one allele to be processed earlier than the other. (b) The initial stochastic decision that differentiates between the two k alleles occurs around the time of implantation, when this locus becomes marked by asynchronous DNA replication with either the maternal or paternal allele being copied earlier than the other. In pre-B cells this prior decision serves an instructive role by bringing about monoallelic epigenetic changes that culminate in rearrangement on the early allele. If the initial rearrangement is nonproductive, or yields autoreactive antibodies, rearrangement can occur on the second allele (E, early; L, late).

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Figure 2

Choreography of allelic choice in B cells. This scheme shows a possible step-wise model for a number of epigenetic changes that lead to initiation and maintenance of Ig allelic exclusion. In pre-implantation cells, both the IgH and Igk loci, harboring the variable (V), diversity (D) and joining (J) recombining gene segments, replicate synchronously. Around the time of implantation both loci begin to replicate asynchronously, with one allele replicating early (light colors) and the other replicating later in S-phase (dark colors), and both alleles undergo de novo methylation. In pro-B cells both the Ig heavy and Igk loci become associated with acetylated histones (Ac), but to different degrees. The Rag genes are activated (orange background) leading to D-to-J rearrangement on both alleles, and V-to-DJ exclusively on the early replicating allele (blue lines indicate ongoing transcription). This is Current Opinion in Immunology 2008, 20:308–317

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[54]. Moreover, germline transcription occurs biallelically [55,56] and experiments on exogenously introduced transgenes clearly show that the rearrangement machinery can potentially operate at very high frequency [57,58]. Rather, it appears that rearrangement itself is enabled by previous epigenetic events, and in most cases, this occurs in a monoallelic manner. Any model for explaining the binary choice of one out of two alleles must involve a stochastic step that differentiates between the two homologous copies. The only remaining question concerns the timing of this event. According to the fortuitous mechanism, this choice takes place in a probabilistic manner at some early time during B cell development, while the instructive model proposes that this choice takes place during early embryogenesis and is then maintained in a stable manner in all cell types (Figure 1).

Once pro-B cells have undergone rearrangement of the heavy chain locus, they enter into a proliferative stage that is fueled by IL7 signaling. During the transition to small pre-B cells, however, this pathway is down regulated, causing a marked decrease in phosphorylated STAT5, which evidently sets in motion a process of histone deacetylation at the IgH locus [59–62]. This then leads to a decrease in accessibility that serves to prevent any further recombination even though these same cells are actively undergoing IgL rearrangement. In T cells, E47-binding at the TCRb locus is originally responsible for the histone acetylation and increased accessibility that enables rearrangement in DN cells. At later T-cell stages, however, this same locus becomes deacetylated and resistant to recombination in a process that is mediated by a reduction in E47 levels and removal of this factor from the TCRb locus [63].

Maintenance of allelic exclusion

Secondary decisions

Once an initial rearrangement event has occurred, there appear to be several different feedback mechanisms that play a role in preventing simultaneous recombination of the second allele, as well as further rearrangements during later stages of B or T cell development. At the IgH locus, for example, successful V(D)J rearrangement of one allele brings about membrane signaling through the generation of a pre-B cell receptor that sets in motion a number of key molecular mechanisms (reviewed in [11]). First, and most directly, this feedback system brings about the downregulation of factors needed for recombination, such as RAG1, RAG2 and TdT (reviewed in [20]). Other changes resulting from pre-B cell signaling involve reorganization of the heavy chain locus itself, including repositioning of the DJH rearranged allele to repressive pericentric chromatin and reversal of locus compaction [47] which serves to physically separate the distal VH genes from the proximal IgH domain, thus preventing further rearrangement of the second DJH allele (Figure 2). In a similar manner, the TCRb locus, which initially contracts in DN cells, then undergoes decontraction during transition to the DP stage. This serves to distance the Vb gene cluster from the proximal DJCb domain, thereby lowering the probability for further rearrangement on the second DJb-rearranged allele [50]. In both cases, this mechanism seems to target mainly distal variable genes, and, as a result, VH or Vb segments positioned close to the DJC locus actually have a tendency to escape allelic exclusion.

In the event that the first rearranged allele is either nonproductive or generates an autoreactive receptor, lymphocytes still have the potential for carrying out secondary rearrangements, and it is interesting to ask whether this takes place with equal probability on both alleles, or whether the initially rearranged early replicating allele is the preferred substrate. Earlier studies had suggested that autoreactive B cells are edited primarily by deletional recombination [64,65]. However, in a more recent study utilizing mice heterozygous for the hCk allele (Igkm/h), it was shown that 25% of all developing B cells actually undergo light chain editing, and that this can occur with equal probability either by deletion or allelic inclusion [66]. These results led the authors to suggest that although V-Jk rearrangements initially target a single selected allele, induction of autoreactivity or extension of the pre-B cell stage may promote accessibility to the recombination machinery on both alleles, perhaps by providing additional time for the second allele to undergo de-heterochromatinization, histone acetylation, methylation of H3K4 and DNA demethylation. While this idea is consistent with the data, the results themselves may be better explained by taking into consideration the fact that the pre-recombined allele is early replicating in about 50% of the cells and late replicating in the others. In the event that the targeted gene is on the early-replicating allele, it is highly likely that it will be preferred for secondary rearrangement, and this would

facilitated by large-scale contraction (vertical rectangle). In large pre-B cells, pre-BCR signaling inhibits Rag expression and induces decontraction of the IgH loci and repositioning of one IgH allele to repressive centromeric domains (grey area), and this state is then maintained in small pre-B and early immature B cells. During the transition to small pre-B cells (pre-B1), the Igk locus undergoes a similar contraction process (vertical rectangles) that is maintained until the immature B cell stage. In small pre-BII, the later-replicating Igk allele is sequestered to heterochromatin, while the early replicating allele becomes highly enriched with acetylated histones. In late small pre-B cells (pre-BIII), the recombination machinery is re-activated (orange background), and the early replicating, hyperacetylated k allele undergoes monoallelic demethylation and recombination. In immature B cells both Ig loci are centrally located and transcribed. Finally, in mature B cells the Igk locus undergoes decontraction and becomes a substrate for somatic hypermutation (striped V region). www.sciencedirect.com

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result in deletions of the pre-rearranged VJk. However, in the 50% of cells where it is on the late replicating allele, secondary rearrangement will probably occur on the germline early replicating allele, thereby giving rise to allelically included cells. This alternative interpretation would clearly fit in nicely with the instructive model of rearrangement. Another post-rearrangement adjustment in the immune system involves somatic hypermutation of variable regions which takes place in germinal centers. In order to test whether this process occurs stochastically on both alleles of the light chain, targeted mice carrying two almost-identical allelically marked pre-rearranged light chain genes were employed. Despite this pre-existing recombined state, one allele in each B cell was still found to be early replicating, while the second allele was late replicating and methylated in the Jk region. Interestingly, somatic mutations were found to take place preferentially on the unmethylated allele. These results clearly indicate that the same system that marks a single allele in each cell for preferential rearrangement continues to serve as a distinguishing feature for directing later somatic hypermutation events [67].

Monoallelic expression at other loci The olfactory receptor genes make up a large class of sequences that appear to be regulated in a manner similar to that of the immune receptor loci. About 1000 of these genes are distributed amongst distinct clusters spread over almost all of the chromosomes in both man and mouse. The expression of these genes in olfactory neurons is subject to a process of single gene selection that involves clear-cut allelic exclusion, resulting in the appearance of only one receptor per cell [3]. Although the mechanism for this phenomenon has not been completely elucidated, there is no question that its basic foundations are analogous to those that operate at the immune receptor loci. All of the olfactory receptor loci, for example, have been found to undergo asynchronous replication, and this epigenetic mark appears to be established early in development and maintained in all cell types in a clonal manner with some cells having the paternal allele early replicating, while in others it is the maternal allele [42]. Within olfactory neurons, the final selection of which unique receptor gene becomes activated involves some sort of counting mechanism that utilizes chromosome interactions within the nucleus and specific changes in local DNA methylation [68,69] and the use of feedback inhibition [70], all suggesting that allelic exclusion in both the olfactory and immune systems use common molecular pathways aimed at generating cell expression diversity. It is clear from these examples that asynchronous replication timing represents one of the most basic components of the allelic exclusion process, and it is likely that all of these loci are subject to common regulatory mechanisms. One indication that this may be the case is the observation Current Opinion in Immunology 2008, 20:308–317

that replication timing of many monoallelically expressed loci is highly coordinated at the level of each chromosome [55]. Thus, it appears that the olfactory or immune receptor loci on any given chromosome replicate in a coordinate manner with the full set being copied early during S-phase in some cells, and late in others, in a pattern similar to that seen on the X chromosome in female cells. While the mechanism for establishment of asynchronous replication is not known, recent genetic experiments in ES cells indicate that the Polycomb group protein Eed may play a role in this process [71].

Widespread monoallelic expression It was always assumed that almost all genes in diploid organisms are expressed biallelically, but two recent studies using genome wide approaches have challenged this concept by showing that between 1 and 5% of autosomal genes in both human and mouse are actually subject to various forms of random monoallelic expression [72,73]. A relatively large fraction of these newly identified genes encode cell-surface proteins, suggesting that monoallelic expression may play a prominent role in controlling the diversity of cell–cell interactions. Unlike the imprinted gene loci or the X chromosome where the expression of a single allele in each cell is stably maintained in a clonal manner, many of these newly discovered genes appear to be subject to allele switching or conversion to biallelic expression in a manner similar to what has previously been observed for the lymphokines, IL2 and IL4 [74]. Furthermore, this type of monoallelic expression does not appear to be regionally coordinated, and, as a result, adjacent genes in any given cell may be monoallelically expressed, but on different chromosomes. In these cases, the mechanisms for allelic expression most likely involve stochastic interactions with limiting protein factors that may operate at the level of histone acetylation and chromatin accessibility. Studies on the IgL chain demonstrated that the asynchronous replication-time marking system initiated during early development [52], is associated with differential accessibility of the two alleles in pre-B cells [44], with one being more likely (about threefold) than the other to undergo the initial epigenetic changes required for rearrangement [53]. In the light of this relatively modest difference, it is very likely that continued exposure to factors that enable the rearrangement machinery would ultimately result in recombination at the second allele, as well. Assuming that other monoallelic loci in the genome are also associated with asynchronous replication, individual genes would be subject to random monoallelic expression, but this pattern is likely to be unstable over time, resulting in allele switching or biallelic transcription. From this perspective, it appears that feedback mechanisms designed to prevent additional rearrangement events in the immune system probably play a key role in stabilizing the initial decision www.sciencedirect.com

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Conclusions Even though it has been known for a long time that productive rearrangement in the immune system occurs on only one of the two alleles that make up each antigen receptor locus, the mechanisms involved in this process have not yet been fully elucidated. Earlier results had suggested that both alleles may be equally accessible in target B or T cells, and the choice is made stochastically by random hits of the recombination machinery. More recent studies, however, demonstrate that recombination is actually preceded by allele-specific epigenetic changes (Figure 2), clearly indicating that recombination competent cells in the immune system already carry instructive information on which allele will be preferentially chosen for rearrangement as well as other genetic alterations that contribute to immune diversity. Whatever the mechanism for the initial allelic choosing process, it is now quite clear that this decision is then stabilized by feedback mechanisms that protect the alternate allele from the recombination machinery. There appear to be many examples of genomic loci that are subject to monoallelic choice during development, and it is very likely that they are regulated by the same common molecular components as those used in the immune system, with asynchronous replication timing being a major theme. Once it is taken into consideration that alleles can be marked as more or less accessible by stable epigentic mechanisms, it is much easier to understand how monoallelic choice events are accomplished within the nucleus. The major unsolved question, however, has to do with the initial choice of allele. In some manner there must be a toggle-switch mechanism that allows the cell to always choose one out of two identical alleles. In the case of X chromosome inactivation, recent experiments seem to indicate that this is accomplished by ‘interactive pairing’, a space-based process that would clearly enable the choosing of one allele at the exclusion of the other. Deciphering this fundamental mechanism clearly stands at the foundation of understanding allelic exclusion in general.

Acknowledgements This work was supported by grants from the Israel Academy of Science (Y.B. and H.C.), Philip Morris USA Inc. and Philip Morris International (Y.B. and H.C.), the National Institutes of Health (Y.B. and H.C.) and the Israel Cancer Research Fund (Y.B. and H.C.).

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23. Chowdhury D, Sen R: Stepwise activation of the immunoglobulin m heavy chain gene locus. EMBO J 2001, 20:6394-6403. 24. Matthews AG, Kuo AJ, Ramon-Maiques S, Han S, Champagne KS,  Ivanov D, Gallardo M, Carney D, Cheung P, Ciccone DN et al.: RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination. Nature 2007, 450:1106-1110. 25. Liu Y, Subrahmanyam R, Chakraborty T, Sen R, Desiderio S: A  plant homeodomain in RAG-2 that binds Hypermethylated lysine 4 of histone H3 is necessary for efficient antigenreceptor-gene rearrangement. Immunity 2007, 27:561-571. 26. Ramon-Maiques S, Kuo AJ, Carney D, Matthews AG,  Oettinger MA, Gozani O, Yang W: The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2. Proc Natl Acad Sci U S A 2007, 104:18993-18998. These three reports [24–26] provide new insight into the molecular basis for chromatin-dependent Ig recombination by showing both biochemically and structurally that the PHD domain of RAG2 recognizes trimethylation of K4 together with dimethylation of R2 on histone H3. Reducing the levels of H3K4me leads to a decrease in V(D)J recombination and mutation in the PhD domain is actually linked to Omenn Syndrome. 27. Abarrategui I, Krangel MS: Regulation of T cell receptor-alpha  gene recombination by transcription. Nature Immunol 2006, 7:1109-1115. 28. Abarrategui I, Krangel MS: Noncoding transcription controls  downstream promoters to regulate T-cell receptor alpha recombination. EMBO J 2007, 26:4380-4390. Using gene-targeting in the TCRa locus, they showed that a transcriptional terminator introduced downstream of Ja56 gene segment or the TEA lead to silencing of promoters downstream to the integration sites, but to the activation of Ja promoters located further 30 in the locus. This is accompanied by suppressed Va-to-Ja recombination, changes in chromatin structure and by retargeting of Va-to-Ja recombination events to more 30 Ja segments. These studies [27,28] explain the role of noncoding transcription in controlling the ordered progression of TCRa locus recombination events, thus maximizing a robust TCRa repertoire. 29. Bolland DJ, Wood AL, Johnston CM, Bunting SF, Morgan G, Chakalova L, Fraser PJ, Corcoran AE: Antisense intergenic transcription in V(D)J recombination. Nat Immunol 2004, 5:630637. 30. Bolland DJ, Wood AL, Afshar R, Featherstone K, Oltz EM,  Corcoran AE: Antisense intergenic transcription precedes Igh D-to-J recombination and is controlled by the intronic enhancer Emu. Mol Cell Biol 2007, 27:5523-5533. This study demonstrates that antisense intergenic transcription occurs throughout the IgH DHJH region before D-to-J rearrangement. Since the intronic enhancer regulates both DH-to-JH recombination as well as DH antisense synthesis, it is suggested that this transcription plays a positive regulatory role in V(D)J recombination. 31. Chakraborty T, Chowdhury D, Keyes A, Jani A, Subrahmanyam R,  Ivanova I, Sen R: Repeat organization and epigenetic regulation of the DH-Cmu domain of the immunoglobulin heavy-chain gene locus. Mol Cell 2007, 27:842-850. This study reports on the different chromatin status of DH gene segments. The 50 - and 30 - most gene segments are associated with acetylated histones, whereas the intervening DSP2 gene segments are associated with heterochromatic marks such as histone H3K9me2 that are maintained by ongoing histone deacetylation. Interestingly, the centrally located DHs form a tandem array that expresses tissue-specific, antisense oriented transcripts. It is proposed that the intervening DHs are suppressed by heterochromatinization, thus explaining their infrequent rearrangement.

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32. Perlot T, Alt FW, Bassing CH, Suh H, Pinaud E: Elucidation of IgH intronic enhancer functions via germ-line deletion. Proc Natl Acad Sci U S A 2005, 102:14362-14367.

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33. Afshar R, Pierce S, Bolland DJ, Corcoran A, Oltz EM: Regulation of IgH gene assembly: role of the intronic enhancer and 50 DQ52 region in targeting DHJH recombination. J Immunol 2006, 176:2439-2447.

49. Sayegh C, Jhunjhunwala S, Riblet R, Murre C: Visualization of looping involving the immunoglobulin heavy-chain locus in developing B cells. Genes Dev 2005, 19:322-327.

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Pericentromeric recruitment (similar to the Igk) and decontraction (similar to the IgH locus) were shown to contribute to the initiation and maintenance of allelic exclusion at the TCRb locus. Thus, locus decontraction may serve as a general mechanism for preventing V-to-DJ rearrangement on the second DJ-rearranged allele. 51. Liu H, Schmidt-Supprian M, Shi Y, Hobeika E, Barteneva N,  Jumaa H, Pelanda R, Reth M, Skok J, Rajewsky K et al.: Yin Yang 1 is a critical regulator of B-cell development. Genes Dev 2007, 21:1179-1189. YY1, an evolutionary conserved zinc finger protein that has been suggested to function as a PcG protein during development was shown to bind to the intronic Eim enhancer and modulate IgH locus contraction which in turn facilitates rearrangement of VH distal DHJH gene segments in pro-B cells. 52. Mostoslavsky R, Singh N, Kirillov A, Pelanda R, Cedar H, Chess A, Bergman Y: k chain monoallelic demethylation and the establishment of allelic exclusion. Genes Dev 1998, 12:18011811. 53. Goldmit M, Schlissel M, Cedar H, Bergman Y: Differential accessibility at the kappa chain locus plays a role in allelic exclusion. EMBO J 2002, 21:5255-5261. 54. Gorman JR, Alt FW: Regulation of immunoglobulin light chain isotype expression. Adv Immunol 1998, 69:113-181. 55. Singh N, Bergman Y, Cedar H, Chess A: Biallelic germline transcription at the k immunoglobulin locus. J Exp Med 2003, 197:743-750. 56. Jia J, Kondo M, Zhuang Y: Germline transcription from T-cell receptor Vbeta gene is uncoupled from allelic exclusion. EMBO J 2007, 26:2387-2399. 57. Ferrier P, Krippl B, Blackwell TK, Furley AJ, Suh H, Winoto A, Cook WD, Hood L, Costantini F, Alt FW: Separate elements control DJ and VDJ rearrangement in a transgenic recombination substrate. EMBO J 1990, 9:117-125. 58. Alvarez JD, Anderson SJ, Loh DY: V(D)J recombination and allelic exclusion of a TCR beta-chain minilocus occurs in the absence of a functional promoter. J Immunol 1995, 155:11911202. 59. Goetz CA, Harmon IR, O’Neil JJ, Burchill MA, Farrar MA: STAT5 activation underlies IL7 receptor-dependent B cell development. J Immunol 2004, 172:4770-4778. 60. Stanton ML, Brodeur PH: Stat5 mediates the IL-7-induced accessibility of a representative D-Distal VH gene. J Immunol 2005, 174:3164-3168. 61. Chowdhury D, Sen R: Transient IL-7/IL-7R signaling provides a mechanism for feedback inhibition of immunoglobulin heavy chain gene rearrangements. Immunity 2003, 18:229-241. 62. Bertolino E, Reddy K, Medina KL, Parganas E, Ihle J, Singh H: Regulation of interleukin 7-dependent immunoglobulin heavychain variable gene rearrangements by transcription factor STAT5. Nat Immunol 2005, 6:836-843. 63. Agata Y, Tamaki N, Sakamoto S, Ikawa T, Masuda K,  Kawamoto H, Murre C: Regulation of T cell receptor Beta gene rearrangements and allelic exclusion by the helix-loop-helix protein, E47. Immunity 2007, 27:871-884. The dosage of the helix-loop-helix protein, E47, was shown to play a dual role both at the initiation of Vb-to-DJb gene rearrangement, as well as in the pre-TCR feedback inhibition mechanism to suppress continued TCRb gene rearrangement.

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