Chromatin Architecture and the Generation of Antigen ...

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Review

Chromatin Architecture and the Generation of Antigen Receptor DiversitySuchit Jhunjhunwala,1 Menno C. van Zelm,1,2 Mandy M. Peak,1 and Cornelis Murre1,*1Division of Biological Sciences, Department of Molecular Biology, University of California, San Diego, La Jolla, CA 92093, USA2Department of Immunology, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands*Correspondence: [email protected] 10.1016/j.cell.2009.07.016

The adaptive immune system generates a specific response to a vast spectrum of antigens. This remarkable property is achieved by lymphocytes that each express single and unique antigen receptors. During lymphocyte development, antigen receptor coding elements are assembled from widely dispersed gene segments. The assembly of antigen receptors is controlled at multiple levels, including epigenetic marking, nuclear location, and chromatin topology. Here, we review recently uncovered mechanisms that underpin long-range genomic interactions and the genera-tion of antigen receptor diversity.

IntroductionThe lymphocyte compartment consists of cells that express a diverse repertoire of antigen receptors, which enables organ-isms to mount an immune response specifically tailored to invading pathogens. Dreyer and Bennett (1965) first proposed that antigen receptor diversity is generated by DNA recombi-nation. Later studies confirmed this original insight, revealing that antigen receptor loci are organized into distinct genomic regions that contain variable (V), diversity (D), and/or join-ing (J) and constant (C) coding elements (Brack et al., 1978; Seidman et al., 1978; Weigert et al., 1978). Since this early work, the understanding of the biochemical and molecular mechanisms that underpin the assembly of antigen receptors has blossomed. Excellent reviews have described the findings generated by these studies in great detail (Jung and Alt, 2004; Schatz and Spanopoulou, 2005). Here, we will briefly introduce the basics of polymer science in order to illuminate some of the physical considerations of chromatin structure that come into play upon exploring the nature of long-range genomic interac-tions. We then discuss how epigenetic marking, nuclear loca-tion, and chromatin topology modulate DNA recombination. Finally, we describe what has been learned about the actual topology of antigen receptor loci and how it relates to long-range genomic interactions and antigen receptor gene rear-rangement. The main goal of this review is to bring together the seemingly unrelated concepts of polymer science, nuclear organization, long-range genomic interactions, and the assem-bly of antigen receptor loci.

Chromatin StructureThe antigen receptor loci are not merely linear chromosomal structures but posses a three-dimensional configuration. They must fold into an elaborate pattern of loop arrangements to permit antigen receptor gene segments to encounter each other with the appropriate frequencies. Resolving this ques-tion requires insight into long-range chromatin structure and dynamics through polymer physics.

The unit of the chromatin fiber is the nucleosome. A nucleosome consists of a 146 base pairs (bp) DNA segment wrapped around an octamer that has two copies each of his-tones H2A, H2B, H3, and H4. The nucleosomes form a 10 nm fiber creating a structure resembling “beads on a string.” A naked DNA fiber without any histones contains approximately 3 bp per nm if stretched linearly. Addition of histones compacts this value to 20 bases/nm in the 10 nm fiber. The 10 nm fiber, in the presence of histone H1, condenses into a more compact 30 nm fiber, of which the precise structure is still not completely resolved (Schalch et al., 2005). The 30 nm fiber contains ?100 bases/nm.

How the chromatin fiber is folded into higher order struc-tures beyond the 30 nm fiber remains largely unknown. In the late 1970s and early 1980s, distinct folding patterns for chro-mosome structure were proposed, including topologies involv-ing helical, radial, or combined loop-helical folding (Sedat and Manuelidis, 1977; Paulson and Laemmli 1977; Rattner and Lin, 1985). Using electron microscopic analyses of chromo-some spreads, Laemmli and collaborators showed that chro-mosomes appeared to be composed of loops of ?90 kbp in size. It was postulated that such loops interact with a putative nuclear matrix during mitosis and cluster further into rosettes containing on average ?18 loops, yielding ?100 rosettes per mitotic chromosome (Paulson and Laemmli, 1977; Pienta and Coffey, 1984). More recently, serial thin-section electron microscopy has suggested a different topology for chromatin structure, namely a “chromonema” fiber, where a chain with a diameter of 60–130 nm is interspersed by more loosely folded segments that have a diameter of 30 nm (Belmont and Bruce, 1994). Recent advances in technology, including structured-illumination (SIM) and photoactivatable localization micros-copy (PALM), will permit higher resolution imaging and should increase our still-rudimentary knowledge of genome structure and the ensemble of topologies that are adopted by antigen receptor, olfactory, globin, and Hox genes and other large regions of the genome.

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Chromatin Dynamics and TopologyThe chromatin fiber is in continuous motion. To what extent is this motion random, and to what degree is it directed? Is it shaped, and how can such a structure be described? In essence, the words “shape” and “structure” are only attempts to provide order to a wide spectrum of confor-mations that are adopted by the chromatin fiber. Thus, the shape of a chromatin fiber can best be discussed in terms of its average properties, rather than the precise location of each nucleotide. A chromatin fiber, because of its repeti-tive nature, resembles a polymer, and many of its physical

Figure 1. Flexible Polymer Chain Models in Free and Confined Environments(A) Freely jointed chain. A freely jointed chain consists of a series of rigid seg-ments connected by flexible hinges.(B) Self-avoiding random walk. In a self-avoiding chain, a segment cannot intersect any other segment.(C) Worm-like chain. In contrast to the freely jointed chain, which is flexible within the hinges that separate the segments, the worm-like polymer chain is continuously flexible.(D) Random walk/giant loop model. Giant loops of 3–5 Mbp are tethered to a backbone. The DNA within the loops and the backbone itself follow a random walk.(E) Multiloop subcompartment model. Chromatin is organized into 1–2 Mbp subcompartments. Each subcompartment consists of a bundle of loops that are attached to a common loop base. Linkers connect the chromatin subcom-partments. Both loops and linkers undergo random walk behavior.(F) Random loop model. Dynamic loops of large and small sizes are formed at random intervals on the chromosome. Both individual loops and bundles of loops are shown.

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properties can be analyzed in terms of random walk models, established by the illustrious Kuhn, Flory, and de Gennes (Morawetz, 1985). In a random walk model, the chromatin fiber can be imagined as a series of rigid and nonflexible segments, connected by flexible hinges. Several types of random walk models have been used to describe the struc-ture and dynamics of polymer chains (Figure 1). In a freely jointed chain model, the hinges connecting adjacent seg-ments, named Kuhn segments, are free to rotate, and the polymer segments are allowed to overlap with each other; that is, the orientation of one segment is independent of the orientation of its two adjacent segments (Figure 1A). A more realistic model, frequently applied to describe polymer chain behavior, is the self-avoiding chain (Figure 1B). A self-avoiding chain is similar to the freely jointed chain, except that the chain cannot cross its own path or that of another chain, which is to say that the Kuhn segments cannot inter-sect with each other (de Gennes, 1979). Yet another model is the worm-like or Kratky and Porod chain, which consid-ers the polymer as a continuously flexible chain rather than freely jointed discrete segments (Kratky and Porod, 1949) (Figure 1C). Recent studies have described the dynamics of the yeast chromatin fiber in terms of the worm-like chain (Bystricky et al., 2004).

In the absence of confinement effects, the effective volume occupied by the chain is determined by its contour length (genomic distance separating the ends of a chromatin fiber), attractive and repulsive intrachain interactions, molecular crowding effects, and the intrinsic flexibility of the fiber. The most important parameter determining the flexibility of a poly-mer is the “persistence length,” which is defined as the length of the polymer at which the ends become decorrelated, mean-ing that the polymer becomes flexible. The persistence length for naked DNA is ?50 nm. The persistence length for a chro-matin fiber remains controversial but, depending on the experi-mental design by which it is measured, varies between 30 and 200 nm (Langowski and Heermann, 2007).

In eukaryotic nuclei, the total DNA contained in the chro-mosomes has a combined length of approximately 2 m, which fits inside a nucleus with a diameter of the order of 10 µm, implying that the eukaryotic genome must have adopted strat-egies that permit its folding into a highly condensed state. Indeed, chromosomes show a confined geometry, indicated by the presence of chromosome arms as well as bands, argu-ing against free random walk behavior. Furthermore, distance measurements demonstrate that the spatial distance scales as a function of genomic separation over 4 Mbp, with exponents that are incompatible with that of free random walk statistics (Warrington and Bengtsson, 1994; Sachs et al., 1995; Münkel and Langowski, 1998).

As a first approach to describing the configuration and dynamics of the chromatin fiber in vertebrate nuclei, a chro-matin topology named the random walk/giant loop model (RW/GL) was proposed (Yokota et al., 1995; Sachs et al., 1995). The RW/GL model assumes that the chromatin fiber is subject to random motion but is spatially confined as large loops (2–5 Mbp), tethered to loop attachment points (Figure 1D). How-ever, measurements of spatial distances between genomic

markers, spaced less than 4 Mbp apart, did not agree well with the RW/GL model (Münkel and Langowski, 1998). In the late 1990s, the multiloop subcompartment (MLS) model was proposed as an alternative configuration to describe long-range chromatin folding (Münkel and Langowski, 1998; Knoch et al., 2000). The MLS model assumes that the chro-matin fiber is folded into 1 Mbp compartments, containing loops that are clustered as rosettes, connected by flexible linkers (Figure 1E). Most recently, yet another topology, the random loop (RL) model, has been proposed to describe the long-range chromatin folding (Figure 1F). Whereas loops

in the RW/GL and MLS models are assumed to be uniform in size, the RL model permits loops of variable sizes that dynamically associate and dissoci-ate from loop attachment points (Bohn et al., 2007). The development of new computational methods to model chro-matin topologies should help to define experimental strategies that evaluate model predictions. Furthermore, as will be discussed below, modeling of long-range eukaryotic chromatin structure has already suggested new avenues of investigation.

Genetic Organization of Antigen Receptor LociIn each B cell, the antigen receptor or antibody consists of two heavy and light chain polypeptides, encoded on sepa-rate loci. The murine immunoglobulin heavy chain locus (Igh) which codes for the Ig heavy chain, manifests itself as a single massive stretch of DNA (3 Mbp) in length, which is divided into distinct DNA elements encoding the variable, diversity, joining, and constant regions (Figure 2A). Each subregion displays much complex-ity. For example, 15 partially dispersed V region families encode approximately 195 VH gene segments, depending on the genetic background, each of which is approximately 500 bp in size. The den-sity of gene segments within the V region cluster is relatively low, containing large intergenic regions up to 50 kbp in size.

Downstream of the VH regions, are 10–13 DH and four JH gene segments, as well as eight CH regions that encode the various Igh isotypes, including Cµ, Cδ, Cγ1, Cγ2a, Cγ2b, Cγ3, Cα, and Cε.

The light chain of immunoglobulins is produced by one of two loci, Igκ or Igλ. The Igκ locus is composed of approxi-mately 120 Vκ gene segments that span almost 3 Mbp, a Jκ cluster, and a single constant region positioned within very close proximity (2.5 kbp) to the Jκ cluster (Figure 2B). The orga-nization of the Igλ locus is quite distinct from that of the Igh and Igκ loci. Rather than a common set of J gene segments located upstream of the constant region(s), the four constant regions of

Figure 2. Genomic Organization of Antigen Receptor LociGenetic structures of antigen receptor loci are shown. Variable (V) gene segments are shown in blue; diversity (D) gene segments, if present in pur-ple, and joining (J) gene segments in red; constant (C) regions are in black, and enhancers in green. Note that the TCRδ locus is interspersed within the TCRα locus. Genomic distances (Mbp or Kbp) are indicated for each of the loci.


Igλ each contain their own unique Jλ gene segment. Moreover, only two V region gene segments, Vλ1 and Vλ2, are frequently utilized. Vλ2 is located approximately 60 kbp from Jλ2, and it will generally not recombine with other Jλ gene segments. On the other hand, Vλ1, located 22 kbp from the Jλ1, will form joints with either Jλ1 or Jλ3 (Figure 2C). Thus, each chain of an antibody is produced using a similar theme of combining distinct gene segments.

The organization of genes encoding the T cell receptor (TCR) gene segments is strikingly similar to that observed for the immunoglobulin loci (Figures 2D–2F). Two distinct T cell lineages characterized by the antigen receptor expressed on their cell surface, αβ and γδ T cells, develop in the thymus from early T lineage progenitor cells. The TCRβ locus spans approx-imately 650 kbp of genomic DNA. It contains 31 Vβ gene seg-ments, of which 20 are functional and located upstream from two DβJβ clusters and two Cβ regions. Each of the DβJβ clus-ters contains a single Dβ and six Jβ gene segments.

The TCRα locus hews to a similar theme in that it is com-prised of approximately 100 V gene segments located within a 1.5 Mbp region (Figure 2D). At least 200 kbp separates the Vα regions from the Jα cluster. The TCRα locus is unusual in that it contains many more J regions as compared to other antigen receptor loci, with 61 Jα gene segments that span 65 kbp. Nested within the TCRα locus is the TCRδ locus, contain-ing numerous Vδ, two Dδ, and Jδ elements and one Cδ region. Unlike the TCRα, TCRβ, and TCRδ loci, the TCRγ locus is small (less than 200 kbp), containing few Vγ and Jγ gene segments (Figure 2F). Thus, the majority of antigen receptor loci are com-prised of large numbers of V regions that span a vast genomic region and numerous clustered D or J gene segments.

Lymphocyte Development and Antigen Receptor AssemblyPluripotent hematopoietic stem cells, which are capable of self-renewal, develop into lymphoid-primed multipotent pro-genitors (LMPPs) that lack long-term self-renewal capacity and have myeloid- or lymphoid-restricted differentiation potential. LMPPs have the ability to develop into common lymphoid pro-genitors, which, in turn, have the potential to differentiate to pre-pro-B cells. Pre-pro-B cells, in turn, develop into commit-ted pro-B cells, that initiate and complete Igh V(D)J gene rear-rangement. Rearrangement of antigen receptor loci is mediated by RAG-1 and RAG-2, which act to cleave DNA at recognition sites that flank the V, D, and J gene segments (Schatz and Spanopoulou, 2005). At the pro-B cell stage, DHJH joining pre-cedes that of VHDHJH gene rearrangement. Once a productive VHDHJH gene rearrangement has been generated, a pre-B cell receptor (pre-BCR) is assembled that acts, in turn, to inhibit the expression of RAG-1 and RAG-2 and promotes the survival and expansion of developing large pre-B cells. This prolifera-tion phase is followed by exit from the cell cycle, during which RAG gene expression is reinduced to enable Igκ gene rear-rangement. At the pre-B cell stage, Igκ VJ gene rearrangement is initiated. If the rearrangement is nonproductive or results in Igκ deletion, B cells can undergo Igλ rearrangement. B cells that express autoreactive receptors maintain RAG expression and promote secondary Igκ VJ rearrangements, a process


termed “receptor editing.” These secondary rearrangements can lead to replacement of rearranged Igκ loci with secondary productive rearrangements. Alternatively, nonfunctional rear-rangement or deletion of both Igκ alleles would permit Igλ VJ locus rearrangement to ensue. Predominantly, B cells express-ing Igλ can be generated from pre-B cells with two nonpro-ductive Igκ rearrangements or via receptor editing from Igκ+ cells (Nemazee, 2006). Thus, the Igh and Ig light chain loci are assembled sequentially during early B cell development.

The development of αβ and γδ T cells in the thymus is a process characterized by the sequential rearrangement of the gene segments of the T cell antigen receptor loci (TCR). Shortly after arriving in the thymus, T cell progenitors initiate TCRβ, TCRγ, and TCRδ loci rearrangement. The rearrangement of the TCRβ locus is initiated and completed at a developmen-tal stage that lacks the expression of the coreceptors for the TCR, CD4, and CD8, a population of cells commonly referred to as the double-negative stage. Upon rearrangement and expression of a productive TCRβ chain and its assembly into a pre-TCR complex, RAG expression is suppressed and thymo-cytes undergo developmental progression, characterized by rapid cellular expansion. During this phase, thymocytes begin to express CD8, followed by CD4. Thymocytes that express CD4 and CD8, referred to as double-positive cells, undergo cell-cycle arrest and initiate TCRα VJ gene rearrangement. VαJα rearrangements can be initiated multiple times, such that secondary TCRα rearrangements progressively can replace primary VαJα joints. The primary rearrangements predomi-nantly utilize the Vα gene segments positioned toward the 3′ end of the locus and the most 5′ Jα elements. Double-positive thymocytes then progress through the processes of positive and negative selection, allowing the maturation of only those cells that express TCRs with moderate affinity for self-major histocompatibility complexes expressed by thymic epithelial cells. Positively selected thymocytes decrease expression of either CD4 or CD8 to develop into mature CD8 or CD4 single-positive (SP) progeny.

Two distinct mechanisms have been described that ensure monoallelic antigen receptor rearrangement. First, antigen receptor rearrangement is monoallelically activated, which has been well characterized for the Igκ locus (Cedar and Bergman, 2008). Second, once a productive Igh or TCRβ V(D)J gene rear-rangement has been generated, signaling mediated by the pre-BCR or pre-TCR antagonizes continued rearrangement by a feedback mechanism (Jung and Alt, 2004; Krangel, 2007). As a result, only one copy of a functional antigen receptor gene is produced in a single lymphocyte. Thus, the adaptive arm of the immune system is generated by distinct cell types, which undergo ordered gene rearrangement, to provide each lym-phocyte with a single and unique antigen receptor.

Epigenetic Marking and Antigen Receptor Locus AssemblyThe antigen receptor chromatin fiber, both DNA and its associ-ated histones, is epigenetically marked during developmental progression. The Igκ locus is monoallelically demethylated at the DNA prior to the onset of VκJκ gene rearrangement, and the demethylated Igκ allele is also selectively targeted in germinal

center B cells by activation-induced deaminase (AID) (Cedar and Bergman, 2008). The amino-terminal tails of the core his-tones are also marked. These tails can be modified by lysine acetylation, arginine and lysine methylation, lysine ubiquitina-tion, and serine and threonine phosphorylation. Histone acety-lation correlates well with chromatin accessibility. Acetylation is catalyzed by histone acetyltransferases, whereas deacetyla-tion is mediated by the histone deacetylases. Methylation, in turn, serves to promote interactions of histones with factors involved in chromatin remodeling (chromodomains) as well as with plant homeodomain-containing proteins. The methylation state of histone lysine residues is mediated by methyltrans-ferases and histone demethylases.

Recent studies have demonstrated that the regulation of ordered and lineage-specific Igh locus assembly correlates well with chromatin structure and DNA recombination. Iso-lated lymphoid nuclei, upon incubation with the recombinase, show cleavage of recombination signal sequences in a lineage and developmental stage specific fashion, linking chromatin structure, RAG1 and RAG2 activity, and V(D)J gene rearrange-ment into a common framework (Stanhope-Baker et al., 1996). Although the precise mechanism remains to be determined, the common view is that enhancer elements and/or promoter regions liberate recombination signal sequences from a repres-sive chromatin environment to permit RAG1/RAG2-mediated cleavage (Golding et al., 1999; Kwon et al., 2000).

The ordered rearrangement of antigen receptor loci also correlates well with temporally restricted epigenetic mark-ing (Chowdhury and Sen, 2004). Three patterns of epigenetic marks are particularly interesting: First, prior to DHJH rear-rangement, the DH-CH region becomes acetylated. Second, VH gene segments are not associated with acetylated histones in progenitor B cells that have not undergone DHJH rearrange-ment. Third, Igh loci prone to undergo VH-DHJH rearrangement manifest elevated levels of VH histone acetylation (Chowdhury and Sen, 2004). The pattern of histone acetylation across the DH-JH region, however, is not uniform and is mainly restricted to the most 5′ and 3′ located DH elements. Interestingly, these DH gene segments are used most frequently in DHJH junctions (Chakraborty et al., 2007). Thus, it appears that Igh locus his-tone acetylation is regulated in a sequential manner. The DH-JH domain is hyperacetylated first, followed by DHJH rearrange-ment. Subsequently, VH gene segments become activated by acetylation to permit VHDHJH joining.

The observations described above indicate a correlation between epigenetic marking and antigen receptor assembly. Recently, a direct link between these two processes has been established in a series of experiments that are particularly illu-minating. Specifically, methylation of histone H3 (H3K4me3 and H3R2me2) upon interacting with RAG-2 acts to enhance DNA binding and enzymatic activity of the recombinase (Liu et al., 2007a; Matthews et al., 2007; Ramón-Maiques et al., 2007; Shimazaki et al., 2009). This interaction requires a noncanoni-cal PHD domain that is located within the noncore domain of RAG-2. At first glance, these data raise the possibility that the histone code may act to promote the targeting of the recom-binase to the recombination signal sequences. However, epi-genetic marking of H3K4 and H3R2 by methylation is associ-

ated with transcriptional initiation and definitely not limited to H3 residues that are positioned within close proximity to the recombination signal sequences. Additional specificity is prob-ably provided by the interaction involving RAG1 and recombi-nation signal sequences.

How are histone marks established and removed during devel-opmental progression? Two factors, PAX5 and EZH2, are poten-tial players. PAX5 is a paired homeodomain-containing protein that plays a central role in B cell commitment. It is dispensable for DHJH and proximal VH-DHJH rearrangements, but it is absolutely required for distal VH-DHJH joining (Fuxa et al., 2004). H3K9 methy-lation epigenetically marks VH gene segments in a repressive state in hematopoietic cells that are not committed to the B cell lineage (Johnson et al. 2004). Prior to VHDHJH rearrangement, H3K9 resi-dues become demethylated in the VH locus in a Pax-5 dependent manner. EZH2 is a polycomb group protein acting as a H3K27 histone methyltransferase (Su et al., 2003). EZH2-deficient pro-B cells show a defect in distal VH-DHJH rearrangements. It has yet to be determined how EZH2 mechanistically acts to promote distal VH-DHJH rearrangement.

Recent studies demonstrate a role for noncoding RNAs in modulating histone tails. Among the first noncoding RNAs to be identified were germline transcripts, also named sterile tran-scripts (Yancopoulos and Alt, 1985). A direct role for noncoding RNAs in modulating epigenetic marking and antigen receptor assembly has been demonstrated for the TCRα locus. As men-tioned earlier, the TCRα locus is unusual in that it permits multiple rearrangements to occur, in which secondary VαJα rearrange-ments can progressively replace primary VαJα joints through recombination of 5′ Vα segments with 3′ Jα elements, until an αβ TCR has been generated that is capable of interacting with members of the major histocompatibility complex. The targeting of RAG proteins to the 5′ Jα regions requires the transcription of noncoding RNAs (Abarrategui and Krangel, 2006). Further-more, suppression of transcriptional elongation substantially interferes with the trimethylation of H3K4 at Jα gene segments localized downstream, possibly interfering with the targeting of the recombinase to the recombination signal sequence. These studies are of interest as they directly link noncoding RNAs, epi-genetic marking, and antigen receptor assembly.

Nuclear Topology and Immunoglobulin Locus AssemblyThe localization of genes varies during developmental pro-gression (Schneider and Grosschedl, 2007). Such reposition-ing of loci during differentiation is often conserved through-out evolution. A prominent example involves multiple genes in yeast that appear to cluster at the nuclear membrane-nuclear lamina (Akhtar and Gasser, 2007). A subset of genes that are undergoing active transcription is associated with nuclear pores, whereas others, which are associated with the inner nuclear membrane, appear to be transcriptionally silenced. In mammalian cells, the expression of the β-globin locus is initiated at the nuclear membrane prior to its movement toward more centrally located domains (Ragoczy et al., 2006). A second nuclear compartment involves the pericentromeric heterochromatin. The association of genes with pericentro-meric heterochromatin is often correlated with transcriptional silencing.


Evidence indicating that the assembly of Ig loci is also regulated by nuclear location is accumulating. The Igh locus in hematopoi-etic progenitors associates with the inner nuclear lamina (Kosak et al., 2002). The distal VH region cluster is tethered to the nuclear membrane, whereas the DHJH elements are located away (Fig-ure 3) (Roldan et al., 2005). Notice that in such an arrangement, the spatial orientation of the Igh locus may allow the RAG-1 and RAG-2 proteins access to the DH-JH domain while the VH cluster remains closed. The presence of DHJH rearrangements but the complete absence of VHDHJH joints in hematopoietic progenitors is consistent with such a topology.

Upon commitment to the B cell lineage, the Igh locus undergoes relocation away from the nuclear periphery and large-scale contraction, followed by VH-DHJH gene rearrange-ment (Kosak et al., 2002, Fuxa et al., 2004; Roldan et al., 2005; Sayegh et al., 2005). How are conformational alterations in antigen receptor loci established during developmental pro-

gression? Plausibly, this will involve the introduction of constraints in antigen receptor loci, promoted by tethering. However, we suggest that in differentiat-ing cells, induced changes in the flexibil-ity of the chromatin fiber also contribute. For example, a decrease in the persis-tence length will affect the frequency of interaction between distant genomic regions. Localized reduction in the per-sistence length may be achieved by his-tone modifications spanning the fiber. It is possible that germline transcription, observed when antigen receptor loci are active, may induce such modifica-tions. Rather than targeting histone modifications at a regulatory sequence, germline transcription may introduce epigenetic marks across a large region

as the polymerase scans the fiber. Such changes in flexibility would affect the ensemble of conformations adopted by the chromatin fiber.

Once a productive VH-DHJH rearrangement has occurred, the Igh locus undergoes decontraction. The nonfunctional Igh allele relocates to pericentromeric heterochromatin, where it transiently interacts with an Igκ allele (Roldan et al., 2005; Hewitt et al., 2008) (Figure 3). The interactions between the Igh and Igκ alleles are intriguing. Interphase chromosomes are not dispersed throughout the nucleus but rather are posi-tioned within distinct areas. Small chromosomes appear to preferentially localize to internal locations, while large chro-mosomes seem to localize mainly in peripheral areas (Bolzer et al., 2005). However, individual chromosomes are not entirely separated, given that different chromosomes intermingle, best characterized in nucleoli, heterochromatic regions, and now including the Igh and Igκ loci.

Figure 3. Location and Conformation of Antigen Receptor Loci in Developing LymphocytesNuclear locations of antigen receptor loci during developmental progression are indicated.(A) Indicated are the nuclear positions of the Igh and Igκ alleles at various stages of early B cell development. Pre-pro-B and pro-B cell stage are shown in blue. Large pre-B and small pre-B are indicated in green. Immature-B cells are shown in red. Blue clusters of loops indicate V regions. Red clusters of loops represent D/J/C coding elements. Dark dots represent the pericentromeric hetero-chromatin. The relative degree of Igh and Igκ locus contraction and decontraction is depicted.(B) Indicated are the nuclear positions of the TCRβ and TCRα alleles during thymopoieisis. The dou-ble-negative cell stage is shown in blue. Double-positive cells are in green, and single-positive cells are in red. Blue clusters of loops indicate V regions. Red clusters of loops represent D/J/C coding elements. Dark dots represent the pericen-tromeric heterochromatin. The relative degree of TCRα and TCRβ contraction and decontraction are depicted.


The decontraction of antigen receptor loci has been pos-tulated to suppress further rearrangement and to enforce the allelic exclusion mechanism (Roldan et al., 2005). However, the spatial distances separating the distal and proximal VH regions from the DHJH or JH gene segments have not been directly determined in cells that express a productive rear-rangement (Figure 3). Thus, we are faced with the question, does decontraction take place within the entire VH repertoire or only within the distal VH regions? If it is the latter scenario, then decontraction is not an attractive mechanism to ensure allelic exclusion given that after feedback inhibition the major-ity of proximal VH regions would have the same probabilities of encountering a DHJH or JH gene segment as prior to pre-BCR expression. Perhaps a more efficient mechanism to ensure the allelic exclusion process is the recruitment of the non-productive Ig allele to the repressive pericentromeric region (Goldmit et al., 2005; Roldan et al. 2005; Hewitt et al., 2008).

The Igκ locus is also relocated during B cell development. In pre-pro-B cells, the Igκ locus interacts with the nuclear membrane (Kosak et al., 2002) (Figure 3A). In committed, pro-B cells, the Igκ loci move away from the nuclear mem-brane to more centrally located nuclear domains (Figure 3A). Subsequently, in pre-B cells one of the Igκ alleles becomes associated with the repressive environment of pericentro-meric chromatin to favor rearrangement of the euchromatic Igκ allele (Goldmit et al., 2005). How is one of the Igκ alleles selectively recruited to the heterochromatin? Allelic replica-tion asynchrony at the Igh and Igκ loci is established early at the time of implantation as demonstrated by early- and late-replicating alleles (Mostoslavsky et al. 2001). The late-replicating Igκ locus appears to selectively associate with centromeric heterochromatin. Thus, pre-existing epigenetic marks may target one of the Igκ alleles to the centromeric heterochromatin to promote allelic asynchrony.

We are now faced with the question as to how the silenced allele associated with the heterochromatin becomes acti-vated when the first allele is nonproductively rearranged. As all associations are in flux, we suggest that the silenced allele is not permanently associated with the heterochro-matin. Perhaps the equilibrium between the “free” and the “bound” state of Igκ alleles is temporally regulated during the developmental progression from the pre-B to the imma-ture-B cell stage, allowing Igκ VJ rearrangement to proceed on the second allele if the first allele fails to generate a pro-ductive rearrangement. In principle, this could be achieved by a gradient of a transcription factor(s) that modulate the interaction of Igκ alleles with the heterochromatic environ-ment. It is the dynamics of such interactions that are critical and it seems that in vivo imaging approaches should permit a resolution to this intriguing problem.

What are the molecular players that modulate the associa-tion of the Igκ alleles with the heterochromatic environment? One candidate is IRF-4, which directs the Igκ allele away from the pericentromeric heterochromatin (Johnson et al., 2008). Another possible player is the transcriptional regulator Ikaros. Ikaros associates with a regulatory element in the Igκ locus, named Sis. Sis is required for the repositioning of the Igκ locus to the pericentromeric heterochromatin (Liu et al., 2006).

In sum, Ig loci move around in the nucleus during B cell maturation and associate with different nuclear structures. What is the purpose of moving the Ig loci around? Has unambiguous evidence been reported demonstrating that gene activity and nuclear location are linked? The observa-tions described above indicate a correlation between anti-gen receptor location and antigen receptor assembly. How-ever, different results have been documented regarding the physiological roles for positioning of genes near the nuclear membrane versus more centrally located domains. Position-ing of a reporter gene at the nuclear membrane upon inter-action with lamin B did not suppress transcriptional acti-vation upon stimulation (Kumaran and Spector, 2008). On the other hand, upon tethering a reporter construct to the inner nuclear membrane protein emerin, the expression of a reporter was modestly but significantly suppressed (Reddy et al. 2008). These differences likely reflect the different experimental strategies that were employed, but they do indicate that interaction with the nuclear periphery, despite the repressive environment, is not sufficient to completely suppress transcriptional activation. The full meaning of Ig repositioning will need to be addressed by mutational analy-sis of DNA elements that are responsible for the associa-tions with distinct nuclear domains by gene targeting (Hewitt et al., 2008).

Nuclear Location and TCR Locus AssemblyThe assembly of TCR loci also appears to be regulated by nuclear location. TCRα and TCRβ loci undergo contraction in cells that are poised for DNA recombination, and this pattern is reversed upon developmental maturation (Skok et al., 2007) (Figure 3). Additionally, monoallelic association of antigen receptor genes with pericentromeric chromatin has also been observed in TCRβ loci during thymocyte maturation. The ini-tial observations suggested a monoallelic interaction of the TCRβ alleles with pericentromeric heterochromatin (Skok et al., 2007). Another study, however, concludes that both TCRβ alleles associate independently of each other and not strictly monoallelically with the nuclear lamina and pericentromeric heterochromatin (Schlimgen et al., 2008). However, an inher-ent bias exists for one of the alleles to localize to nuclear lamina or pericentromeric heterochromatin throughout early T cell development. The precise mechanism by which TCRβ alleles are recruited to the nuclear lamina or pericentromeric heterochromatin remains to be determined. Either mecha-nism would diminish the probability that TCRβ V(D)J gene rearrangements are initiated simultaneously on both alleles and may buy developing thymocytes sufficient time to induce suppression of rearrangement on the nonrearranged allele by a feedback mechanism. In contrast to the Igh, Igκ, and TCRβ loci, the TCRδ and TCRα loci do not appear to associate with pericentromeric heterochromatin, consistent with the notion that both alleles simultaneously undergo TCR assembly in double-negative and double-positive cells, respectively (Skok et al., 2007) (Figure 3).

What are the molecular players that contribute to the inter-action of antigen receptor loci with pericentromeric hetero-chromatin? The helix-loop-helix protein, E47, is a good can-


didate. E47 dosage is rate-limiting with regard to TCRβ V(D)J rearrangement, and forced E47 expression interferes with pre-TCR-mediated feedback inhibition (Agata et al., 2007).

What is the mechanistic basis for silencing TCRβ VβDβJβ DNA recombination in a heterochromatic environment? Germline transcription at the TCRβ locus is biallelic, that is, functional and nonfunctional alleles are both transcribed (Jia et al., 2007). Igκ germline transcription is also biallellic (Singh et al., 2003). It is intriguing that recruitment to the peri-centromeric heterochromatin does not completely silence antigen receptor noncoding RNA transcription, contrary to expectations. These observations bring into question as to how VβDβJβ as well as VκJκ assembly is suppressed at the pericentromeric heterochromatic environment. Perhaps his-tone marks that permit recruitment of the RAG proteins to the recombination signal sequences or the RAG proteins them-selves are excluded from the heterochromatic environment. Clearly, substantial progress has been made, but the precise mechanism that underpins monoallelic antigen receptor acti-vation remains unresolved.

Ordered Assembly of Antigen Receptor Loci and Chromatin TerritoriesDuring lymphocyte development, the rearrangement of anti-gen receptor loci is ordered. Igh DH-JH and TCR Dβ-Jβ rear-rangement are initiated prior to that of V-DJ gene joining (Alt et al. 1984). Once a productive Igh or TCRβ V(D)J gene rear-rangement has been generated, signaling mediated by the pre-BCR or pre-TCR antagonizes continued rearrangement by a feedback mechanism, ensuring allelic exclusion (Jung and Alt, 2004; Krangel, 2007). Feedback signaling, however, does not suppress rearrangement of the entire Igh region repertoire. The four most proximal VH regions escape the allelic exclu-sion mechanism (Costa et al., 1992). Thus, we are faced with the question, what might explain these differences in V region usage?

Functional chromosomal domains may provide a means by which regions undergoing DNA recombination are distin-guishable from regions not undergoing DNA rearrangement. Such functional domains have been described for the chicken β-globin cluster, which is marked by the presence of bound-ary elements, containing CTCF binding sites (Felsenfeld et al., 2004). To determine whether such functional domains exist within antigen receptor loci, VH regions, normally distally located and subject to the allelic exclusion mechanism, were inserted in a chromosomal position immediately 5′ of the D-J cluster (Bates et al., 2007). Interestingly, the targeted VH regions appear to rearrange with substantially higher frequency than their endogenous counterparts. Furthermore, targeting of the VH region to a location immediately 5′ of the DH-JH region reveals that the ordered rearrangement process is perturbed as well (Bates et al., 2007). As suggested previously, these data are consistent with a model in which the majority of VH and DH domains are located in functionally separate territories (Bates et al., 2007).

Compartmentalization of antigen receptor loci into func-tional domains may also explain the underlying mechanism that permits the stage-specific rearrangement of TCRα and


TCRδ loci. The TCRδ elements are embedded within the TCRα locus. The TCRδ locus undergoes DNA recombination in double-negative thymocytes, whereas TCRα gene rearrange-ments are not induced prior to the double-positive stage. The mutually exclusive rearrangement patterns of TCRα and TCRδ loci suggest that they are located in separate chromatin territories that become accessible at distinct stages of thy-mocyte development.

The process of ordered rearrangement correlates well with the expression of noncoding RNAs. Noncoding RNAs initiate from V region promoters after DHJH rearrangement (Yanco-poulos and Alt, 1985). Furthermore, in pro-B cells, antisense noncoding RNAs traverse across the entire DH-JH region prior to DH-JH rearrangement (Bolland et al., 2007). Once DH-JH rear-rangements have been completed, biallelic antisense tran-scription is initiated across the VH region cluster (Bolland et al., 2004). How does antisense transcription promote ordered Igh locus DNA recombination? It is conceivable that antisense transcription across the Igh locus acts to promote chroma-tin accessibility in a manner similar as that described above for the TCRα VJ gene rearrangement. Alternatively, germline transcripts may alter the three dimensional structure of dis-tinct chromatin territories to promote localized accessibil-ity, independent of epigenetic marking. Interesting work has recently shown an architectural role for a noncoding RNA that is required for the formation of paraspeckels, nuclear struc-tures whose precise function remain to be resolved (Clemson et al., 2009). We think that the potential role of noncoding RNAs in modulating chromatin topology is of particular interest. It is conceivable that they modulate the higher order folding of the antigen receptor loci into separate domains to promote spatial proximity and to establish boundaries. Targeting of noncoding RNAs combined with geometric approaches may reveal their role in the organization of genome structure.

Chromatin Territories as Physical EntitiesFluorescence in situ hybridization studies suggest the pres-ence of chromatin domains within the Igh locus (Jhunjhunwala et al., 2008). In pre-pro-B cells, each allele appears as two to three clusters of fluorescence that are connected by linkers, whereas in the large majority of pro-B cells, only one such cluster is detectable. However, visualization of distinct clusters by fluorescence is limited by the resolution of the technique and does not necessarily imply functional chromatin compart-ments. An alternative strategy would be to define functional compartments in terms of epigenetic histone marks, for exam-ple present at boundary elements, as described for the chicken β-globin locus (Felsenfeld et al. 2004). But this also may not be a general strategy to define chromatin domains. Chromatin com-partments can also be viewed as physical units. Two genomic markers are localized in one compartment if they coordinately motion toward or away from an anchor localized in a separate compartment (Figure 4). Here, we would like to consider three distinct possible arrangements, named the V, L, and O config-urations (Figure 4). In a V configuration, two DNA segments are located in one compartment, whereas an anchor is localized in a separate compartment. In a V arrangement, two markers move coordinately away from or toward the anchor (Figure 4).

In an L configuration, the anchor is located in a compartment together with another genomic marker, whereas the second marker is positioned in a separate compartment. In an L con-figuration, both markers motion independently toward or away from the anchor (Figure 4). In an O configuration, the anchor and both genomic markers are located in one compartment or alternatively in three distinct compartments and motion inde-pendently from each other (Figure 4). Thus, chromatin territo-ries could be viewed as physical units in which DNA regions move coordinately toward or away from an anchor located in another unit.

Using triple-point spatial distance measurements, as out-lined above, it should be possible to determine whether genomic markers within the most 5′ VH regions show coordi-nate movement toward or away from an anchor located in a dif-ferent compartment. Boundaries that separate compartments could also be identified with this approach. Such a strategy would permit the resolution of a few interesting questions: Do chromatin boundaries separate VH regions that are allelically excluded from other VH regions that are not subject to the allelic exclusion mechanism? Do these boundaries correlate with the cluster of CTCF binding sites that separate the DH from the VH domains? Are boundaries, separating the V, D, or J domains, also present in other antigen receptor loci? Are the TCRα and TCRδ loci located in distinct functional chromosomal domains? By defining compartments as physical units, it should be pos-sible to determine to what degree antigen receptor loci are compartmentalized and where the borders are located that separate such domains.

Immunoglobulin Heavy Chain Locus TopologyThe antigen receptor loci are more than just linear DNA sequences. Thus, we are faced with these questions: How are the antigen receptor loci organized in three dimensional space? What is the fundamental mechanism that gives rise to antigen receptor locus topology? How do the topologies of antigen receptor loci relate to function? Recent studies have provided some insight into these questions. As a first approach, spatial distances measurements as a function of genomic separation showed that the Igh locus topology could not be described in terms of the self-avoiding random walk, the worm-like chain, or the RW/GL model (Figure 1) (Jhunjhunwala et al., 2008). Rather, this analysis showed substantial agreement between pre-pro-B Igh locus topology and simulated MLS conformations. The spatial distances as a function of genomic distances in pro-B cells also agreed well with those predicted by the MLS model but only for genomic markers separated by less then 1 Mbp. However, beyond a genomic separation of 1 Mbp, the spatial distances leveled off in pro-B cells and did not compare well with the MLS configuration (Jhunjhunwala et al., 2008). Thus, it appears that the Igh locus topology shows substantial, but not complete, agreement with simulated MLS configurations.

The MLS model predicts a rosette-like configuration for the chromatin fiber. In itself, this notion is not novel. Rosette-like structures have been observed previously with electron micros-copy as well as formaldehyde crosslinking approaches. They were originally observed in preparations derived from both mitotic and interphase chromosomes (Paulson and Laemmli,

1977; Okada and Commings, 1979). Aggregates of loops have also been suggested to underlie the observations obtained by chromosome-conformation capture studies in the T helper type-2 cytokine locus. Notably, SATB1 (special AT-rich-binding protein 1) is proposed to be required for a multiloop-contain-ing structure (Cai et al., 2006). A similar structure, also based on formaldehyde crosslinking studies, has been proposed to underpin the organization of the bithorax complex (Lanzuolo et al., 2007).

The comparison of experimental with simulated data pre-dicts that the chromatin fiber at the Igh locus in pre-pro-B cells is organized into 1 Mbp rosettes, containing on average 120 kbp loops, which are separated by 60 kbp linkers. Does this analysis imply that the Igh locus fiber is structured into evenly sized loops that fold back in a regular and fixed pattern to loop attachment points, forming a perfect rosette? Not likely. Rather, we would like to suggest that the chromatin fiber folds into loops of variable sizes, that loop size is determined by bridg-ing factors, and that loop formation is dynamic. Such a con-figuration has been proposed, for large genomic separations

Figure 4. Genomic Elements and Chromatin TerritoriesPositioning of genomic markers in distinct compartments using triple-point spatial distance measurements in individual cells. Two genomic markers are located in one compartment if they move coordinately toward or away from an anchor. Nuclear subcompartments are depicted as gray discs. Genomic markers within different subcompartments are shown in red, green, or blue. Spatial distances from the anchor (green) are shown as red or blue, respec-tively. Various configurations are indicated. V configuration: Red and blue markers are in the same subcompartment, whereas anchor (green) is posi-tioned in a separate subcompartment. The red and blue markers move coor-dinately away from the anchor. L configuration: One of the markers (blue) is in the same subcompartment as the anchor (green). The distance separating the blue marker from the anchor is independent of the distance separating the red marker from the anchor. O configuration: All three genomic markers are in localized in one compartment or alternatively in three distinct compartments. The markers move independently from each other.


(5–10 Mbp), in the RL model (Bohn et al., 2007). The continuous dynamic folding and unfolding of loops is attractive with regard to VH region gene usage as it would permit increased flexibility for VH regions that are located within close proximity of putative loop attachment points. Clearly, the precise arrangements of loops in the Igh locus are yet to be determined. However, as a working model, we envision that the Igh locus folds into chro-matin compartments, containing aggregates of loops, which fold and unfold in elaborate and dynamic patterns.

Modulators of Long-Range Chromatin StructureAll loops, including those present in rosettes, require a tether at their base. Putative tethers that span the entire Igh locus have been identified. Prominent among these are YY1 and CTCF. YY1 is a zinc-finger protein that is evolutionarily con-served from the fruit fly Drosophila melanogaster to humans. VH-DHJH rearrangement is severely perturbed in YY1-deficient pro-B cells affecting mainly the distal VH gene segments (Liu et al., 2007b). Interestingly, the Igh locus is decontracted in YY1-deficient pro-B cells, raising the possibility that YY1 acts to modulate Igh locus topology to promote distal VH-DHJH rear-rangement (Liu et al., 2007b).

How does YY1 promote Igh locus long-range chromatin contraction? The precise mechanism is unknown, but YY1 is of interest as it has been demonstrated to interact with CTCF (Donohoe et al., 2007). CTCF is a factor previously shown to promote looping within the β-globin locus (Splinter et al., 2006). In addition to interacting with YY1, recent genome-wide binding studies have revealed that CTCF also interacts with cohesins (Parelho et al., 2008). These data are intriguing as during DNA replication, the cohesins form a ring-like structure, surrounding and stabilizing the sister chromatid strands. Inter-estingly, in a recent elegant study, it is demonstrated that the cohesins also interact in cis to confine IFNG locus topology (Hadjur et al., 2009).

Is CTCF a reasonable candidate to act as a bridging fac-tor in antigen receptor loci? The binding pattern of CTCF in the Igh locus in pro-B cells is striking (Degner et al., 2009). A total of 53 CTCF binding sites span the entire VH region cluster but are largely absent within the DH-JH and CH gene segments (Degner et al., 2009). A few CTCF sites are located immediately upstream of the DHJH cluster and may form a boundary between the VH cluster and the DHJH gene segments. The binding of CTCF to sites present in the Igh locus is not pro-B cell specific, and it is unlikely by itself to account for the differences observed in pre-pro-B and pro-B Igh locus topology. However, Rad21, a component of the cohesin complex, was found to preferentially bind with CTCF in pro-B cells as compared to thymocytes and pre-B cells (Parelho et al. 2008; Degner et al. 2009). CTCF expres-sion has been conditionally ablated in developing thymo-cytes and peripheral T cells (Heath et al. 2008; Ribeiro de Almeida et al., 2009). Surprisingly, TCR antigen receptor assembly appears normal in the absence of CTCF (Heath et al., 2008).

Other candidates have emerged that might be involved in directly modulating Igh locus topology. Among these is Pax-5, a homeodomain protein that plays an essential role in B cell


commitment. Interestingly, the distal VH cluster in Pax5-defi-cient pro-B cells is decontracted, and it is this region that is severely perturbed in VHDHJH rearrangement (Fuxa et al., 2004; Roldan et al., 2005). How Pax5 promotes locus contraction remains to be established. It seems unlikely that it acts by itself, given that forced Pax5 expression in T lineage cells does not promote Igh locus contraction (Fuxa et al., 2004). Furthermore, in pre-B cells, Pax-5 abundance is high, whereas the Igh alleles are decontracted, arguing against a direct and dominant role for Pax-5 in locus contraction (Roldan et al., 2005). Ikaros is yet another player involved in modulating Igh locus topology. Ikaros not only activates Rag expression and Igh locus acces-sibility, but also appears to promote Igh locus contraction (Reynaud et al., 2008).

A protein involved in DNA repair, 53BP1, has recently been implicated in modulating antigen receptor topology. 53BP1 initially targets double-strand DNA breaks upon interact-ing with H2AX and methylated histone H4K20 or possibly H3K79, which are constitutive histone marks exposed upon break formation. 53BP1 promotes VαJα gene rearrangement (Difilippantonio et al., 2008). Interestingly, the TCRα locus is in an extended state in 53BP1-ablated as compared to wild-type thymocytes. These data raise the question as to how 53BP1 permits DNA elements, separated by large genomic distances, to interact with relatively high probability. Two fundamentally different models have been suggested to underpin 53BP1 activity. 53BP1 may accumulate at double-strand DNA breaks, and upon homo-oligomerization may act as a bridging factor (Difilippantonio et al., 2008). Alter-natively, 53BP1 may directly modulate chromatin dynamics, possibly by affecting the persistence length of the chromatin fiber (Dimitrova et al., 2008).

As pointed out above, changes in the flexibility of the chro-matin fiber may also contribute to conformational alterations. The majority of factors known to modulate Igh locus chro-matin structure, including Pax5, YY1, and Ikaros, also epi-genetically mark chromatin. As such marking may affect the persistence length and overall conformation, it is critical to determine the precise mechanism by which these factors act to regulate chromatin topology. Live-cell imaging and mea-surement of chromatin dynamics may resolve the question of localized changes in the flexibility of the chromosomal fiber at the Igh locus.

Thus, a few molecular components have been identified that modulate antigen receptor locus topology. The candi-dates identified likely represent only the tip of the iceberg. The challenge will be now to identify others and to find out how they collaborate to modulate chromatin structure and how they promote encounters between close and remote gene segments.

An Even Playing FieldApart from the four most proximal V regions, VH region usage is random and unbiased (Yancopoulos et al., 1984; Malynn et al., 1990; Gu et al., 1991; Love et al., 2000). Distal VH regions rearrange often and the frequency of VH region usage scatters uniformly throughout the distal cluster (Gu et al., 1991; Love et al., 2000). Thus, with the exception of VH

regions located within the immediate proximity of the DHJH cluster, no correlation has been established between Igh V region usage and genomic location. Consequently, nearby and remote V regions must have equal opportunities to make a connection with a DJ or J element. Thus, we are faced with the question as to how the gigantic range of antigen receptor diversity is generated during lymphocyte develop-ment. Specifically, why do V elements scattered over a vast genomic region rearrange with similar frequencies?

Recent studies have allowed insight into this question. Spa-tial distance measurements were performed across the entire Igh locus and analyzed to present a statistical view of Igh locus structure. Specifically, a geometric approach, named trilateration, was applied to determine the relative average three-dimensional (3D) coordinates of the VH, DH, JH, and CH gene segments in pre-pro-B and pro-B cells (Jhunjhunwala et al., 2008). Spatial distance measurements of different genomic markers across the Igh locus from a common set of four “reference” markers were used to determine the average 3D coordinates of all the genomic markers. The most strik-ing features of the topology are as follows: (1) Large-scale Igh locus conformational changes appear to accompany the transition from the pre-pro-B to the pro-B cell stage. (2) Most telling, in pro-B cells, the proximal and distal VH regions seem to merge and to be juxtaposed to the DHJH elements, enabling the entire VH region repertoire access to the DH-JH elements. It is important to emphasize that the variation in the chromatin fiber configuration is more fundamental than a statistical representation of the structure as revealed by tri-lateration. Ultimately, it is the ensemble of conformations that permit equal opportunities for V regions to connect with a DJ or J segment. Nevertheless, the geometric analysis leads to one cardinal conclusion: if it is assumed that spatial proximity directly relates to the probability of encounter, the entire VH region repertoire has similar access to the DHJH gene seg-ments in cells prone to undergo DNA recombination, provid-ing an equal playing field.

The comparison of cumulative frequency from experimen-tally derived spatial distributions and theoretical distribu-tions reveals that DH-JH gene segments undergo free random walk behavior, whereas the VH regions are spatially confined (Jhunjhunwala et al., 2008). Gene-targeting approaches have suggested that the large number of V regions directly compete for close encounters with DJ elements (Bassing et al., 2008). Thus, we suggest a scenario in which DJ or J elements wander freely, searching for a connection with competing V regions, until a productive rearrangement has been generated.

Are other antigen receptor loci structured in a similar fash-ion? Probably. The V gene segments in the Igκ, TCRβ, and TCRα loci are also scattered over a vast genomic region. Apart from a Vβ element located 3′ of the TCRβ Dβ-Jβ region, usage of the Vβ region is also randomized. The TCRβ locus has been shown to undergo contraction in the double-negative thymo-cyte compartment, but becomes decontracted upon devel-opmental progression into double-positive cells (Skok et al., 2007). Where the contraction and decontraction occur within the TCRβ locus remains to be resolved, but it is conceivable

that the Vβ regions have merged into one compartment in cells poised to undergo TCRβ gene rearrangement, perhaps in a manner similar to that described for the Igh locus.

In contrast to the Igh and TCRβ loci, Vα gene usage is not random (Krangel, 2007). It is conceivable that the TCRα chro-matin fiber folds into distinct territories to permit the develop-mental regulation of early versus late Vα and/or Jα regions. Alternatively, epigenetic marking may regulate or contribute to the ordered rearrangement of TCRα V gene segments. Related epigenetic marking may also underpin the nonrandom usage of Vκ elements (Feeney et al., 1997). Thus, the fundamental point is that antigen receptor topology permits equal opportu-nities for all V regions that are trying to make a connection with DJ or J gene segment, but epigenetic marking may contribute to the nonrandom usage of V regions.

Long-Range Genomic InteractionsIt is plausible that the topology described for the Igh locus is lim-ited to that of antigen receptor loci, given that these loci undergo DNA recombination and may have special requirements for close spatial proximity of paired DNA elements. However, we consider this unlikely, as spatial distances also level off as a function of genomic separation in a region centromeric of the Igh locus, as well as in other loci (Jhunjhunwala et al. 2008; Rauch et al., 2008; Mateos-Langerak et al., 2009). Thus, although a detailed analysis is required, we propose that the Igh topology reflects a general feature of antigen receptor loci, and possibly of large portions of the mammalian chromatin fiber. Such a topology mechanistically permits regulatory elements that are separated by large genomic distances, including enhancer and promoter elements, to be localized in close spatial proximity. This is not to say that the statistical structure described for the Igh locus is uniform throughout the genome. Likely, chromatin compart-ments will vary in the number of loops, the sizes of loops and linkers, the spacing between the bases of the loops, dynamics of loop formation, and the number of clusters of loops. Resolv-ing antigen receptor loci topologies at high resolution would be a major step forward. Furthermore, with respect to antigen recep-tor diversification and promoter-enhancer interactions, it is the seemingly aimless wandering of the eukaryotic chromatin fiber that is especially intriguing. Within a given lymphocyte popula-tion, a wide spectrum of conformations must exist that permit equal opportunities for the entire repertoire of antigen recep-tor gene segments. In his eloquent and prescient book “What is Life?” the physicist Erwin Schrödinger likened chromosome structure to a finely embroidered Raphael tapestry. Indeed such “Raphael-like trajectories” are not completely randomly struc-tured. Rather these patterns reflect “an elaborate, coherent and meaningful design” that underpins the generation of antigen receptor diversity.

ACknOwLEDGMEnTs

We are grateful to D. Forbes, A. Goldrath, M. Levine, G. Arya, B. McGinnis, R. Riblet, anonymous reviewers, and members of the Murre laboratory for com-ments on the manuscript. We especially thank T. Knoch for many stimulating discussions, T. Hwa for suggesting that we perform triple-point measure-ments and define chromatin compartments as physical units, and S. Cutchin for lessons in geometry. We thank D. Heermann for making us aware of the “random loop” model. We thank the National Institutes of Health for support.


REFEREnCEs

Abarrategui, I., and Krangel, M.S. (2006). Regulation of T cell receptor-alpha gene recombination by transcription. Nat. Immunol. 7, 1109–1115.

Agata, Y., Tamaki, N., Sakamoto, S., Ikawa, T., Masuda, K., Kawamoto, H., and Murre, C. (2007). Regulation of T cell receptor beta gene rearrangements and allelic exclusion. Immunity 27, 871–884.

Akhtar, A., and Gasser, S.M. (2007). The nuclear envelope and transcriptional control. Nat. Rev. Genet. 8, 507–517.

Alt, F.W., Yancopoulos, G.D., Blackwell, T.K., Wood, C., Thomas, E., Boss, M., Coffman, N., Rosenberg, N., Tonegawa, S., and Baltimore, D. (1984). Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 3, 1209–1219.

Bassing, C.H., Whitlow, S., Mostoslavsky, R., Yang-Iott, K., Ranganath, S., and Alt, F.W. (2008). Vβ cluster sequences reduce the frequency of primary Vβ2 and Vβ14 rearrangements. Eur. J. Immunol. 38, 2564–2572.

Bates, J.G., Cado, D., Nolla, H., and Schlissel, M.S. (2007). Chromosomal position of a Vh gene segment determines its activation and inactivation as a substrate for V(D)J recombination. J. Exp. Med. 204, 3247–3256.

Belmont, A.S., and Bruce, K. (1994). Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure. J. Cell Biol. 127, 287–302.

Bohn, M., Heermann, D.W., and van Driel, R. (2007). Random loop model for long polymers. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 76, 051805.

Bolland, D.J., Wood, A.L., Afshar, R., Featherstone, K., Oltz, E.M., and Corc-oran, A.E. (2007). Antisense intergenic transcription precedes Igh D-to-J re-combination and is controlled by the intronic enhancer Emu. Mol. Cell. Biol. 27, 5523–5533.

Bolland, D.J., Wood, A.L., Jhonston, C.M., Buntling, S.F., Morgan, G., Chaka-lova, L., Fraser, P.J., and Corcoran, A.E. (2004). Antisense intergenic transcrip-tion in V(D)J recombination. Nat. Immunol. 5, 630–637.

Bolzer, A., Kreth, G., Solovei, I., Koehler, D., Saracoglu, K., Fauth, C., Müller, S., Eils, R., Cremer, C., Speicher, M.R., and Cremer, T. (2005). Three-dimen-sional maps of all chromosomes in human male fibrobast nuclei and prometa-phase rosettes. PLoS Biol. 3, e157.

Brack, C., Hirama, M., Lenhard-Schuffer, R., and Tonegawa, S. (1978). A complete immunoglobulin gene is created by somatic recombination. Cell 15, 1–14.

Bystricky, K., Heun, P., Gehlen, L., Langowski, J., and Gasser, S.M. (2004). Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques. Proc. Natl. Acad. Sci. USA 101, 16495–16500.

Cai, S., Lee, C.C., and Kohwi-Shigematsu, T. (2006). SATB1 packages dense-ly looped, transcriptionally active chromatin for coordinated expression of cytokine genes. Nat. Genet. 38, 1278–1288.

Cedar, H., and Bergman, Y. (2008). Choreography of Ig allelic exclusion. Curr. Opin. Immunol. 20, 308–317.

Chakraborty, T., Chowdhury, D., Keyes, A., Jani, A., Subrahmanyam, R., Ivanova, I., and Sen, R. (2007). Repeat organization and epigenetic regulation of the DH-Cmu domain of the immunoglobulin heavy chain locus. Mol. Cell 27, 842–850.

Chowdhury, D., and Sen, R. (2004). Regulation of immunoglobulin heavy chain gene rearrangements. Immunol. Rev. 200, 182–196.

Clemson, C.M., Hutchinson, J.N., Sara, S.A., Ensminger, A.W., Fox, A.H., Chess, A., and Lawrence, J.B. (2009). An architectural role for a nuclear non-coding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol. Cell 33, 717–726.

Costa, T.E., Suh, H., and Nussenzweig, M.C. (1992). Chromosomal position of rearranging gene segments influence allelic exclusion in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 2205–2208.


de Gennes, P.G. (1979). Scaling Concepts in Polymer Physics (Ithaca, NY: Cornell University Press).

Degner, S.C., Wong, T.P., Jankevicius, G., and Feeney, A.J. (2009). Develop-mental stage-specific recruitment of cohesin to CTCF sites throughout immu-noglobulin loci during B lymphocyte development. J. Immunol. 182, 44–48.

Difilippantonio, S., Gapud, E., Wong, N., Huang, C.Y., Mahowald, G., Chen, H.T., Kruhlak, M.J., Callen, E., Livak, F., Nussenzweig, M.C., et al. (2008). 53BP1 faciliates long-range DNA end-joining during V(D)J recombination. Nature 456, 529–533.

Dimitrova, N., Chen, Y.M., Spector, D.L., and de Lange, T. (2008). 53BP1 pro-motes non-homologous end joining of telomeres by increasing chromatin mo-bility. Nature 456, 524–528.

Donohoe, M.E., Zhang, L.F., Xu, N., Shi, Y., and Lee, J.T. (2007). Identification of a CTCF cofactor, YY1, for the X chromosome binary switch. Mol. Cell 25, 43–56.

Dreyer, W.J., and Bennett, J.C. (1965). The molecular basis of antibody forma-tion: a paradox. Proc. Natl. Acad. Sci. USA 54, 864–869.

Feeney, A.J., Lugo, G., and Escuro, G. (1997). Human cord blood kappa rep-ertoire. J. Immunol. 158, 3761–3768.

Felsenfeld, G., Burgess-Beusse, B., Farrell, C., Gaszner, M., Ghirlando, R., Huang, S., Jin, C., Litt, M., Magdinier, F., Mutskov, V., et al. (2004). Chromatin boundaries and chromatin domains. Cold Spring Harb. Symp. Quant. Biol. 69, 245–250.

Fuxa, M., Skok, J., Souabni, A., Salvagiotto, G., Roldan, E., and Busslinger, M. (2004). Pax5 induces V-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev. 18, 411–422.

Golding, A., Chandler, S., Ballestar, E., Wolffe, A.P., and Schlissel, M.S. (1999). Nucleosome structure completely inhibits in vitro cleavage by the V(D)J re-combinase. EMBO J. 18, 3712–3723.

Goldmit, M., Ji, Y., Skok, J., Roldan, E., Jung, S., Cedar, H., and Bergman, Y. (2005). Epigenetic ontogeny of the IgK locus during B cell development. Nat. Immunol. 6, 198–203.

Gu, H., Tarlington, D., Muller, W., Rajewsky, K., and Forster, I. (1991). Most peripheral B cells in mice are ligand selected. J. Exp. Med. 173, 1357–1371.

Hadjur, S., Williams, L.M., Ryan, N.K., Cobb, B.S., Sexton, T., Fraser, P., Fish-er, A.G., and Merkenschlager, M. (2009). Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus. Nature 10.1038/nature09079.

Heath, H., Ribeiro de Almeida, C., Sleutels, F., Dingjan, G., van de Nobe-len, S., Jonkers, I., Ling, K.W., Gribnau, J., Renkawitz, R., Grosveld, F., et al. (2008). CTCF regulates cell cycle progression of alphabeta T cell in the thymus. EMBO J. 27, 2839–2850.

Hewitt, S.L., Farmer, D., Marszalek, K., Cadera, E., Liang, H.E., Xu, Y., Schlis-sel, M.S., and Skok, J.A. (2008). Association between the Igk and Igh immu-noglobulin loci mediated by the 3′ Igk enhancer induces ‘decontraction’ of the Igh locus in pre-B cells. Nat. Immunol. 9, 396–404.

Jia, J., Kondo, M., and Zhuang, Y. (2007). Germline transcription from T-cell re-ceptor Vβ gene is uncoupled from allelic exclusion. EMBO J. 26, 2387–2399.

Johnson, K., Pflugh, D.L., Yu, D., Hesselin, D.G., Lin, K., Bothwell, A.L.M., Thomas-Tikhonenko, A., Schatz, D.G., and Calame, K. (2004). B cell-specific loss of histone 3 lysine 9 methylation in the VH locus depends on Pax5. Nat. Immunol. 5, 853–861.

Johnson, K., Hashimshony, T., Sawal, C.M., Pongubala, J.M.R., Skok, J., Aifantis, I., and Singh, H. (2008). Regulation of immunoglobulin light chain recombination by the transcription factor IRF-4 and the attenuation of inter-leukin-7 signaling. Immunity 28, 335–345.

Jhunjhunwala, S., van Zelm, M.C., Peak, M., Cutchin, S., Riblet, R., van Don-gen, J.J.M., Grosveld, F., Knoch, T.A., and Murre, C. (2008). The 3D-structure of the immunoglobulin heavy chain locus: implications for long-range genom-ic interactions. Cell 133, 265–279.

Jung, D., and Alt, F.W. (2004). Unraveling V(D)J recombination: insights into gene regulation. Cell 116, 299–311.

Knoch, T.A., Münkel, C., and Langowski, J. (2000). Three-dimensional orga-nization of chromosome territories in the human interphase nucleus. In High Performance Computing in Science and Engineering 1999, E. Krause and W. Jäger, eds. (Heidelberg, Germany: Springer), pp. 229–238.

Kosak, S.T., Skok, J.A., Medina, K.L., Riblet, R., Beau, M.M.L., Fisher, A.G., and Singh, H. (2002). Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296, 158–162.

Krangel, M.S. (2007). T cell development: better living through chromatin. Nat. Immunol. 8, 687–694.

Kratky, O., and Porod, G. (1949). Röntgenuntersuchung gelöster Faden-moleküle. Recueil des Travaux Chimiques des Pays-Bas 68, 1106–1133.

Kumaran, R.I., and Spector, D.L. (2008). A genetic locus targeted to the nu-clear periphery in living cells maintains its transcriptional competence. J. Cell Biol. 180, 51–65.

Kwon, J., Imbalzano, A.N., Matthews, A., and Oettinger, M.A. (2000). Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleav-age of nucleosomal DNA. Mol. Cell 6, 1037–1048.

Langowski, J., and Heermann, D. (2007). Computational modelling of the chromatin fiber. Semin. Cell Dev. Biol. 18, 659–667.

Lanzuolo, C., Roure, V., Bantignies, F., and Orlando, V. (2007). Polycomb re-sponse elements mediate the formation of chromosome higher-order struc-tures in the bithorax complex. Nat. Cell Biol. 9, 1167–1174.

Liu, Z., Widlak, P., Zou, P., Xiao, F., Oh, M., Li, S., Chang, M.Y., Shay, J.W., and Garrard, W.T. (2006). A recombination silencer that specifies heterochromatin positioning and Ikaros association in the immunoglobulin K locus. Immunity 24, 405–415.

Liu, Y., Subrahmanyam, R., Charaborty, T., Sen, R., and Desiderio, S. (2007a). A plant homedomain in Rag-2 that binds hypermethylated lysine 4 of histone H3 is necessary for efficient antigen-receptor-gene rearrangement. (2007). Immunity 27, 561–571.

Liu, H., Schmidt-Supprian, M., Shi, Y., Hobeika, E., Barteneva, N., Jumaa, H., Pelanda, R., Reth, M., Skok, J., Rajewsky, K., and Shi, Y. (2007b). Yin Yang 1 is a critical regulator of B cell development. Genes Dev. 21, 1179–1189.

Love, V.A., Lugo, G., Merz, D., and Feeney, A.J. (2000). Individual Vh promot-ers vary in strength, but the frequency of rearrangement of those Vh genes does not correlate with promoter strength nor enhancer-independence. Mol. Immunol. 37, 29–36.

Malynn, B.A., Yancopoulos, G.D., Barth, J.E., Bona, C.A., and Alt, F.W. (1990). Biased expression of JH-proximal Vh genes occurs in the newly generated repertoire of neonatal and adult mice. J. Exp. Med. 171, 843–859.

Mateos-Langerak, J., Bohn, M., de Leeuw, W., Giromus, O., Manders, E.M., Verschure, P.J., Indemans, M.H., Gierman, H.J., Heermann, D.W., van Driel, R., and Goetze, S. (2009). Spatially confined folding of chromatin in the inter-phase nucleus. Proc. Natl. Acad. Sci. USA 106, 3812–3817.

Matthews, A.G., Kuo, A.J., Ramón-Maiques, S., Han, S., Champagne, K.S., Ivanov, D., Gallardo, M., Carney, D., Cheung, P., Ciccone, D.N., et al. (2007). RAG2 PHD finger couples histone H3 lysine 4 trimethylation with VDJ recom-bination. Nature 450, 1106–1110.

Morawetz, H. (1985). Polymers. The Origins and Growth of a Science (New York: Dover Publications, Inc.).

Mostoslavsky, R., Singh, N., Tenzen, T., Goldsmit, M., Gabay, C., Elizur, S., Qi, P., Reubinoff, B.E., Chess, A., Cedar, H., and Bergman, Y. (2001). Asyn-chronous replication and allelic exclusion in the immune system. Nature 414, 221–225.

Münkel, C., and Langowski, J. (1998). Chromosome structure described by a polymer model. Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 57 (5B), 5888–5896.

Nemazee, D. (2006). Receptor editing in lymphocyte development and central

tolerance. Nat. Rev. Immunol. 6, 728–740.

Okada, T.A., and Commings, D.E. (1979). Higher order structure of chromo-somes. Chromosoma 72, 1–14.

Parelho, V., Hadjur, S., Spivakov, M., Leleu, M., Sauer, S., Gregson, H.C., Jar-muz, A., Canzonetta, C., Webster, Z., Nesterova, T., et al. (2008). Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell 132, 422–433.

Paulson, J.R., and Laemmli, U.K. (1977). The structure of histone depleted metaphase chromosomes. Cell 12, 817–828.

Pienta, K.J., and Coffey, D.S. (1984). A structural analysis of the nuclear matrix and DNA loops in the organization of the nucleus and chromosome. J. Cell Sci. Suppl. 1, 123–135.

Ramón-Maiques, S., Kuo, A.J., Carney, D., Matthews, A.G., Oettinger, M.A., Gozani, O., and Yang, W. (2007). The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2. Proc. Natl. Acad. Sci. USA 104, 18993–18998.

Ragoczy, T., Bender, M.A., Telling, A., Byron, R., and Groudine, M. (2006). The locus control region is required for association of the murine beta globin locus with engaged transcription factories during erythroid maturation. Genes Dev. 20, 1447–1457.

Rattner, J.B., and Lin, C.C. (1985). Radial loops and helixcal coils coexist in metaphase chromosomes. Cell 42, 291–296.

Rauch, J., Knoch, T.A., Solovei, I., Teller, K., Stein, S., Buiting, K., Horsthemke, B., Langowski, J., Cremer, T., Hausmann, M., and Cremer, C. (2008). Light optical precision measurements of the activate and inactive Prader-Willi syn-drome imprinted regions in human cell nuclei. Differentiation 76, 66–82.

Reddy, K.L., Zullo, J.M., Bertolino, E., and Singh, H. (2008). Transcriptional repression mediated by repositioning of genes to the nuclear lamina. Nature 452, 8846–8854.

Reynaud, D., Demarco, I.A., Reddy, K.L., Schjerven, H., Bertolino, E., Chen, Z., Smale, S.T., Winandy, S., and Singh, H. (2008). Regulation of B cell com-mitment and immunoglobulin heavy-chain gene rearrangements by Ikaros. Nat. Immunol. 9, 927–936.

Ribeiro de Almeida, C., Heath, H., Krpic, S., Dingjan, G.M., van Hamburg, J.P., Bergen, I., van de Nobelen, S., Sleutels, F., Grosveld, F., Galjart, N., and Hendriks, R.W. (2009). Critical roles for the transcriptional regulator CCCTC-binding factor in the control of Th2 cytokine expression. J. Immunol. 182, 999–1010.

Roldan, E., Fuxa, M., Chong, W., Martinez, D., Novatchkova, M., Busslinger, M., and Skok, J. (2005). Locus ‘decontraction’ and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat. Immunol. 6, 31–41.

Sachs, R.K., van den Engh, G., Trask, B., Yokota, H., and Hearst, J.E. (1995). A random-walk/giant-loop model for interphase chromosomes. Proc. Natl. Acad. Sci. USA 92, 2710–2714.

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

Schalch, T., Duda, S., Sargent, D.F., and Richmond, T.J. (2005). X-ray struc-ture of a tetranucleosome and its implications for the chromatin fibre. Nature 436, 138–141.

Schatz, D.G., and Spanopoulou, E. (2005). Biochemistry of V(D)J recombina-tion. Curr. Top. Microbiol. Immunol. 290, 49–85.

Schlimgen, R.J., Reddy, K.L., Singh, H., and Krangel, M.S. (2008). Initiation of allelic exclusion by stochastic interaction of TCRβ alleles with repressive nuclear compartments. Nat. Immunol. 9, 802–809.

Schneider, R., and Grosschedl, R. (2007). Dynamics and interplay of nucle-ar architecture, genome organization and gene expression. Genes Dev. 21, 3027–3043.

Sedat, J. and Manuelidis, L. (1977). A direct approach to the structure of eu-


karyotic chromosomes. Cold Spring Harb. Symp. Quant. Biol. 42, 331–350.

Seidman, J.G., Leder, A., Edgell, M.H., Polsky, F., Tilghman, S.M., Tiemeier, D.C., and Leder, P. (1978). Multiple related immunoglobulin variable-regioin genes identified by cloning and sequence analysis. Proc. Natl. Acad. Sci. USA 75, 3881–3885.

Shimazaki, N., Tsai, A.G., and Lieber, M.R. (2009). H3K4me3 stimulates the V(D)J RAG complex for both nicking and hairpinning in trans in addition to tethering in cis: implications for chromosomal translocations. Mol. Cell 34, 535–544.

Singh, N., Bergman, Y., Cedar, H., and Chess, A. (2003). Biallelic germline tran-scription at the kappa immunoglobulin locus. J. Exp. Med. 197, 743–750.

Skok, J.A., Gisler, R., Novatchkova, M., Farmer, D., de Laat, W., and Bus-slinger, M. (2007). Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes. Nat. Immunol. 8, 378–387.

Splinter, E., Heath, H., Kooren, J., Palstra, R.J., Klous, P., Galjart, N., and Grosveld, F. (2006). CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus. Genes Dev. 20, 2349–2354.

Stanhope-Baker, P., Hudson, K.M., Shaffer, A.L., Constantinescu, A., and Schlissel, M.S. (1996). Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell 85, 887–897.


Su, I.H., Basavarah, A., Krutchinsky, A.N., Hobert, O., Ullrich, A., Chait, B.T., and Tarakhovsky, A. (2003). Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement. Nat. Immunol. 4, 124–131.

Warrington, J.A., and Bengtsson, U. (1994). High-resolution physical mapping of human 5q31-q33 using three methods: radiation hybrid mapping, inter-phase fluorescence in situ hybridization, and pulsed-field gel electrophoresis. Genomics 24, 395–398.

Weigert, M., Gatmaitan, L., Loh, E., Schilling, J., and Hood, L. (1978). Re-arrangement of genetic information may produce immunoglobulin diversity. Nature 276, 785–790.

Yancopoulos, G.D., Desiderio, S.V., Paskind, M., Kearney, J.F., Baltimore, D., and Alt, F.W. (1984). Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines. Nature 331, 727–733.

Yancopoulos, G.D., and Alt, F. (1985). Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell 40, 271–281.

Yokota, H., van den Engh, G., Hearst, J.E., Sachs, R.K., and Trask, B.J. (1995). Evidence for the organization of chromatin in megabase pair-sized loops ar-ranged along a random walk path in the human G0/G1 interphase nucleus. J. Cell Biol. 130, 1239–1249.

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