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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: Curr Opin Immunol. 2012 Mar 15;24(2):153–159. doi: 10.1016/j.coi.2012.01.003

The role of CTCF in regulating V(D)J recombination

Julie Chaumeil 1, Jane A Skok 1
PMCID: PMC3444155  NIHMSID: NIHMS351169  PMID: 22424610

Abstract

V(D)J recombination in B and T cells is required for the generation of receptors with a broad spectrum of specificity to foreign antigen. A total number of three immunoglobulin (Ig) and four T cell receptor (Tcr) loci can be targeted by the recombinase enzyme (RAG1/2) in a defined series of recombination events, which drive the progression of B and T cell development. This process is regulated at multiple levels to ensure lineage specific, ordered rearrangement and allelic exclusion [1]. One key component of this is modulation of chromatin looping and locus contraction, which is important in bringing widely separated gene segments into close contact with each other to enable synapse formation for lineage and stage specific V gene rearrangement [2,3,4•,5,6•]. Recent studies provide new insight into looping and its role in these processes. In this review we focus on the contribution of the 11 zinc finger nuclear protein, CTCF, in mediating loop formation and conformational changes that are important for the regulation of Ig and Tcr rearrangement.

Introduction

Chromatin organization in eukaryotic cells is known to have a fundamental impact on gene regulation [7]. Classical studies combining genetic analyses and DNA/RNA FISH provided the first insights into the organization of genes relative to each other and to nuclear compartments and chromosome territories. The antigen receptor loci have afforded a rich model system for studying locus dynamics. DNA FISH studies first demonstrated that large-scale locus contraction (which occurs through chromatin looping) is known to be a common feature of V(D)J recombination [2,3]. Several factors, including the B cell specific transcription factor, Pax5, the polycomb group protein Ezh2, Ikaros and Yin Yang 1 (YY1) were identified as being required for Igh contraction and distal VH gene rearrangement [4•]. Further painstaking FISH analyses by Kees Murre’s lab elucidated the fine details of the Igh looping substructure and they proposed a model in which the contracted Igh locus adopts a rosette like structure containing clusters of loops separated by linkers [8].

The advent of chromosome conformation capture and genome wide sequencing has enabled us to look in greater detail at inter- and intra-chromosomal interactions. One particular protein that has emerged as a key player in implementing chromatin conformation is the CCCTC-binding factor, CTCF. It can act both as a transcriptional repressor or activator, depending on the context. To explain these contrasting effects it has been proposed that CTCF is an insulator protein that plays a role in establishing regulatory domains. Indeed, a recent study examining CTCF in a global setting confirmed and extended the observations of many other labs showing that it connects and separates different chromatin domains through loop formation [9]. Handoko et al. combined ChIP, chromatin conformation capture (3C) and high throughput genome wide sequencing (ChIA-PET) and found five distinct chromatin domains associated with CTCF in mouse embryonic stem cells (Figure 1). These indicate the various functions of CTCF in higher-order nuclear organization.

Figure 1.

Figure 1

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CTCF as a genome organizer: 5 types of chromatin domains. CTCF has several functions in higher-order nuclear organization. It has a role in (i) creating active or repressive domains which contain coordinately regulated genes, (ii) insulator activity which prevents the spread of active or repressive chromatin into neighboring domains, and in (iii) the cross-talk between regulatory sequences and their targets. (I) Active chromatin domains enriched in H3K4me1/2, H3K36me3, RNA PolII and p300. (II) Repressive chromatin domains enriched in H3K27me3, H3K9me3, H4K20me3. (III) Enhancer–promoter hubs with loops enriched in H3K4me1/2 and boundaries particularly enriched in H3K4me3, RNA PolII and p300. (IV) Domain barriers physically separate active and repressive domains. (The type V is not represented as it does not show any specific chromatin and gene expression pattern).

Identification of CTCF sites in the Igh locus

In the V(D)J recombination field, several groups have shown that CTCF sites within antigen receptor loci have a fundamental regulatory role. Using ChIP-chip, the Feeney lab found CTCF sites throughout the VH regions of the Igh locus and identified two additional sites close to the most 5′ DH gene, DFL16.1 [10] (Figure 2a). However, CTCF binding was similar in pro-B, pre-B and thymocytes, so this could not explain their hypothesis that these elements were involved in regulating changes in locus conformation, which is distinct in the three cell types. To analyze this further they looked at changes in the binding pattern of the CTCF cofactor, cohesin, which is known to play a role in holding sister chromatids together during mitosis and in regulating gene expression through binding to CTCF sites in a lineage and stage specific manner [11•,12•,13]. Degner et al. [10] discovered that cohesin binding at CTCF-sites in the Igh locus follows a more cell-type specific pattern than CTCF alone (Figure 2a), which lent support to their proposal that CTCF regulates locus contraction.

Figure 2.

Figure 2

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CTCF and the architecture of the Igh locus. (a) Schematic presentation of the Igh locus with CH, DH, JH and VH regions as well as the 3′ regulatory region (3′RR) and the Igh enhancer (Eμ). CTCF- and cohesin-binding sites are shown below [10]. (b) Location of the 14 PAIR elements with CTCF- and Pax5-binding sites [14•]. The green box contains the details of epigenetic characteristics of 3 PAIR elements (4,6,7). (c) Detail of the 3′ region of the Igh locus from the 3′RR to the proximal VH segments showing CTCF-binding sites: (i) the two hypersensitive sites (HS4/5) corresponding to the two CTCF-binding sites (CBE1/2) located upstream of DFL16.1 [16•,18••], and the IGCR1 deletion [18••] and (ii) the CTCF-binding outside of the 3′ RR. At the pre–pro-B cell stage, CTCF bound to CBE1/2 acts as an insulator, which stops antisense transcription and active chromatin from the DH region spreading to the VH region, thus limiting recombination to DH–JH. At the subsequent pro-B cell stage, the barrier is released and transcription and active chromatin can spread to the VH region, allowing VH–DJH recombination. (d) Model of the two-types of CTCF-mediated loops in the Igh locus [19]. At the early stage of B cell development, the Eμ-dependent loop involves the CTCF-sites at the 3′ RR and CBE1/2, the Eμ enhancer and the DH and JH segments (upper panel). Later, two types of Eμ-independent loops create two chromatin domains in the VH segments, one with the proximal and one with the distal VH genes (lower panel).

PAIR elements link CTCF with Pax5, a key mediator of Igh locus contraction

To investigate the role of CTCF in locus contraction the Busslinger lab performed ChIP-chip to map active chromatin within intergenic regions of the Igh locus [14•]. They identified 14 Pax5-activated intergenic repeat elements (PAIR elements) within the distal VH gene region, most of which contain functional CTCF, E2A and Pax5 binding sites (Figure 2b). 11 out of the 14 PAIR elements were found immediately upstream of VH3609 genes interspersed within the distal VHJ558 gene family. Detailed analysis of three PAIR elements (PAIRs 4, 6 and 7) show binding of Pax5, E2A and CTCF in pro-B cells (Figure 2b, green box). Ebert et al. demonstrated that the presence of Pax5 at these elements is essential for an active chromatin signature and antisense transcription within this region. In pre-B cells depletion of Pax5 binding correlates with loss of antisense transcripts, although E2A and CTCF remain bound. These transcription sites are distinct from those previously proposed by the Corcoran lab as being important in opening up the locus [15]. Since locus contraction and decontraction follow the same pattern as Pax5 binding at the pro-B and pre-B cell stage, respectively, these PAIR elements are implicated in Pax5 mediated changes in locus conformation that are known to be essential for most VH–DJH rearrangements [3,4•].

Conserved CTCF sites within the Igh locus act as insulators

Additional insight into the role of CTCF in the Igh locus came from studies performed by the Corcoran lab. They demonstrated that two conserved CTCF binding sites located upstream of the DFL16.1 gene are embedded in adjacent DNase hypersensitive sites (HS4/5) that have enhancer blocking activity in the Igh locus, and their presence marks a sharp boundary of antisense transcription that stops at least 40 kb from the VH genes [16•]. At the pre–pro-B cell stage and in T cells the two CTCF sites separate regions of active and inactive chromatin in the DH and VH regions, respectively (Figure 2c). Antisense transcription in this intergenic region occurs at high levels early in pre–pro-B cells (where the locus is undergoing DH–JH rearrangement) and is subsequently reduced in pro-B cells (where VH–DJH recombination takes place). Antisense transcription is also observed in T cells where some DH–JH rearrangement of the Igh locus occurs. Featherstone et al. propose that the presence of these two conserved CTCF sites could prevent antisense transcripts from extending into the VH region during DH–JH rearrangement. This could then act as a mechanism to ensure ordered rearrangement (DH–JH prior to VH–DJH) in B cells and to limit DJH recombination in T cells. However, the Feeney lab showed that although knocking down CTCF mildly increased transcription at DFL16.1, it did not extend antisense transcription into the proximal VH regions [17]. Of course, an absence of CTCF is toxic to the cells so it is difficult to perform experiments in cells where CTCF is totally absent. Thus, the full effects of CTCF may not have been unmasked with this approach and this could explain why knockdown only has a mild effect on Igh locus contraction [17]. Also, there could be residual binding of CTCF to its target genes so once there it may be difficult to get rid of, at least in certain places.

CTCF sites regulate ordered, lineage specific rearrangement of the Igh locus

To circumvent these issues the Alt lab took a genetic approach and deleted the region containing the two conserved CTCF binding elements (CBE1/2) located adjacent to the DFL16.1 gene (discussed above) to investigate their role in regulating recombination [18••] (Figure 2c). The deleted 4.1 kb fragment (named IGCR1) encompasses both CBE sites alongside potential binding sites for other regulators (YY1 and PU.1). To be certain that phenotypic changes associated with deletion of this fragment could be attributed to the two CBE elements, they also scrambled these sequences to inhibit binding of CTCF. Guo et al. found a marked upregulation of proximal VH7183 and VHQ52 transcripts and an enrichment of active histone marks in RAG deficient IGCR1 mutant Abelson transformed pro-B cells that they generated. Increased accessibility/transcription of proximal VH genes was linked to preferential rearrangement of VH7183 and VHQ52, at the expense of distal VHJ558 gene rearrangement in recombination competent IGCR1 targeted cells isolated from mice. In addition, deleted IGCR1 and mutant CBE alleles could undergo VH–DH rearrangement prior to DH–JH rearrangement, indicating a role for these elements in regulating ordered recombination. Further, mutant Igh alleles could also undergo VH–DJH rearrangement in developing thymocytes, in contrast to wild-type counterparts which normally only undergo DH–JH rearrangement. Thus, the two CTCF binding sites also have a role in regulating lineage specific VH rearrangement. These results confirmed predictions made by the Corcoran lab about the insulating function of the two CBEs in regulating accessibility [16•]. Guo et al. propose that regulation of Igh could be mediated by chromatin loops that form between the CBEs and CTCF sites located downstream of the 3′ regulatory region (3′RR), which involve the intronic enhancer (Eμ). These loops, which were detected by both the Feeney and Alt labs, create a DH–JH–Eμ–CH domain that is separated from the VH domain during DH–JH rearrangement [17,18••] (Figure 2d, upper panel). The CTCF dependent nature of these loops was confirmed by knockdown experiments and analysis of IGCR1 mutant alleles in RAG deficient Abelson cells by the Feeney and Alt labs, respectively.

Eμ dependent and independent loops at the Igh locus

New insights into loop formation at the Igh locus and the factors involved come from studies performed by the Sen lab [19]. This group examined the entire locus and defined two forms of loops that alter the conformation of Igh. The first, which creates the DH–JH–Eμ–CH domain that is found at the earliest stages of B cell development (discussed above), is Eμ dependent, while two other domains that form loops at the 5′ end of the locus are Eμ independent (Figure 2d).

Locus contraction, which brings mid and distal VH gene segments together at the pro-B cell stage, is known to be dependent on YY1 although the mechanism for this has not been elucidated [20]. Using chromatin immunoprecipitation Guo et al. determined that YY1 and CTCF colocalize at the 3′RR and sites 5′ of DFL16.1. YY1 also binds Eμ, but CTCF is absent from this site [19]. Furthermore, Sen and co-workers find YY1 binds selectively to the 5′7183 and 3′J558 gene segments but there is little enrichment on other VH genes, which they argue could explain their selective interaction with Eμ. To determine whether CTCF is involved in looping they performed a CTCF ChIP loop assay and looked at CTCF bound regions that interact with VH3 (within VH7183 genes) and VH10 (in distal VHJ558 genes). VH3 contacts other proximal VH genes as well as the 5′ DFL16.1 site while VH10 makes several contacts within the distal VHJ558/3609 region. The two regions do not interact with each other indicating that they exist as two different chromatin domains (Figure 2d, lower panel). Interaction sites within the two regions bind CTCF but enrichment is higher in the VH3 versus the VH10 region. The VH3 Eμ independent looped domain is brought into contact with the DH–JH–Eμ–CH domain through an interaction with Eμ. They propose a model in which loops between the different Eμ dependent and independent domains come together and separate in a dynamic manner. It is interesting to note that although the VH10 domain lies in the same region as the newly identified PAIRs [14•] none of the interaction sites within this region correspond to CTCF-binding sites within PAIR elements. Thus it is clear that there are more loops to be discovered.

As a result of their recent ChIP-seq analysis [21] the Murre lab has also been able to equate CTCF binding with loop formation [22]. Lucas et al. [22] propose that CTCF organizes the proximal VH segments into rosettes with the inner cavity containing the DH–JH region associated in a recombination center. They suggest that these rosette-structures would be of a dynamic nature, with loops rapidly associating and dissociating, in order to allow equal access to all the VH segments in the recombination center.

Interestingly, it has recently been reported that mb1-Cre mediated conditional deletion of Ctcf in pre–pro-B cells has little effect on Igh recombination [23]. These data do not reconcile with the results of CTCF knockdown experiments [17] that impact on the formation of CBE dependent loops [18••], which would be expected to have an effect on distal versus proximal VH gene rearrangements [18••]. Contradictory data from the two systems highlight a need for further investigation.

Implications for CTCF in regulating Igk recombination

At the Igk locus CTCF is bound in both pro- and pre-B cells [10]. Degner et al. reported that fewer sites are bound on Igk relative to Igh, but binding to the CTCF sites in Igk is more stage-specific in pre-B cells than CTCF binding to the Igh locus in pro-B cells (Figure 3a). The only other CTCF site identified is located in the silencer (Sis) within the Vk–Jk intron (Figure 3a). This site is enriched for CTCF in pro- and pre-B cells as well as in T cells, with the highest binding found in pre-B cells. Binding of CTCF to this site was confirmed by the Garrard lab who deleted the entire Sis element [24]. Xiang et al. found that an absence of Sis skews the usage of Vk genes so that proximal genes are rearranged at the expense of distal Vk genes. In this respect the CTCF site within the Igk Sis imitates the function of the CBE sites located adjacent to the DFL16.1 gene in the Igh locus. However, an absence of Sis does not impact on lineage specific regulation, as Igk rearrangements are not detected in thymocytes from Sis–/– mice.

Figure 3.

Figure 3

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CTCF/cohesin and the architecture of the Igk and Tcra loci. (a) Schematic presentation of the Igk locus with Ck, Jk and Vk regions as well as the 3′ Igk enhancer (3′Ek) and the Igk silencer (Sis). CTCF- and cohesin-binding sites identified by Degner et al. [10] are shown below. Detail of the 3′ region including the regulatory elements (3′Ek, intronic Igk enhancer (iEk) and Sis) with the CTCF-binding sites shown below [10,24]. (b) Regions of low (L1-4) and high (H1-5) CTCF-occupancy over the Vk region as well as the 3′ and 5′ CTCF-boundaries (3′B and 5′B) are shown below the Igk schematic [23]. Model of the CTCF-mediated loops: (i) separation of the enhancers from the proximal Vk genes through CTCF-binding to Sis, (ii) separation of the enhancers from the outside 3′ sequences, (iii) separation of the proximal and distal Vk gene segments, and (iv) interactions between the C-Jk region and distal Vk segments [23]. (c) Schematic presentation of the 3′ region of the Tcra locus, from the Dad1 gene to the first Vα genes, including the Tcra locus control region (LCR), the Tcra enhancer (Eα), the C-Jα region, the T early α promoter (TEA), and the V–C–E–J and Dδ elements of Tcrd. Cohesin and CTCF-binding sites are shown below [25•]. Model of the cohesin/CTCF-mediated loops in the 3′ Tcra region that allow interactions of Eα with TEA and the LCR in order to insulate the C-Jα region from Dad1 and Tcrd.

The Hendriks lab reports that mb1-Cre mediated conditional deletion of Ctcf in the presence of the VH81X Igh transgene also promotes preferential proximal versus distal Vk usage [23]. This group reports more coverage of CTCF at Igk than Degner et al., most probably due to the greater sensitivity of ChIP-seq versus ChIP-chip. They identified approximately 60 unevenly distributed binding sites throughout the locus, with regions of high (H1-5) and low (L1-4) CTCF-occupancy (Figure 3b). These include the major CTCF binding sites at the 5′ and 3′ boundaries of Igk as well as the Sis element (Figure 3b). Ribeiro de Almeida et al. find that proximal Vk genes that are rarely used in wild-type alleles lack CTCF occupancy, while those Vk genes that bind CTCF are the most frequently used. To examine CTCF mediated interactions within Igk they performed 3C-seq using the Sis element as bait in targeted mice bred onto a RAG deficient background. They showed that Sis interacts with regions of high CTCF occupancy through-out the locus. These interactions are not affected by an absence of CTCF however, although interactions with the proximal Vk regions are increased in line with increased proximal Vk usage. These data are also consistent with the data published by Garrard and co-workers [24] showing that deletion of Sis increases proximal Vk usage at the expense of distal Vk genes but has no apparent effect on locus contraction as judged by DNA FISH analyses. Interestingly, in a 3C-seq assay the intronic and 3′ enhancer bait sequences give a similar spectrum of interactions as the Sis bait, but in contrast to Sis, deletion of CTCF decreases interaction of the enhancers (particularly the 3′ Ek) with the distal Vk genes. Loss of CTCF also alters interaction of the Igk enhancers with regions upstream and downstream of 3′ and 5′ boundaries of the Igk locus. In light of these data they propose a model in which Sis forms CTCF dependent loops that separate the enhancers (in one loop) from proximal Vk genes (in a second loop) (Figure 3b). Separation of the proximal Vk genes favors enhancer–promoter interactions on distal Vk genes which impacts on the choice of Vk genes that are rearranged.

Cohesin has a role in regulating Tcra locus conformation and V(D)J rearrangement

Rearrangement of Tcra takes place in non-cycling CD4+CD8+ double positive (DP) T cells so it is possible to deplete the cells of cohesin and monitor the effects on the Tcra locus independent of cell division. The Merkenschlager lab took advantage of this and conditionally deleted Rad21, a component of cohesin, using a CD4-Cre transgene [25•]. They showed that cohesin binds to the Tcra locus control region (LCR), the Eα enhancer, the Jα49 promoter, the TEA promoter, sites located between Tcrd and the first Vα segments and to Vα gene promoters (Figure 3c). In CD4-Cre Rad21lox/lox thymocytes, primary Jα rearrangements involving 5′ proximal Jα gene segments occur at normal levels, probably because some Rad21 molecules remain bound to these regions before complete depletion by CD4-Cre. By contrast, cohesin is depleted at distal 3′ Jα gene segments and this is linked to impaired secondary rearrangements, decreased transcription, decreased H3K4me3 deposition, RAG binding and a reduction in double strand DNA breaks within this region. An absence of cohesin also affects locus conformation resulting in significantly lowered interaction between the TEA and Eα as well as between Eα and the Dad1 cohesin site, which is located within the Tcra LCR and is known to be a CTCF-dependent insulator (Figure 3c). The absence of Rad21 is linked to an increase in Dad1 transcription, suggesting that the CTCF-insulator function is impaired. Cohesin also mediates interactions during sequential rearrangements, with Eα contacting the Jα49 and 37 promoters as well as Vα promoters. Finally, Seitan et al. show that thymocyte differentiation is impaired in CD4-Cre Rad21lox/lox mice with reduced diversity of TCRA molecules. However, functionally rearranged Tcr genes rescue differentiation indicating that this particular defect can be attributed to defects in the Tcra locus.

Conclusions

The combined data from the papers discussed here indicate a fundamental role for CTCF and its cofactor, cohesin, in regulating locus conformation and V(D)J rearrangement of the antigen receptor loci. It remains to be seen how other cofactors such as YY1 participate in this control. Moreover, additional genetic experiments targeting individual and/or multiple CTCF binding sites will determine which elements are the most functionally relevant within each of the loci. This will present the greatest challenge given the number of binding sites that are present within each locus.

Acknowledgments

We would like to thank Susannah Hewitt and the rest of the Skok lab for discussions and critical reading of this manuscript. This work is supported by the National Institute of Health: R01GM086852, RC1CA145746 and an LLS scholar (J.A.S.). JC is an Irvington Institute Fellow of the Cancer Research Institute.

References and recommended reading

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• of special interest

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