An ectopic CTCF-dependent transcriptional insulator influences the choice of Vβ gene segments for VDJ recombination at TCRβ locus

Insulators regulate transcription as they modulate the interactions between enhancers and promoters by organizing the chromatin into distinct domains. To gain better understanding of the nature of chromatin domains defined by insulators, we analyzed the ability of an insulator to interfere in VDJ recombination, a process that is critically dependent on long-range interactions between diverse types of cis-acting DNA elements. A well-established CTCF-dependent transcriptional insulator, H19 imprint control region (H19-ICR), was inserted in the mouse TCRβ locus by genetic manipulation. Analysis of the mutant mice demonstrated that the insulator retains its CTCF and position-dependent enhancer-blocking potential in this heterologous context in vivo. Remarkably, the inserted H19-ICR appears to have the ability to modulate cis-DNA interactions between recombination signal sequence elements of the TCRβ locus leading to a dramatically altered usage of Vβ segments for Vβ-to-DβJβ recombination in the mutant mice. This reveals a novel ability of CTCF to govern long range cis-DNA interactions other than enhancer–promoter interactions and suggests that CTCF-dependent insulators may play a diverse and complex role in genome organization beyond transcriptional control. Our functional analysis of mutated TCRβ locus supports the emerging role of CTCF in governing VDJ recombination.


INTRODUCTION
Insulators prevent interactions between promoters and enhancers in a position-dependent manner due to their ability to influence higher order chromatin structure (1). CTCF (CCCTC-binding factor) appears to be important for their activity as it can facilitate both intrachromosomal and interchromosomal interactions and thus define chromatin domains that may be independently regulated (2). A large number of CTCF binding sites have been identified in mammalian genomes suggesting their extensive involvement in governing cis-DNA interactions among regulatory elements (3)(4)(5). Whether CTCF defined domains restrict specifically the enhancerpromoter communication relevant for transcriptional regulation or can influence other types of cis-DNA interactions in the genome is not currently known.
In this context, antigen receptor loci like IgH, TCRa/d and TCRb that code for the immunoglobulin (Ig) and T-cell receptors (TCR), are particularly interesting. By exhibiting long range interactions between different types of elements, they present a useful framework to explore the role of CTCF in defining independently regulated chromatin domains. Enhancer-promoter interactions are necessary for defining the developmental stage specificity of RAG-mediated VDJ recombination at these loci (6). Additionally, recombination requires physical interaction between recombination signal sequence (RSS) elements associated with the V, D and J segments in a combinatorial manner. These segments are located at large distances from each other on the chromosome and higher order chromatin reorganization, manifested as locus contraction, is necessary to bring them together prior to recombination in a lineage specific manner (7,8).
Recent analysis of the IgH and Igk loci demonstrates that interactions between CTCF binding sites indeed organize the higher order chromatin structure relevant for regulation of transcription and recombination. Multiple CTCF binding sites at these loci were postulated to organize the chromatin in concert with cohesin, Pax5, YY1 etc. (9,10). As predicted, some of the sites are important for enhancer blocking at the IgH locus (11,12). An additional role of CTCF and cohesin in locus contraction seems evident by the multiple interactions between the distantly placed CTCF binding sites at the IgH locus in Pro-B cells (12,13). Knockdown of CTCF led to a partial reduction in these long range interactions thereby supporting the role of CTCF in higher order chromatin organization (14). A conditional knockdown of CTCF in ProB cells demonstrated that CTCF may not be necessary for VDJ recombination per se but plays an important role in restricting Igk enhancer activity and thus influences recombination at this locus (15). While these studies underscore the importance of CTCF for appropriate chromatin organization relevant to VDJ recombination, the manner by which CTCF may impact this process needs further elucidation (16).
To gain further insights into the nature of chromatin domains organized by CTCF and their control on nuclear processes like transcription and VDJ recombination, we adopted a 'gain-of-function' genetic approach. A CTCF binding element [H19 imprint control region (H19-ICR) of the Igf2/H19 locus] was inserted to the murine TCRb locus by genetic manipulation. We present evidence to show that introduction of ectopic CTCF binding sites from an unrelated locus is sufficient to drastically alter the transcription and recombination patterns at the TCRb locus.
Organization and regulation of murine TCRb locus, encoding b chain of a/b T cell receptor (a/b TCR), is useful to delineate the interactions of diverse elements. Unlike the relatively large IgH and Igk loci, TCRb locus spans only about 700 kb in the genome. It has 31 Vb gene segments (TRBV1 to TRBV31). Majority of the Vb segments are located upstream to the two clusters of Db and Jb segments, i.e. Db1-Jb1.1-1.7-Cb1 (DJCb1) and Db2-Jb2.1-2.7-Cb2 (DJCb2) while the TRBV31 is located downstream to the DJCb regions ( Figure 1A) (17). A developmentally regulated enhancer (Eb) and promoters PDb1 and PDb2 are the key regulatory elements that interact functionally during early double negative (DN, CD8 À CD4 À ) stage of T cell development (18)(19)(20)(21). Their interaction, achieved by formation of a holocomplex comprising enhancer Eb and promoter PDb1 and/or PDb2, leads to generation of chromatin accessibility restricted to about 25 kb region encompassing the DJCb1 and DJCb2 clusters (22,23). Consequently, PDb1 and PDb2 initiate germline transcripts from DJCb1 and DJCb2 clusters, respectively. The altered chromatin also becomes accessible to RAG proteins that orchestrate the RSS-mediated Db-to-Jb recombination at the DJCb clusters in accordance with the accessibility hypothesis (24). The alleles that have undergone successful Db-to-Jb recombination, act as substrates for RSSmediated Vb-to-DbJb recombination that generates the functional TCRb gene. Chromatin structure of Vb segments is also altered in DN T cells but is independent of regulation by Eb (25). Juxtapositioning of RSS elements associated with Vb and DbJb segments is achieved by locus contraction prior to recombination (8). Keeping these observations in mind, we argued that the insertion of a CTCF-dependent insulator at the TCRb locus could potentially interfere with different aspects of VDJ recombination that may be regulated by Eb and/or be otherwise influenced by chromatin organization.
We exploited the CTCF-binding activity of the H19-ICR of the murine Igf2/H19 locus ( Figure 1B) for interfering with TCRb locus regulation. H19-ICR regulates the parent-of-origin specific monoallelic expression of H19 and Igf2 in vivo (26)(27)(28). It binds CTCF and organizes an insulator that prevents Igf2 promoter activation by the downstream enhancers on the maternally inherited allele. Loss of CTCF binding due to methylation of CpG residues prevents binding of CTCF and abrogates enhancer blocking on the paternally inherited allele leading to activation of Igf2 promoter by the enhancers (29,30). Loss of CTCF binding due to targeted mutations of CTCF binding sites on the maternal allele activates maternal Igf2 gene emphasizing the crucial link between enhancer blocking and CTCF (31). We chose H19-ICR as the insulator to perturb the TCRb locus because the enhancer blocking by H19-ICR is clearly dependent on CTCF. Also, when inserted at the Afp locus in mice, H19-ICR was able to interact with enhancer and Igf2/H19 TCRβ-del  promoter as it does at the endogenous Igf2/H19 locus (32). Although the organization of the regulatory elements at the Afp locus precluded an unambiguous functional readout of enhancer blocking, these observations suggested that the H19-ICR was likely to function as an enhancer blocker in a heterologous context in vivo.
Our analysis demonstrates that the inserted H19-ICR is capable of curtailing functional enhancer-promoter interaction required not only for transcriptional activation but also for Db-to-Jb recombination in the heterologous context of TCRb.
Most intriguingly, we find that the usage of Vb gene segments in the TCRb chains of the mutant mice is altered. This indicates that the inserted H19-ICR can perturb not only enhancer-promoter interactions but can also reorganize the chromatin to influence Vb-to-DbJb recombination. These results demonstrate the ability of a CTCF-dependent transcriptional insulator to influence long range DNA interactions necessary for VDJ recombination and provide useful insights into the multiple roles of CTCF in light of the topological model of interactions of regulatory elements in the genome.

MATERIALS AND METHODS
All experiments using mice were conducted as approved by Institutional Animal Ethics Committee. Sequences of all primers are given in Supplementary Table S1.
Targeting the H19-ICR and H19-ICR-mut to the TCRb locus To generate the TCR-ins ( Figure 1A), a targeting vector was constructed (Supplementary Figure S1), that had a 5.7 kb BamHI-HindIII fragment and a 4.7 kb HindIII-BamHI fragment from the TCRb locus as the 5 0 flank and 3 0 flank, respectively, which originated from 129S6/ SvEvTac gDNA. The 2.4 kb BglII fragment carrying the H19-ICR fragment with all four CTCF binding sites and a 'neomycin resistance gene' cassette (Neo-r) were cloned between the flanks along with three Lox P sites in tandem orientation. Additionally, 'diphtheria toxin-A' (DTA) gene was also cloned that served as the negative selection marker. The construct was linearized with NotI and electroporated in R1 embryonic stem (ES) cells derived from mouse strain 129X1/SvJ x 129S1 and the clones were selected for G418 resistance. Genomic DNA from G418 resistant clones was digested with BglII and subjected to Southern hybridizations with the 5 0 and 3 0 probes. The correctly targeted alleles (Tins) generate 6.4 and 7.5 kb bands upon hybridization with the 5 0 and 3 0 probes, respectively, compared to the 12.6 kb band of the wild-type allele in each case. The Neo-r cassette was subsequently removed by electroporation with a plasmid pBS185 transiently expressing 'Cre recombinase'. The correct clones (TCR-ins), which had excised Neo-r gene but retained the H19-ICR, were screened using a PCRbased strategy that detected the insulator-TCRb junctions on either end. Further, the TCR-ins clones having excision of Neo-r gene were confirmed by Southern hybridization of BglII digested gDNA. The BglII fragment size, detected by 3 0 probe, in the non-excised Tins clones is 7.5 kb and changes to 6.4 kb in Neo-r excised TCR-ins clones. Correctly manipulated TCR-ins ES cells were microinjected into C57bl/6 blastocysts to derive founder chimeric mice that were bred to derive TCR-ins mutant mice. The germline transmission of the mutation in the mice was confirmed by Southern hybridization and PCR analysis (Supplementary Figure S1).
Exactly similar strategy was used for targeting of H19-ICR-mut to generate TCR-mut mice. The H19-ICR-mut had all the four CTCF binding sites mutated (31).

Generation of the mouse strain TCR-cas
The congenic mouse strain TCR-cas was generated that carried wild-type TCRb locus derived from Mus castaneus castaneus in a Mus musculus domesticus genetic background. The female F1 progeny of Mus castaneus castaneus Â Mus musculus domesticus was backcrossed to Mus musculus domesticus males for eight generations to derive this strain. At each generation, the presence of castaneus sequences upstream and downstream to TCRb allele was verified by PCR using oligonucleotide primers DMit274 and D6Nds4.

Allele specific transcriptional analysis by SNAPSHOT assay
Allele specific expression of germline transcripts was analyzed in thymocyte RNA of 4-5 weeks old RAG deficient mice which had maternally inherited domesticus allele (wild-type or TCR-ins) and paternally inherited castaneus allele (TCR-cas). Random primers were used for the reverse transcription and gene-specific primers were used to amplify DJb1, DJb2, TRBV13.2 and TRBV31. The amplified products were subjected to SNAPSHOT analysis (Snapshot multiplex kit, ABI) that relied on incorporation of fluorescently labelled dideoxynucleotides by single nucleotide primer extension (SNuPE) based on specific nucleotide differences between domesticus and castaneus alleles. Detection of incorporation was by ABI Genetic Analyzer 3130x1. Genomic DNA of TCR-cas, C57Bl/6 and C57Bl/6/TCR-cas (F1) were used as controls to verify incorporation of specific nucleotides by castaneus and domesticus alleles. The incorporation of dideoxynucleotides by F1 gDNA was used to normalize the values for their incorporation in test amplicons (DJb1, DJb2, TRBV13.2 and TRBV31) before calculating allele specific contribution of the doemsticus (TCR-ins or wild-type) and castaneus alleles to total mRNA.

Transcriptional analysis by quantitative RT-PCR
Thymocytes from 6-8 weeks old TCR-ins, TCR-mut and control mice were used to derive total RNA. The RNA was reverse transcribed using random primers and used for quantitative PCR to detect transcripts for DJb1, DJb2, Thy1.2, TRBV13-Db-Jb1.1, TRBV31-Db-Jb1.1, TRBV13-Db-Jb2.7 and TRBV31-Db-Jb2.7 using gene specific primers. The forward TRBV13 primer was designed to detect TRBV13.1, TRBV13.2 and TRBV13.3. SYBR-Green chemistry based Real time PCR analysis on ABI PRISM-7000 Sequence Detection System, ABI, USA was used for the quantitative PCR. The amount of transcripts in mutant mice were calculated normalized to the endogenous reference (Thy1.2) and relative to a calibrator (control mice) using the standard curve method for relative quantification.

Db-to-Jb recombination analysis by PCR
Genomic DNA isolated from thymocytes was subjected to PCR and analyzed for the appearance or disappearance of recombination specific bands as described.

DJCb usage analysis
RNA from thymocytes was reverse transcribed using random primers followed by PCR amplification using gene specific primers to detect rearranged TCRb genes having TRBV12 (including TRBV12.1 and TRBV12.2), TRBV13 (including TRBV13.1, TRBV13.2 and TRBV13.3) and TRBV31 and any of the two Cb (Cb1 or Cb2). The amplified products were cloned. Sequence analysis from at least 36 clones of each genotype was carried out and fraction of clones using DJCb1 or DJCb2 determined.

Genetic manipulation of the TCRb locus
We used a 'knock-in' approach to insert the H19-ICR at the TCRb locus in mouse ES cells such that the 2.4 kb H19-ICR element, carrying all four CTCF binding sites and known to be sufficient for enhancer blocking in vitro (30,33), was introduced immediately downstream to the PDb1-regulated transcription unit comprising Db1-Jb1.1-1.7-Cb1 (DJCb1; Figure 1A). This positioned the H19-ICR between the promoter PDb1 and the enhancer Eb. PDb2 dual promoters, responsible for the expression of the second transcription unit Db2-Jb2.1-2.7-Cb2 (DJCb2), are located downstream to the H19-ICR insertion site. Correctly targeted ES cells (TCR-ins) were used to derive mutant mice that carried the H19-ICR inserted at the TCRb locus. To unambiguously ascertain the importance of CTCF in the H19-ICR function in the heterologous context, we also generated mutant mice (TCR-mut) that carried a mutant version of H19-ICR (H19-ICR mut) wherein all the four CTCF binding sites were mutated ( Figure 1A). The four sitespecific mutations are known to abrogate CTCF binding by H19-ICR in vitro as well as in vivo (31). All heterozygous and homozygous mutants were viable, fertile and indistinguishable from the wild-type mice in physical appearance.
H19-ICR functions as an enhancer blocker specifically upon maternal inheritance at the endogenous Igf2/H19 locus. Hence, the transcription and recombination analysis was carried out in an allele specific manner. Thymocytes for the analysis were derived from mutant mice that maternally inherited TCR-ins (M, TCR-ins/ TCRb-del), TCR-mut (Mx, TCR-mut/TCRb-del) or wild-type TCRb allele (C, +/TCRb-del). In each case, the presence of TCRb-del ( Figure 1A), carrying 15 kb deletion (encompassing DJCb1 and DJCb2) as the paternal allele, ensured that it would not contribute to any analysis as it could neither generate transcripts in the deleted region nor undergo recombination (34,35). Alternatively, using a congenic mouse strain, TCR-cas (see 'Materials and Methods' section for details), mutant mice were obtained that maternally inherited domesticus TCR-ins (M*, TCR-ins/TCR-cas) or domesticus wild-type TCRb allele (C*, +/TCR-cas). In each case, the paternally inherited allele was castaneus wild-type TCRb (TCR-cas). Nucleotide differences between the domesticus and castaneus alleles afforded allele-specific transcriptional analysis of thymocytes in these mice.

H19-ICR insertion at the TCRb locus does not arrest overall thymocyte development
Thymocyte development proceeds sequentially via double negative (DN, CD4 À CD8 À ), double positive (DP, CD4 + CD8 + ) and single positive (SP, CD4 + CD8 À and CD4 À CD8 + ) stages characterized by the presence of cell surface markers CD4 and CD8. The development beyond DN stage depends on the synthesis of a functional TCRb chain consequent to successful VDJ recombination of TCRb locus (35). Since the inserted H19-ICR could potentially interfere in the regulation of transcription and/or recombination at TCRb locus, we first examined the effect of our TCRb manipulation on overall thymocyte development.
The cellularity of the thymus in TCR-ins (M), TCR-mut (Mx) and control (C) mice as well as their thymocyte developmental profiles were comparable ( Figure 2). As described above, the TCRb-del background allele is not capable of supporting recombination and contributing to T-cell development beyond DN stage (35). Therefore, T-cell development beyond DN stage to generate DP and eventually SP thymocytes in the mutant mice demonstrated the ability of the TCR-ins and TCR-mut alleles to undergo VDJ recombination and generate functional TCRb chains. Thus, the inserted H19-ICR neither completely abolished VDJ recombination nor arrested thymocyte development. The ability of TCR-ins and TCR-mut mutant alleles to support VDJ recombination cannot be used to simplistically infer that regulation of VDJ recombination was not affected. Multiple Vb, Db and Jb segments are used in a combinatorial manner during VDJ recombination. Hence, it was plausible that the inserted position-dependent H19-ICR insulator influenced transcription and recombination in a gene-segment-specific manner. Thymocytes isolated from the mutant mice were, therefore, utilized to unambiguously investigate gene-segment-specific transcription as well as Db-to-Jb and Vb-to-DbJb recombination events at the manipulated alleles.

Effective CTCF-dependent enhancer blocking by inserted H19-ICR
We hypothesized that if the H19-ICR organized a functional insulator between promoter PDb1 and enhancer Eb in the TCR-ins allele, it would reduce the generation of DJb1 germline transcripts that depend on activation of PDb1 by Eb. An assay was designed wherein RT-PCR was followed by SNAPSHOT analysis. Based on this, single nucleotide differences between the domesticus and castaneus alleles were utilized to determine the contribution of the mutant (TCR-ins) or the wild-type (+) allele to the germline transcripts originating from various promoters of the TCRb locus in thymocytes of TCR-ins mutants (M*) and control mice (C*). The analysis was performed in RAG deficient mice (Rag À/À ) to prevent VDJ recombination and avoid heterogeneity in DNA templates that could interfere in accurate detection of germline transcripts.
A drastic reduction in the DJb1 transcription from the mutant TCR-ins allele was evident as it contributed to less than 5% of the total DJb1 transcripts in the mutant mice (M*; Figure 3A). The control mice (C*) exhibited nearly equal contribution of the domesticus and castaneus alleles (biallelic expression) for DJb1 as expected. We also observed that the expression of DJb2 transcripts continued to be biallelic in mutants as in the control mice ( Figure 3A). Further, the Vb germline transcripts (TRBV13.2 and TRBV31) also continued to be expressed from both alleles in mutants as in control mice ( Figure 3A).

CD4-PE
CD4-PE  The significant impairment in DJb1 transcripts indicated that the inserted H19-ICR abrogates Eb and PDb1 interaction necessary for transcriptional activation. Insulators curtail enhancer-based promoter activation only when located between the enhancer and the promoter. Our finding of abrogation of Eb-PDb1 interaction taken together with no alteration in Eb-PDb2 interaction unambiguously demonstrated that the inserted H19-ICR acts as an effective position-dependent enhancer blocking insulator when inserted at the TCRb locus. The Vb promoters are known not to be affected by Eb (25). An unaltered transcriptional profile of Vb germline transcripts indicated that H19-ICR insertion specifically abrogated the Eb-PDb1 interaction rather than lead to generalized alterations in germline transcription at the TCRb locus.
It was important to confirm the abrogation of Eb-PDb1 transcription in RAG sufficient (Rag +/+ ) mice since normal RAG expression in the developing T cells was necessary for the analysis of interference of inserted H19-ICR with VDJ recombination. To minimize the influence of recombination on detection of germline transcription, DN thymocytes at DN2/3 stage of development were isolated. Such cells have a high proportion of alleles that have not undergone recombination and hence are in the germline configuration. A quantitative RT-PCR assay was designed to specifically estimate the abundance of DJb1 germline transcripts arising due to Eb-PDb1 interaction prior to recombination ( Figure 3B). Further, the RT-PCR primer design ensured that the DJb1 transcripts arising from the paternal TCRb-del allele were not detected. In this assay also, the DJb1 transcripts in mutant mice (M) were observed to be drastically reduced compared to controls (C; Figure 3B, left graph). Further, the Eb-PDb2 interaction-dependent DJb2 germline transcripts continued to be expressed at high levels in TCR-ins mutants (M) like the control mice (C) in the sorted DN2/3 thymocytes (Supplementary Figure S2). It was evident, therefore, that the inserted H19-ICR functions as a position-dependent enhancer blocker in RAG sufficient mice also. All subsequent experiments were performed on mice with RAG sufficient background. Finally, to ascertain the requirement of CTCF for the insulator organized by H19-ICR, we analyzed the abundance of DJb1 germline transcripts in TCR-mut mice (Mx). Clearly, in these mice, the Eb-PDb1 interactiondependent DJb1 transcription was not affected ( Figure 3B, right graph). This demonstrated that the enhancer blocking organized by the inserted H19-ICR at the TCRb locus is CTCF dependent.

Severe abrogation of Eb-PDb1 interaction-mediated recombination
Deletion analysis has indicated that the interaction of Eb with the promoters PDb1 and PDb2 leads to generation of chromatin accessibility both for transcription and recombination (22). Since the inserted H19-ICR was able to prevent Eb-PDb1 interaction-mediated transcriptional activation, we examined its ability to interfere in Db-to-Jb recombination by PCR based on genomic DNA from thymocytes ( Figure 4A).
Amplification products of 450 and 318 bp arising due to the recombination of Db1 with Jb1.1 and Jb1.2, respectively, were evident in control mice (C) but were absent in TCR-ins mutants (M). Further, the 1.1 kb band representing the non-rearranged germline configuration was robustly amplified in mutants ( Figure 4A, left panels). This indicated a severe abrogation of recombination at the DJCb1 cluster. The block in recombination was CTCF dependent as TCR-mut mice (Mx) did not exhibit such an abrogation. In accordance with our observation that H19-ICR did not influence Eb-PDb2 interaction for transcription, the PDb2-regulated recombination of Db2 with Jb2 was also not significantly altered in TCR-ins mutants. Both mutants (M) and control littermates (C) exhibited the recombination of Db2 with Jb2.1 and Jb2.2, evidenced by appearance of 411 and 208 bp band and reduction in amplification of 991 bp band ( Figure 4A, right panels). These results suggest that enhancerpromoter interaction-dependent recombination can also be curtailed by an insulator in a position-dependent manner.  Thymocyte RNA was used for RT-PCR using forward primers specific to TRBV12 or TRBV13 or TRBV31 (black arrows on the locus diagram) and a reverse primer common to exon I of both Cb1 and Cb2 (grey arrows on the locus diagram). The amplification products were cloned and sequenced to determine the fraction of clones with DJCb1 or DJCb2 as depicted in the graph. At least 36 clones were sequenced for each genotype. For PCR and RT-PCR analysis, thymocytes were isolated from mice that maternally inherited the wild-type TCRb allele (C, +/TCRb-del) or TCR-ins (M, TCR-ins/TCRb-del) or TCR-mut (Mx, TCR-mut/TCRb-del).

Preferential usage of DJCb2 cluster for VDJ recombination
The TCRb alleles that have successfully recombined DbJb regions act as a substrate for RSS-mediated recombination between Vb and DbJb segments. The severe reduction in the recombination at DJCb1 cluster upon maternal inheritance of TCR-ins allele ( Figure 4A), was therefore expected to substantially reduce the usage of DJCb1 region in TCRb chains in these mutants. To verify this, RNA was isolated from thymocytes of TCR-ins mutant and control mice. TRBV12 and TRBV13 together contribute to TCRb chains on about 30% of thymocytes (36). Primers for RT-PCR were designed such that the forward primer could bind to specific Vb regions (TRBV12, TRBV13 or TRBV31) while the reverse primer was common to exon I of Cb1 and Cb2 and avoided any inherent bias for amplification of Cb1 or Cb2 ( Figure 4B). Amplified products after RT-PCR were cloned and sequenced to estimate the usage of DJCb1 or DJCb2 in transcripts encoding TCRb chain. Sequence analysis revealed a very strong bias in favor of DJCb2 usage in mice that had maternally inherited TCR-ins (M) and exhibited severely compromised Db1-to-Jb1 recombination compared to the mice that had a wildtype allele (C; Figure 4B). Again, CTCF dependence was clearly evident as the DJCb2 usage in TCR-mut mice (Mx) was comparable to control mice.
The enhanced usage of DJCb2 cluster for Vb-to-DbJb recombination was a logical consequence of efficient enhancer blocking by the inserted H19-ICR that curtailed Eb-PDb1 interaction based events, i.e. DJb1 transcription and Db1-to-Jb1 recombination and hence reduced the usage of DJCb1 for Vb-to-DbJb recombination.

Altered usage of Vb segments for VDJ recombination
VDJ recombination at TCRb locus involves long range cis-DNA interactions of RSS elements between the recombined DbJb segments and the Vb segments located a large distance away. In the TCR-ins allele, in addition to the two DJCb clusters, Vb regions also get partitioned by the insulator such that TRBV1-TRBV30 are located upstream to the DJCb1 cluster and inserted H19-ICR while TRBV31 is located downstream to the H19-ICR and DJCb2 cluster ( Figure 5A). Having established that the inserted H19-ICR organizes a CTCF-dependent functional insulator at TCRb locus, we analyzed the choice of Vb gene segments used for Vb-to-DbJb recombination in TCR-ins mutant mice.
Thymocytes were analyzed for surface expression of TCRb that use Vb gene segments present on either side of the H19-ICR. We first analyzed the TRBV31 specific TCRb by immunostaining thymocytes from TCR-ins mice that organize a functional insulator. We observed an enormous increase ($8-to 10-fold), in the number of CD4-SP thymocytes (CD4 + CD8 À ) that had TRBV31 in the TCRb chain in TCR-ins mutants (M; Figure 5B) compared to control littermates (C). Also, in the same thymocyte samples, there was a drastic reduction (about 5-fold) in the usage of several other Vb tested (TRBV2,   TRBV4, TRBV12, TRBV13 and TRBV19). A similarly altered Vb usage was observed in CD8-SP cells (data not shown). Importantly, such a drastic alteration was not observed for TCR-mut mice (Mx; Figure 5B) that are unable to organize an insulator due to mutated CTCF binding sites.
This suggested that the presence of the ectopic H19-ICR insulator is responsible for a decrease in usage of Vb segments located upstream to the inserted H19-ICR and a concomitant increase in the usage of TRBV31 located downstream to the inserted H19-ICR. Thus, Vb choice for Vb-to-DbJb recombination was influenced by H19-ICR in a CTCF-dependent manner. In conjunction with the increased DJCb2 usage (Figure 4B), these results suggest a strong bias in favor of TRBV31 recombination specifically with DJCb2 cluster in the TCR-ins allele. In sharp contrast, recombination of all other Vb segments with DJCb2 cluster, i.e. interactions across H19-ICR, appear to be hindered significantly.
To confirm our finding, we directly tested the choice of recombination of DJCb1 and DJCb2 clusters with TRBV13 and TRBV31. An RT-PCR assay was designed such that the forward primers, in independent PCR reactions, recognize TRBV13 or TRBV31 while the reverse primer recognizes Jb1.1 in both these reactions. Jb1.1 was taken as a representative Jb for the use of DJCb1 cluster as we observed it to be used maximally in the sequence based analysis of Vb-to-DbJb recombination described earlier. In the mice that inherited the TCR-ins maternally (M), there was a drastic reduction in TRBV13-Db-Jb1.1 as well as TRBV31-Db-Jb1.1 transcripts ( Figure 6A) consistent with the observation of a reduced ability of DJCb1 to support Vb-to-DbJb recombination ( Figure 4A and B).
In a similar assay, by designing the reverse primer in Jb2.7, we estimated the choice of DJCb2 for recombination with TRBV13 and TRBV31. In TCR-ins mutants (M), the TRBV31-Db-Jb2.7 transcripts were increased by more than 10-fold while the TRBV13-Db-Jb2.7 transcripts were about 50% less abundant compared to control mice (C; Figure 6B). Thus, effectively, the usage of TRBV31 increased more than 20-fold relative to usage of TRBV13 for recombination to DJCb2 cluster in the presence of a functional insulator ( Figure 6C, upper graph). Unlike TCR-ins mutants (M), TCR-mut mice (Mx) did not exhibit any enhancement in the TRBV31-Db-Jb2.7 to TRBV13-Db-Jb2.7 transcript ratio compared to control mice (C; Figure 6C, lower graph).
Our results indicate that the H19-ICR significantly alters the regulation of transcription as well as recombination at the TCRb locus and this effect is not merely due to the presence of an inserted 2.2 kb DNA fragment. Ability of inserted H19-ICR to bind CTCF was essential for its influence on these processes. Just as it prevents the enhancer-promoter communication when present between them, the H19-ICR insulator also prevented RSS elements from interacting with each other in a CTCF-and positiondependent manner, i.e. when located between them and led to an alteration in the choice of Vb segments for recombination ( Figure 6D).

DISCUSSION
In this investigation, we took advantage of the precisely regulated transcription and recombination at the TCRb locus to understand the ability of a CTCF-dependent insulator to interfere in the interactions between several cis-acting regulatory elements. By organizing an ectopic insulator at TCRb locus, we demonstrate a novel ability of CTCF to modulate cis-DNA interactions between RSS elements during VDJ recombination.
Our analysis of the TCR-ins and TCR-mut alleles indicated that H19-ICR acts as an efficient CTCF-dependent enhancer blocker when inserted to the TCRb locus and prevents Eb-regulated transcription and recombination. H19-ICR interacts with several CTCF and cohesin binding sites at the Igf2/H19 locus (37) for organizing insulator function. However, it also interacts with enhancer and promoter elements at the Igf2/H19 locus and when inserted at the Afp locus suggesting context independence (32). Our analysis confirms the context independence functionally and clearly establishes the ability of the H19-ICR to act as a position-dependent enhancer blocker in the absence of any specific cis-acting elements of the Igf2/H19 locus in vivo.
Effective organization of an insulator by the inserted H19-ICR at the TCRb locus also led to a marked reduction in the Eb-PDb1-dependent recombination between Db1 and Jb1 segments. As predicted by the accessibility hypothesis (24), deletion of accessibility control elements (ACE), i.e. either Eb or PDb1, has been shown to lead to loss of accessibility generation for transcriptional activation and subsequent recombination (22). Our results provide a strong support for the accessibility hypothesis by relying on a novel approach of altering the ACE activity rather than deletion. Modified histones acetyl-H3K9 and trimethyl-H3K4 have been observed to be associated with chromatin which is accessible for RAG mediated recombination at the antigen receptor loci (38,39). Predictably, an altered epigenetic landscape of TCRb locus due to the ectopic insulator is the basis for the observed curtailment of Eb-dependent transcription and recombination in TCR-ins mutant alleles. Hence, an extensive comparison of the epigenetic landscape in wildtype TCRb and TCR-ins alleles is likely to provide useful insights into the nature of ACE interactions and their ability to alter the chromatin accessibility necessary for transcription and RAG-mediated recombination. Further, the promoters PDb1, 5 0 PDb2 and 3 0 PDb2 are all regulated by Eb but exhibit subtle differences in temporal regulation (21). Due to the drastic reduction in Eb-PDb1 interaction, the TCR-ins allele provides a useful model to understand the basis for the possible competition between the promoters for the shared enhancer as well as their temporal regulation.
The most remarkable feature that emerged in this study was an alteration in the Vb usage during VDJ recombination due to introduction of the four ectopic CTCF binding sites. An important role of CTCF in VDJ recombination by defining functional domains was recently demonstrated at IgH and Igk loci. At these loci, CTCFdefined chromatin loops seem to insulate the proximal V regions from the influence of enhancers Em and iEk. Consequently, the usage of V segments was altered at IgH locus upon deletion of CTCF binding sites (12,40) and at Igk locus upon loss of CTCF (15) or deletion of the CTCF and Ikaros binding element Sis (41). In the present investigation, we show that the normal chromatin interactions for ordered VDJ recombination can be altered simply by the ectopic insertion of non-locus specific CTCF binding sites. Specifically, we found that TRBV31 usage was drastically enhanced in TCR-ins allele while the usage of Vb segments located upstream was concomitantly reduced for recombination with DJCb2. Unlike the V regions of IgH and Igk, the Vb segments of TCRb are not regulated by Eb (25). Accordingly, the germline transcription of upstream Vb segment (TRBV13.2) continued to be unaffected by the (C) Abundance of recombined transcripts V31-DJ2 relative to V13-DJ2. In each case, relative abundance was estimated by real-time quantitative RT-PCR analysis. Error bars denote ± SEM. (D) Schematic of TCRb locus in wild-type (upper) and TCR-ins (lower) mice. Arrows indicate the long range interactions necessary for promoter activation (black arrows) and RSS-mediated recombination (grey arrows) between Vb and DJCb regions. H19-ICR presence in TCR-ins drastically reduces Eb based PDb1 activation. It also defines chromatin domains such that 'intradomain' interaction (TRBV31 to DJCb2 recombination) is significantly increased at the expense of 'interdomain' interaction (TRBV13 to DJCb2 recombination).
insertion of an enhancer blocker. Also, the transcriptional status of TRBV31, located relatively close to Eb, was not altered in the TCR-ins allele. Yet the Vb-to-DbJb recombination profile was completely altered. The most plausible explanation for the altered Vb usage in TCR-ins allele appears to be the CTCF-directed chromatin domain organization such that the RSS-mediated V-to-DJ recombination is efficient only when V and DJ segments share the same domain defined by CTCF ( Figure 6). This suggests that insulators may have the potential to organize specific functional domains with respect to recombination at the antigen receptor loci in addition to their well known ability to define transcriptionally distinct domains. Taken together with the recent evidence from IgH and Igk loci, these data present an emerging picture of CTCF and insulator elements governing multiple chromatin interactions at the recombining loci. Chromatin loop organization is mediated by multiple CTCF and cohesin binding sites at the recombining loci (16,42). Based on analysis of IgH locus, it has been proposed that CTCF organizes dynamic rosette structures to facilitate access to all Vh segments for recombining with DhJh segments (43). CTCF manifests its influence by organizing chromatin loops (2). Consequently, the interactions between DNA elements can be promoted as well as hindered depending on their positions relative to the CTCF-binding insulator (2,44). Keeping this in mind, we designed the TCR-ins allele such that the H19-ICR insulator effectively partitioned the TCRb locus. We observed increased intradomain and reduced interdomain functional interactions between promoter-enhancer regulatory elements as well as between RSS elements across the H19-ICR insertion in the TCR-ins allele. Thus, CTCF appears to have three activities that impact VDJ recombination: (i) enhancer blocking that regulates ordered assembly of genes (12), (ii) contraction of loci prior to recombination (13,14,16) and (iii) organization of chromatin domains that define the choice of recombining gene segments as observed during this investigation. These activities complement each other and emphasize the importance of CTCF for VDJ recombination. More detailed investigations of the interactions between the ectopic and endogenous CTCF binding sites at the TCRb locus as well as at other antigen receptor loci will be meaningful to elucidate these roles in future.
Finally, our results also provide useful insights into the nature of interactions between distantly placed cis-acting regulatory elements which have, so far, been considered only in the context of transcriptional regulation using chromosome conformation capture (3C) analysis (32,(45)(46)(47). Enhancers can activate their cognate promoters in a contact-dependent and contact-independent manner according to the topological loop domain and tracking models, respectively. Accordingly, the intervening insulator may either organize the chromatin domains to abrogate enhancer-promoter contact or prevent the tracking signal from reaching the cognate promoter (48). A fundamental assumption of the topological model is that 'intradomain' contacts are favored over 'interdomain' contacts when an insulator defines the distinct domains. This has been difficult to test functionally as reduction in interdomain promoterenhancer contacts by the intervening insulator cannot be unambiguously discriminated from the block in tracking by an intervening insulator; both the situations would lead to a loss in transcriptional activation.
In this context, it was intriguing to note that the functional H19-ICR insulator was able to hinder RSS contact mediated Vb-to-DbJb recombination across itself just as it prevents enhancer-promoter functional interaction. VDJ recombination unambiguously relies on a contact based RSS interaction and 'captures' the physical juxtaposition of the interacting RSS elements in vivo reporting it as a specific recombination event. When H19-ICR defined the chromatin domains at the TCRb locus, the contact between RSS elements and consequent Vb-to-DbJb recombination, was strongly favored between RSS elements sharing the same chromatin domain (intradomain) rather than those separated by the insulator (interdomain; Figure 6D). Mechanistically, RSS-mediated recombination and enhancer-mediated promoter activation are entirely distinct. The only common feature between them is that both require interaction of specific cis-acting DNA elements. The ability of the insulator to interfere in a position-dependent manner with each of these distinct processes provides functional evidence to support the topological model of interaction of cis acting regulatory elements.
In conclusion, our analysis clearly demonstrates a previously unreported ability of a CTCF-dependent insulator to modulate interactions between RSS elements during VDJ recombination. This emphasizes that the CTCF binding sites may have a complex role in influencing functional interactions between regulatory elements beyond modulating enhancer-promoter interactions and defining transcriptionally independent domains in the genome.