tcra gene recombination is supported by a tcra enhancer- and … · chromosome conformation capture...

Tcra gene recombination is supported by a Tcraenhancer- and CTCF-dependent chromatin hubHan-Yu Shiha, Jiyoti Verma-Gaurb, Ali Torkamanic, Ann J. Feeneyb, Niels Galjartd, and Michael S. Krangela,1

aDepartment of Immunology, Duke University Medical Center, Durham, NC 27710; bDepartment of Immunology and Microbial Science, The Scripps ResearchInstitute, La Jolla, CA 92037; cDepartment of Molecular and Experimental Medicine, The Scripps Research Institute, The Scripps Translational Science Institute,La Jolla, CA 92037; and dDepartment of Cell Biology and Genetics, Erasmus Medical College, 3000 CA Rotterdam, The Netherlands

Edited by Frederick W. Alt, Howard Hughes Medical Institute, Harvard Medical School Children’s Hospital Immune Disease Institute, Boston, MA, andapproved October 24, 2012 (received for review August 14, 2012)

Antigen receptor locus V(D)J recombination requires interactionsbetween widely separated variable (V), diversity (D), and joining(J) gene segments, but the mechanisms that generate these inter-actions are not well understood. Here we assessed mechanismsthat direct developmental stage-specific long-distance interactionsat the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd genesegments in CD4−CD8− double-negative thymocytes and Tcra genesegments in CD4+CD8+ double-positive thymocytes. Initial Vα-to-Jαrecombination occurs within a chromosomal domain that displaysa contracted conformation in both thymocyte subsets. We usedchromosome conformation capture to demonstrate that the Tcraenhancer (Eα) interacts directly with Vα and Jα gene segments dis-tributed across this domain, specifically in double-positive thymo-cytes. Moreover, Eα promotes interactions between these Vα andJα segments that should facilitate their synapsis. We found thatthe CCCTC-binding factor (CTCF) binds to Eα and to many locuspromoters, biases Eα to interact with these promoters, and is re-quired for efficient Vα–Jα recombination. Our data indicate that Eαand CTCF cooperate to create a developmentally regulated chro-matin hub that supports Vα–Jα synapsis and recombination.

T-cell development | T-cell receptor | thymus

Tand B cells produce diverse antigen receptors through therecombination of variable (V), diversity (D), and joining (J)gene segments at the T-cell receptor (Tcra, Tcrb, Tcrg, and Tcrd)and Ig (Igh, Igκ, and Igλ) loci. This V(D)J recombination is ini-tiated by the lymphoid-specific recombination-activating gene-1(RAG-1) and RAG-2 proteins, which recognize the recombi-nation signal sequences (RSSs) that flank all V, D, and J genesegments and then cleave the DNA between the RSSs and theadjacent coding gene segments (1). A critical feature of the re-action is the assembly of a synaptic complex composed of twoRSSs before the generation of RAG-dependent DNA double-strand breaks (DSBs). As such, lineage- and developmental stage-specific V(D)J recombination events can be regulated not onlyby changes in RAG protein expression and RSS accessibility toRAG proteins but also by the ability of those RSSs to undergosynapsis (2).Conformational changes of antigen receptor loci are believed

to support V(D)J recombination events because they can bringdistant RSSs into proximity and therefore increase the proba-bility of RSS synapsis (2, 3). Studies using 3D-FISH have dem-onstrated that lineage- and development stage-specific locus con-traction marks the recombination windows at antigen receptorloci (3). For example, the 3-Mb Igh locus contracts specificallyin pro-B cells to support VH-to-DHJH recombination (4–7). Thiscontracted conformation brings distal and proximal VH seg-ments, which are separated by megabases in the linear DNAsequence, to the vicinity of the DHJH cluster, presumablyallowing all VH segments a similar opportunity for recom-bination (8). In addition, the mapping of Igh locus DNA–DNAcontacts by chromosome conformation capture (3C) and relatedmethods identified three domains, each composed of multipleDNA loops (9). The Igh enhancer, Eμ, was found to promote DNA

contacts within the 3′ domain and to promote large-scale con-traction of the Igh locus, perhaps by mediating interdomaincontacts. However, our understanding of the molecular mecha-nisms regulating locus contraction and long-distance DNA con-tacts within antigen receptor loci remains rudimentary.The CCCTC-binding factor (CTCF) is a highly conserved

multifunctional zinc finger protein (10). CTCF not only insulatesgene activity by blocking enhancer–promoter interaction or de-marcating boundaries between active and inactive chromatin butalso functions as a chromatin organizer that mediates DNA con-tacts and loop formation (11). CTCF is essential for the formationof higher-order chromatin architecture at various loci, includingthe H19/Igf2 (12–14), β-globin (15, 16), MHC class II (17), andIfng (18) loci. These conformations are believed to facilitatedevelopmental stage- or cell activation-specific gene expression.Global mapping of CTCF-binding sites and CTCF-associatedchromatin loops also suggests that CTCF can facilitate long-dis-tance interactions for coordinated gene expression, demarcatechromatin boundaries, or facilitate enhancer–promoter commu-nication (19–23). However, it remains difficult to predict howCTCF might function at any individual locus.Recently, several studies have suggested a role for CTCF in

the regulation of V(D)J recombination at antigen receptor loci(9, 24–28). For example, knockdown of CTCF in a pro–B-cell linereduced DNA contacts between regulatory elements that flankthe DHJHCH region, increased antisense transcription throughthe DH and VH regions, and partially suppressed contractionof the Igh locus (25). Deletion of a pair of CTCF-binding siteslocated between VH and DH segments caused high-frequencyrearrangement of the DH-proximal VH gene segments and led todevelopmentally inappropriate VH gene recombination in B cellsand thymocytes (24). In addition, conditional knockout of CTCFallowed the intronic Igk enhancer to interact more frequentlywith proximal Vκ segments and promoted a bias toward proximalVκ recombination (26). These studies suggested that CTCFmay influence V(D)J recombination by mediating DNA contactsthat insulate or restrict enhancer activity to defined portions ofthe Igh and Igk loci. Whether CTCF impacts V(D)J recombi-nation by playing a more direct role in facilitating RSS synapsisremains unclear.The Tcra/Tcrd locus is a complex genetic locus that contains

both Tcra and Tcrd gene segments that are regulated distinctly

Author contributions: H.-Y.S., J.V.-G., A.J.F., N.G., and M.S.K. designed research; H.-Y.S.and J.V.-G. performed research; N.G. contributed new reagents/analytic tools; H.-Y.S., J.V.-G.,A.T., A.J.F., and M.S.K. analyzed data; and H.-Y.S., A.J.F., and M.S.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The ChIP-Seq data reported in this paper have been deposited in the GeneExpression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE41743).1To whom correspondence should be addressed. E-mail: [email protected].

See Author Summary on page 20192 (volume 109, number 50).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1214131109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1214131109 PNAS | Published online November 19, 2012 | E3493–E3502

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during thymocyte development (29). In CD4−CD8− double-negative (DN) thymocytes, the Tcrd enhancer (Eδ) activates theDδ and Jδ gene segments to promote Vδ-to-Dδ-to-Jδ rearrange-ment. In CD4+CD8+ double-positive (DP) thymocytes, the Tcraenhancer (Eα) activates 3′ Vα promoters and the TEA promoter(TEAp) at the 5′ end of the Jα array to initiate Vα-to-Jα rear-rangement (Fig. 1A). Recent work revealed that Vα-to-Jα re-combination is compromised in mice that are conditionallydeficient in cohesin (30). Cohesin is known to regulate sister

chromatid segregation during mitosis (31). It now is appreciatedthat cohesin has additional roles in nondividing cells, mediatedin part by its ability to interact physically and functionally withCTCF at many genomic sites (32–34). Notably, cohesin was foundto bind to Eα and the TEAp and to facilitate their long-distanceinteraction in DP thymocytes (30). Thus, cohesin appears to reg-ulate Tcra recombination by promoting contacts between cis-reg-ulatory elements that are needed to generate accessible Jα RSSs.Whether CTCF is an important regulator of Tcra locus recom-bination is currently unknown.We previously used 3D-FISH to show that the 5′ portion of

the Tcra/Tcrd locus is contracted in DN thymocytes but extendedin DP thymocytes, whereas the 3′ end of the locus is contractedsimilarly in both thymocyte populations (35). The unique 5′-ex-tended and 3′-contracted conformation in DP thymocytes washypothesized to be significant, in that it could bias initial Tcrarecombination events to use 3′ Vα gene segments, thereby saving5′ Vα gene segments for subsequent rounds of Vα-to-Jα recom-bination that replace the initial recombination events. Althoughthe 3′ end of the Tcra/Tcrd locus is contracted in both DN andDP thymocytes, Eα activity and Vα-to-Jα recombination occurspecifically in DP thymocytes. Therefore, we predicted that theremust be DP thymocyte-specific DNA looping and DNA contactswithin the contracted 3′ region that were not resolved by 3D-FISH. Here, we used 3C to document an Eα-dependent chro-matin hub at the 3′ end of the Tcra/Tcrd locus that forms spe-cifically in DP thymocytes. This chromatin hub brings 3′ Vα and5′ Jα gene segments into proximity to facilitate initial Tcra re-arrangement. We found that the Tcra/Tcrd locus is rich in CTCF-binding sites and that CTCF marks many locus cis-regulatoryelements. Moreover, the loss of CTCF in DP thymocytes dys-regulates long-distance interactions among these elements, sup-presses chromatin hub formation, and impairs initial Vα-to-Jαrearrangement. Our data suggest that Eα and CTCF cooperateto organize a DP thymocyte-specific chromatin hub that setsthe stage for synapsis and recombination of 3′ Vα and 5′ Jα genesegments.

ResultsIdentification of a DP Thymocyte-Specific Chromatin Hub at the 3′ Endof the Tcra/Tcrd Locus. Previous studies indicated that Eα regulatesup to 500 kb of the Tcra/Tcrd locus, including the Eα-proximal3′ Vα gene segments and the entire Jα array (36, 37). Activationof the TEAp at the 5′ end of the Jα array is believed to occurthrough direct enhancer–promoter interaction across 75 kb inDP thymocytes (30). However, it is unclear how the Eα–TEApinteraction is regulated during thymocyte development andwhether Eα activates 3′ Vα gene segments through directinteractions as well. To answer these questions, we performed3C assays to assess long-distance interactions across the Eα-proximal 400 kb of the Tcra/Tcrd locus (Fig. 1A). In 3C, inter-acting DNA sequences are trapped by formaldehyde cross-link-ing; after digestion with restriction enzymes, interacting DNAsequences are ligated and then detected by quantitative PCR(qPCR). To map interactions in cells carrying homogeneous,unrearranged Tcra/Tcrd alleles, we harvested DN thymocytesfrom recombinase-deficient Rag2−/− mice in which thymocytedevelopment is blocked at the CD44−CD25+ DN3 stage (38,39). We harvested DP thymocytes by injecting Rag2−/− mice withanti-CD3 antibody to stimulate DN-to-DP differentiation (40).To address the role of Eα, we analyzed Eα

−/−Rag2−/− mice insimilar fashion. As a control, we also analyzed splenic B cells.We first measured DNA interactions across the Jα–Cα region,

spanning 120 kb from Jδ2, a gene segment 90 kb upstream of Eα,to Dad1, a gene extending 30 kb downstream of Eα (Fig. 1 B andC). Using an Eα-containing HindIII fragment (Fig. 1B, fragment14) as an anchor, in control splenic B cells (Fig. 1B, black dashedline) we detected strong signals from neighboring fragments (Fig.

Fig. 1. Developmentally regulated interactions between Eα and the Jα-Cαregion. (A) The 1.6-Mb Tcra/Tcrd locus of strain 129 mice, with the 3′ 450 kbexpanded to show several V segments and the DδJδCδ and JαCα regions. Eδand Eα (ovals) and V and TEA promoters (bent arrows) are indicated. (B)Interactions between Eα and the Jα region. WT DN thymocytes (n = 3–8), WTDP thymocytes (n = 4–10), Ea

−/− DP thymocytes (n = 3–9), and B cells (n = 2–4)were analyzed by 3C using an Eα anchor. Interactions of the Eα-containingHindIII fragment (no. 14, black bar) with other HindIII fragments (nos.1–13and 15–18, gray bars) are plotted as means ± SEM with normalization to thenearest neighbor fragment, no. 15. White circles indicate CTCF-binding sites.(C) WT DN thymocytes (n = 3), WT DP thymocytes (n = 2–4) Ea

−/− DP thy-mocytes (n = 2), and B cells (n = 2) were analyzed by 3C using the TEAp as ananchor. Interactions of the TEAp-containing HindIII fragment (no. 4, blackbar) with other HindIII fragments (nos. 9–18, gray bars) are plotted withnormalization as in B. All thymocyte preparations were from Rag2−/− back-ground mice.

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1B, fragments 13 and 15) but very low signals at increasing dis-tances from Eα, reflecting the absence of long-distance inter-actions in this cell type. In DP thymocytes (Fig. 1B, red line), Eαwas found to interact strongly with the TEAp (Fig. 1B, fragment4) and to display gradually diminishing interactions moving from5′ to 3′ across the Jα region (Fig. 1B, fragments 6–11). Broadinteractions between Eα and 5′ Jα gene segments likely reflect thepresence of several minor Jα promoters that are distributed acrossthis region (41). In addition to these interactions, we noted rela-tively strong interactions, above the B-cell background, at sitesflanking the anchor (Fig. 1B, fragments 12 and 16). This resultsuggests that the whole region may be more compact in DP thy-mocytes than in B cells.DN thymocytes displayed substantially reduced Eα interac-

tions across the entire Jα array (Fig. 1B, blue line). Interactionswith the TEAp region (Fig. 1B, fragments 3–5) were reduced butwere clearly elevated above the B-cell background. To verifythis point, we also analyzed interactions between Eα and theTEAp using the TEAp-containing fragment (Fig. 1C, fragment4) as an anchor. Indeed, the TEAp interacted with Eα (Fig. 1 Band C, fragment 14) strongly in DP thymocytes and weakly inDN thymocytes compared with background interaction in B cells.To assess the role of Eα in mediating interactions across the

Jα–Cα region, we analyzed Eα−/− DP and Eα

−/− DN thymocytes.In Eα

−/− DP thymocytes, all identified Eα interactions were re-duced to the level of the B-cell background, suggesting that theseinteractions are strictly Eα dependent (Fig. 1 B and C, blackline). In addition, the weak Eα–TEAp interaction detected inDN thymocytes was also Eα dependent (Fig. S1). Because Eα isnot active in DN thymocytes, the weak Eα–TEAp interaction inthese cells must be mediated by Eα-bound factors independentof ongoing transcription. Overall, our data demonstrate that Eαparticipates in a range of lineage- and developmental stage-specific long-distance interactions across the Jα array in DN andDP thymocytes.We asked whether Eα similarly regulates the 3′ V gene seg-

ments through direct physical contacts in DP thymocytes. Usingthe Eα fragment as an anchor, we found that Eα interacted withseveral V gene segments (TRAV19, TRAV21, TRDV1, TRDV2-1,and TRDV2-2) at distances of 280–400 kb (Fig. 2 A and B, sitesi, iii, iv, vi, and vii, respectively). All these interactions weregreatly diminished in DN thymocytes, in Eα

−/− DP thymocytes,and in B cells, indicating that the interactions were lineage spe-cific, developmental stage specific, and Eα dependent. Similarresults were obtained by analysis using TRAV21 or TRDV2-2 asan anchor (Fig. 2C). However, we failed to detect interactionsbetween Eα and Vα gene segments located at the extreme 5′ endof the Tcra/Tcrd locus (Fig. S2). We conclude that in DP thy-mocytes Eα regulates the proximal 400 kb of the Tcra/Tcrd locusthrough direct interactions.Eα might interact in pairwise fashion with the TEAp and V

segments that are widely separated in nuclear space or mightinteract with these sites when all are in close physical proximity.To test these alternatives, we analyzed interactions between theTEAp and 3′ V segments. Indeed, contacts between the TEApand 3′ V segments (TRAV19, TRAV21, and TRDV2-2) (Fig. 2D and E) and between two 3′ V segments (TRAV21 andTRDV2-2) (Fig. 2F) were high in DP thymocytes but low in DNthymocytes and B cells, suggesting that these elements are inproximity in DP thymocytes. Importantly, all these interactionswere Eα dependent, indicating that Eα not only interacts with 3′V segments and the TEAp through direct pairwise interactionsbut also tethers them together to form a multicomponent chro-matin hub.Notably, both Eα and the TEAp interacted less well with in-

tergenic regions (Fig. 2 A, B, and D, sites ii and v). Eα alsointeracted poorly with Tcrd gene segments upstream of the TEAp(Fig. 1B). Therefore, inactive regions are looped-out from this

Eα-dependent, DP stage-specific chromatin hub. We propose thatthis chromatin hub functions to approximate Vα and Jα RSSs toincrease the probability of RSS synapsis, in this way supportingprimary Vα-to-Jα rearrangement in DP thymocytes.

TEAp Suppresses Tcrd Gene Segments in DP Thymocytes. Because theTEAp region interacts with Eα and multiple V segments in theDP stage-specific chromatin hub, we asked whether the TEApregulates long-distance interactions among these elements. Toanswer this question, we collected DP thymocytes from anti-CD3–injected TEAp−/−Rag2−/− mice (Fig. 3A) and performed

Fig. 2. Developmentally regulated interactions among Eα, the TEAp, and 3′V gene segments. (A) Distribution of 3′ V gene segments spanning 280–400kb upstream of Eα. Filled and open rectangles denote functional V genesegments and pseudogenes, respectively. Analyzed restriction fragments areidentified (i–vii). White circles indicate CTCF-binding sites. (B and C) Inter-actions between Eα and 3′ V gene segments. WT DN thymocytes (n = 3), WTDP thymocytes (n = 4–7), Ea

−/− DP thymocytes (n = 2–4), and B cells (n = 2–4)were analyzed by 3C using Eα (B), TRAV21 (C, Left), or TRDV2-2 (C, Right)HindIII fragments as anchors. (D and E) Interactions between the TEAp and3′ V gene segments. WT DN thymocytes (n = 3), WT DP thymocytes (n = 2–5),Ea

−/− DP thymocytes (n = 2–4), and B cells (n = 2–4) were analyzed as aboveusing TEAp (D), TRAV21 (E, Left), or TRDV2-2 (E, Right) HindIII fragments asanchors. (F) Interactions between 3′ V segments. WT DN thymocytes (n = 3),WT DP thymocytes (n = 4), Ea

−/− DP thymocytes (n = 2), and B cells (n = 2–4)were analyzed using TRAV21 (Left) or TRDV2-2 (Right) HindIII fragments asanchors. All thymocyte preparations were from Rag2−/− background mice.Data were plotted as means ± SEM with normalization as in Fig. 1B.

Shih et al. PNAS | Published online November 19, 2012 | E3495

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3C assays using the Eα fragment as an anchor. Compared withWT alleles (Fig. 3B, red line), TEAp-deleted alleles (Fig. 3B,blue line) demonstrated reduced interactions between Eα and 5′Jα segments (Fig. 3B, fragments 5 and 6) and increased Eα

interactions with central Jα segments (Fig. 3B, fragments 8 and9). This shift parallels the documented down-regulation of 5′ Jαpromoters and up-regulation of central Jα promoters on TEAp-deleted alleles (Fig. 3A) (41). Notably, we detected only a slightlyreduced Eα–TEAp interaction on TEAp-deleted alleles (Fig. 3B,fragment 4) but dramatically increased interactions between Eαand sites upstream of the TEAp in the region containing Jδ2, Eδ,Cδ, and TRDV5 (Fig. 3B, fragments 1–3). However, increasedinteractions did not extend as far as the 3′V gene segments (Fig. 3C and D). We suspect that increased interaction between Eα andTRDV5 obscured a diminished Eα–TEAp interaction on TEAp-deleted alleles. Further analysis also revealed that interactionsbetween the TEAp and 3′ V gene segments (TRAV21 andTRDV2-2) were reduced (Fig. 3E), but that interaction betweena Tcrd gene segment (Jδ2) and a 3′ V gene segment (TRDV2-2)was elevated in TEAp-deleted DP thymocytes (Fig. 3F). Takentogether, our data indicate that the TEAp region loops out fromthe Eα-dependent chromatin hub in TEAp-deletedDP thymocytesand that nearby Tcrd gene segments are included more frequentlyin that hub.TEA promoter deletion could impact long-distance interac-

tions through the loss of TEA transcription or through the loss ofTEAp-bound factors. To distinguish these possibilities, we ana-lyzed TEA-terminator (TEA-T) DP thymocytes, which have anactive TEAp but carry an introduced transcription terminatorimmediately downstream (Fig. 3A). TEA-T alleles mimic TEAp-deleted alleles with respect to the shift in promoter activity from5′ Jαs to central Jαs, suggesting that TEA transcription ratherthan the TEAp itself targets Eα to different regions of the Jαarray (42). We found that on TEA-T alleles, as compared withWT, the Eα–TEAp interaction was unperturbed (Fig. 3B, blackline, fragment 4), an Eα–5′ Jα interaction (Fig. 3B, fragment 6)was suppressed, and an Eα–central Jα interaction (Fig. 3B,fragment 8) was increased substantially. We suspect that reducedinteractions with 5′ Jα segments were partially obscured (Fig. 3B,fragment 5) by high-level interaction with the TEAp (Fig. 3B,fragment 4). However, in other respects, Eα interactions acrossthe Jα array were quite similar on TEAp-deleted and TEA-Talleles. Importantly, TEA-T alleles did not display the strongEα–Tcrd gene interactions that were apparent on TEAp-deletedalleles. This finding indicates that TEAp-bound factors, ratherthan TEAp-directed transcription, limit Eα from interactingwith upstream Tcrd gene segments. Our data demonstrate thatthe TEAp limits Eα interactions with Tcrd gene segments andwith central Jα gene segments through distinct mechanisms.To evaluate the biological consequence of increased interac-

tions between Eα and Tcrd gene segments on TEAp-deletedalleles, we analyzed transcriptional activity and chromatin ac-cessibility. Interestingly, TEAp-deleted alleles revealed two- tofivefold increases in transcription at Tcrd gene segments locatedupstream of the TEAp, including TRDV4, Dδ1, Jδ1, Jδ2, andTRDV5 (Fig. 4A). However, increased transcription did not extendto V segments further upstream (TRAV17, TRAV19, TRAV21,and TRDV2-2), suggesting that the impact of the TEAp isregional. We also analyzed histone H3 acetylation, a marker relatedto chromatin accessibility. Indeed, H3 acetylation also was increasedat Tcrd gene segments immediately upstream of the TEAp (Jδ1,Jδ2, and TRDV5) but not at more distant sites (Fig. 4B). Toevaluate whether loss of the TEAp also influences Tcrd genesegment recombination at the DP stage, we compared the fre-quency of TRAV17-Jα49 and TRDV5-Jα49 recombination inWT and TEAp−/− DP thymocytes (Fig. 4C). Remarkably,TRDV5-Jα49 recombination increased more than fourfold inTEAp−/− DP thymocytes, whereas TRAV17-Jα49 recombinationwas unchanged. Hence, the TEAp represses activation and re-combination of the Tcrd gene segments in DP thymocytes.

Fig. 3. Regulation of Eα contacts by the TEAp. (A) WT TEA promoter-de-leted (TEAp−), and TEA-terminator (TEA-T) alleles are diagrammed, withbent arrows denoting active promoters and gray shading denoting regionsof reduced accessibility and promoter activity on mutant alleles. (B) Inter-actions between Eα and Jα regions. WT (data from Fig. 1B), TEAp

−/− (n = 4–11), and TEA-T (n = 3–5) DP thymocytes were analyzed by 3C using an Eαanchor. (C and D) Interactions between Eα and 3′ V segments. WT (n = 5) andTEAp−/− (n = 4) DP thymocytes were analyzed by 3C using Eα (C), TRAV21 (D,Left), or TRDV2-2 (D, Right) as anchors. (E) Interactions between 3′ V genesand the TEAp. WT (n = 5) and TEAp−/− (n = 4) DP thymocytes were analyzedby 3C using TRAV21 (Left) or TRDV2-2 (Right) as anchors. (F) Interactionbetween TRDV2-2 and Jδ2. WT (n = 4) and TEAp

−/− (n = 4) DP thymocyteswere analyzed by 3C using the TRDV2-2 HindIII fragment as an anchor. Allthymocyte preparations were from Rag2−/− background mice. Data areplotted as means ± SEM with normalization as in Fig. 1B.


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Role for CTCF in the Regulation of Tcra Locus Recombination andConformation. Because CTCF has been implicated in long-dis-tance DNA interactions (11), we asked whether CTCF contributesto DNA contacts within the 3′ portion of the Tcra/Tcrd locus. Weused ChIP sequencing (ChIP-Seq) to assess the distribution ofTcra/Tcrd locus CTCF-binding sites in Rag1−/− DN thymocytes,Rag1−/− Tcrb transgenic DP thymocytes, and Rag1−/− pro-B cells.Because of the duplicated nature of the central portion of theTcra/Tcrd locus (Fig. 1A), we focused our analysis on CTCF sitesat the 5′ and 3′ ends of the locus. Within these regions, weidentified CTCF binding at many sites (Fig. 5 A and B). In DPthymocytes, CTCF binding appeared to mark many V segmentpromoters (Fig. 2A, white circles, and Fig. 5 A and B), and inevery case there was an identifiable CTCF consensus sequence(43) within 200 bp of the transcription start site. Notably, amongthe three CTCF-free proximal V gene segments (TRAV22,TRDV4, and TRDV2-1), one (TRAV22) is a pseudogene, andanother (TRDV4) is not used in adult thymocytes. Eα and theTEAp, which were shown to bind cohesin subunit Rad21 in aprevious study (30), also displayed strong CTCF binding; nota-bly, the Eα and TEAp CTCF sites are eliminated on Eα-deletedand TEAp-deleted alleles, respectively (Fig. 1B, white circles, andFig. 5B). The localization of CTCF at many regulatory elementssuggests that CTCF may mediate interactions among these ele-ments and contribute to Tcra/Tcrd locus regulation.Although CTCF binding was observed at many locations in

thymocytes and B cells, some sites appeared to display bindingthat was stronger in or unique to DP thymocytes, including sitesat TRAV18, TRAV19, TRAV20, TRAV21, TRDV3, and Jα42(Fig. 5B). Moreover, the ChIP-Seq experiments described abovewere conducted using samples that were of C57BL/6 origin,whereas our 3C experiments analyzed strain 129 Tcra/Tcrd locus

alleles. Therefore, we used ChIP-qPCR to confirm both consti-tutive and DP stage-specific CTCF binding to strain 129 alleles(Fig. 5C). Indeed, compared with a negative control site, PDβ, alltested C57BL/6 CTCF-binding sites were positive by ChIP-qPCRanalysis of strain 129 DP thymocytes. Also consistent with theChIP-Seq data, ChIP-qPCR revealed CTCF binding to be de-velopmentally regulated at many sites in the 3′ 450 kb of thelocus, including TRAV17-b, TRAV18, TRAV19, TRAV20,TRAV21, TRDV1, TRDV3, Jα42, and Dad1. In contrast, de-velopmentally regulated binding was not apparent at sites at the5′ end of the locus (TRAV3-1, TRAV6-1, and TRAV7-1) or atsome sites at the 3′ end (TRAV17-a, INT1, INT2, TRDV5,TEAp, and Eα). To determine whether DP stage-specific CTCFbinding is Eα dependent, we compared WT and Eα

−/− DP thy-mocytes (Fig. 5C). Interestingly, almost all sites displaying ele-vated CTCF binding in DP thymocytes were Eα dependent,

Fig. 4. Regulation of Tcrd gene segments by the TEAp. (A) qRT-PCR analysisof germ-line transcription in WT (n = 5) and TEAp−/− (n = 3) DP thymocytes(both on a Rag2−/− background). (B) ChIP and qPCR analysis of histone H3acetylation in WT (n = 2) and TEAp−/− (n = 2) DP thymocytes (both ona Rag2−/− background). (C) qPCR analysis of TRAV17-Jα49 and TRDV5-Jα49rearrangement in WT (n = 2) and TEAp−/− (n = 2) recombinase-sufficientCD71− DP thymocytes. Results are expressed as means ± SEM with normal-ization to recombination in WT thymocytes.

Fig. 5. CTCF binding to the Tcra/Tcrd locus. Rag1−/− DN thymocytes, Rag1−/−

Tcrb transgene DP thymocytes, and Rag1−/− pro-B cells (all on a C57BL/6background) were analyzed by ChIP-seq. Results for the 5′ 200-kb of thelocus are depicted in A, and results for the 3′ 450-kb portion are depicted inB. (C) CTCF binding to strain 129 Tcra/Tcrd locus sites was analyzed by ChIP-qPCR using Rag2−/− DN thymocytes (WT DN, n = 3–4), DP thymocytes pre-pared from anti-CD3–injected Rag2−/− (WT DP, n = 5–6) and Rag2−/−Eα

−/−

(Eα−/− DP, n = 4) thymocytes. TRAV17-a and -b are two neighboring CTCF sites

upstream of TRAV17. INT1 and INT2 are two intergenic sites between TRDV3and TRDV4. c-Myc and PDβ served as positive and negative controls, re-spectively. The data are plotted as means ± SEM.


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whereas sites displaying constitutive CTCF binding were not.Thus, Eα stimulates CTCF binding to multiple sites across the 3′portion of the Tcra/Tcrd locus in DP thymocytes. Because CTCF-binding sites colocalized with regions that interact physically withEα (Figs. 1B and 2 B and C) and with each other (Fig. 2 D–F andFig. S3) across the 3′ portion of the locus, we hypothesized thatCTCF is situated at the base of DNA loops and contributes tothe formation of the Eα-dependent chromatin hub.To examine whether CTCF regulates the Tcra/Tcrd locus, we

analyzed DP thymocytes of Ctcf conditional-knockout mice (Fig.6A). To assess the potential influence of CTCF on Tcra re-combination, we bred Ctcf f/f mice with Lck-Cre mice to initiateCtcf deletion in DN thymocytes. We also bred these alleles ontoa Rag2-deficient background to study changes in germ-line tran-scription and locus conformation in the absence of CTCF. InCtcf f/f Lck-Cre mice, thymocyte development was partiallyblocked at the immature single-positive stage during the DN-to-DP transition (Fig. 6B), as described previously (44). We sortedCTCF-deficient DP thymocytes based on their expression of aLacZ marker and compared them with DP thymocytes sortedfrom their WT (Lck-Cre−) littermates (Fig. 6B). We further usedCD71 staining to distinguish early CD71+ from later CD71− DPthymocytes (30). CTCF-deficient DP thymocyte preparationstypically displayed >90% loss of Ctcf alleles and substantiallyreduced CTCF protein (Fig. 6 C and D).To assess an effect of Ctcf deletion on early Vα-to-Jα recom-

bination, we quantified Tcra coding joints by qPCR using primersspecific for 3′ Vα (TRAV17 and TRAV19) and 5′ Jα (Jα61, Jα56,and Jα49) gene segments (Fig. 6E). Analysis of WT DP thymo-cytes revealed low-level, Jα61-biased recombination in early(CD71+) DP thymocytes, with elevated and more uniformlydistributed recombination in late (CD71−) DP thymocytes. Thisresult is consistent with the notion that early rearrangements arepreferentially targeted to Jα61 and that these rearrangementsoften are replaced by subsequent rearrangements to downstreamJα gene segments. Notably, we observed substantial reductionsin all these Vα-Jα recombination events in both early and lateCTCF-deficient DP thymocytes (Fig. 6E). Thus, CTCF is essen-tial for efficient recombination of 3′ Vα and 5′ Jα gene segments.To determine whether CTCF regulates early Tcra rearrange-

ment by mediating loop formation within the 3′ portion of Tcra/Tcrd locus, we used 3C to analyze interactions within the DPstage-specific chromatin hub in LacZ+ DP thymocytes from anti-CD3 antibody–stimulated Ctcf f/f Rag2−/− Lck-Cre mice. Indeed,we observed that loss of CTCF reduced Eα contacts with theTEAp (Fig. 7 A, fragment 4, and Fig. 7B), with Jα segments (Fig.7 A, fragments 5–9, and Fig. 7B), and with certain 3′ V genesegments (TRAV19, TRAV21, and TRDV1) (Fig. 7 C and D).In addition, the loss of CTCF also reduced TEAp contacts withcertain 3′ V gene segments (TRAV19 and TRAV21) (Fig. 7 Eand F), indicating that the Eα-dependent chromatin hub is par-tially but not completely disrupted. However, loss of CTCF in-creased Eα contacts with Tcrd gene segments (Fig. 7A, fragments1 and 2), so that the Eα bias toward 5′ Jα over Tcrd gene segmentsthat characterizes WT DP thymocytes was eliminated in CTCF-deficient DP thymocytes. We suggest that CTCF promotes pri-mary Tcra gene recombination by targeting Eα to 3′ Vα and 5′ Jαsegments and increasing the probability of physical interactionsbetween these gene segments.Because CTCF supports normal chromatin contacts within the

3′ portion of the Tcra/Tcrd locus, we asked whether CTCF alsois involved in the contraction of the 3′ end in DP thymocytes. Weperformed 3D-FISH to measure the distance between foci de-tected by two probes, one hybridizing to V segments in the centralportion of the V gene array and one hybridizing downstream ofCα. These probes had been used previously to detect contractionof the 3′ end in thymocytes compared with B cells (35, 45). In fact,we observed slightly greater contraction in CTCF-deficient than

in WT DP thymocytes (Fig. 7G). Hence, CTCF is not required forTcra/Tcrd locus contraction.We then asked whether the disruption of CTCF-dependent

DNA contacts also impacts Tcra/Tcrd locus germ-line transcrip-tion in CTCF-deficient DP thymocytes. As expected, loss ofCTCF caused reduced transcription at TEA (Fig. 8A). Remark-ably, transcription of Tcrd gene segments (Jδ1, Jδ2, and TRDV5)was increased substantially (Fig. 8A), in a manner nearly identical

Fig. 6. Regulation of Vα-Jα recombination by CTCF. (A) The Ctcf floxed anddeleted alleles are diagrammed, with Ctcf exons numbered, loxP sites rep-resented by triangles, and PGK-puromycin (puror) and splice acceptor-lacZ(SA-LacZ) cassettes indicated. Ctcf-deleted alleles produce a Ctcf-LacZ fu-sion protein. (B) Representative flow cytometric analysis of Ctcf f/f (WT) andCtcf f/f Lck-Cre (KO) littermates. Thymocytes were gated for CD4+CD8+ (Left)and CD71+ or CD71− (Center) and were incubated with lacZ substrate FDGto identify Ctcf-deficient CD71+ or CD71− DP thymocytes (Right; WT, grayshading; KO, black line). (C) qPCR analysis of Ctcf genomic DNA in CD71+

(Left) and CD71− (Right) DP thymocytes sorted from anti-CD3–injected WT(n = 4) and Ctcf-deficient (KO, n = 3) mice. (D) Representative Western blotanalysis of CTCF protein in sorted thymocytes used in C. Analysis of PLCγ1controlled for total protein. (E) Genomic DNA isolated from CD71+ (early)and CD71− (late) DP thymocytes of WT (n = 4) and Ctcf-deficient (KO, n = 4)mice were analyzed by qPCR to measure Vα-to-Jα recombination usingTRAV17 and TRAV19 primers paired with Jα61, Jα56, and Jα49 primers. Resultsare expressed as means ± SEM with normalization to recombination in un-fractionated WT thymocytes.


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to that documented on TEAp-deleted alleles (Fig. 4A). Increasesin Eα-Tcrd gene segment contacts and Tcrd germ-line transcrip-tion in both TEAp-deleted and CTCF-deficient DP thymocytessuggest that Tcrd gene segments are activated by Eα through theloss of CTCF binding at the TEAp.These data imply that CTCF normally may function to limit

Tcrd gene activation in DP thymocytes. Because thymocytes newlyentering the DP compartment may not have undergone Vδ-to-Dδrearrangement on as many as 30% of alleles (Fig. S4), CTCF also

may play an important role in limiting Tcrd gene recombinationin DP thymocytes. To test this notion, we used ligation-mediatedqPCR (LM-qPCR) to detect RAG-mediated DSBs at the 5′ Dδ1and Jδ1 RSSs. Indeed, we observed increased DSBs in both early(CD71+) and late (CD71−) CTCF-deficient DP thymocytes atboth sites (Fig. 8 B and C). This result supports the idea that CTCFspecifies Eα targets to regulate Tcra/Tcrd locus recombination.By simultaneously facilitating Eα–TEAp and suppressing Eα–Tcrdgene segment interactions, CTCF helps commit the locus to V-to-Jα rather than V-to-DδJδ recombination in DP thymocytes.

DiscussionWe previously used 3D-FISH to show that the 3′ end of the Tcra/Tcrd locus is contracted similarly in DN and DP thymocytes, ascompared with its extended configuration in B cells (35). Herewe used 3C to detect a network of interactions between cis-regulatory elements at the 3′ end of the Tcra/Tcrd locus spe-cifically in DP thymocytes, revealing the formation of a develop-mental stage-specific chromatin hub within the contracted portionof the locus (Fig. 9). We found that all detected DNA contactswithin this chromatin hub depend critically on Eα, a potent en-hancer that is known to regulate Tcra/Tcrd locus transcriptionand histone modifications across 500 kb in DP thymocytes. Ourdata reveal that Eα regulates individual Vα and Jα gene segmentsthrough long-distance physical interactions. Moreover, Eα recruitsVα and Jα gene segments into proximity in a manner that shouldfacilitate synapsis of Vα and Jα RSSs.Tcra recombination occurs in multiple cycles starting with

primary recombination events that preferentially use 3′ Vα and 5′

Fig. 7. Regulation of DP thymocyte-specific DNA contacts by CTCF. (A)Interactions between Eα and Jα regions. DP thymocytes isolated from Ctcf

f/f

Lck-Cre−Rag2−/− (WT; n = 2–3) and Ctcff/f Lck-Cre+Rag2−/− (KO; n = 4–6) micewere analyzed by 3C using an Eα anchor. (B) Interaction between Eα and theTEAp. WT (n = 6) and KO (n = 6) DP thymocytes were analyzed using theTEAp fragment as an anchor. (C) Interactions between Eα and 3′ V segments(fragment nomenclature as in Fig. 2A). WT (n = 6) and KO (n = 6) DP thy-mocytes were analyzed using the Eα fragment as an anchor. (D) Interactionsbetween Eα and 3′ V gene segments. WT (n = 5) and KO (n = 5) DP thymo-cytes were analyzed using TRAV21 (Left) and TRDV2-2 (Right) fragments asanchors. (E and F) Interactions between 3′ V gene segments and the TEAp.WT (n = 5) and KO (n = 5) DP thymocytes were analyzed using (E) the TEApfragment or (F) TRAV21 (Left) or TRDV2-2 (Right) fragments as anchors.Data are plotted as means ± SEM. (G) 3D-FISH was used to measure the dis-tance between foci detected by a probe hybridizing to central V gene seg-ments and a probe hybridizing downstream of Cα. Scatter-plots display dis-tances between the centers of probe hybridization in DP thymocytes of Ctcff/f

Lck-Cre−Rag2−/− (WT) and Ctcff/f Lck-Cre+Rag2−/− (KO) mice carrying either the129 or the C57BL/6 Tcra/Tcrd locus. Median values are indicated by horizontallines. Data are from one experiment for each cell type (66–104 alleles).

Fig. 8. Regulation of Tcrd gene transcription and recombination in DPthymocytes by CTCF. (A) qRT-PCR analysis of Tcra/Tcrd locus transcripts in DPthymocytes isolated from Ctcff/f Lck-Cre−Rag2−/− (WT, n = 2–3) and Ctcff/f Lck-Cre+Rag2−/− (KO, n = 4–6) mice. (B and C) LM-qPCR analysis of DSBs in ge-nomic DNA of CD71+ and CD71− DP thymocytes sorted from Ctcff/f Lck-Cre−

(WT, n = 3) and Ctcff/f Lck-Cre+ (KO, n = 3) mice. Specific primers were used todetect DSBs at the RSS 5′ of Dδ1 (B) and at the Jδ1 RSS (C). DSB frequency wasnormalized to the level in Lat−/− DN thymocytes. DP thymocytes isolatedfrom Rag2−/− Tcrb transgene mice served as a negative control (Ctrl). Dataare plotted as means ± SEM.


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Jα gene segments. Because these gene segments are recruitedselectively into the initial chromatin hub by Eα, our data suggestthat this hub serves as a platform to support ordered Tcra re-combination. The 5′ Jα segments previously were shown to sup-port high-level RAG binding to form a recombination center(46). The chromatin hub described here provides direct evidencefor the recruitment of distant V segments into this recombina-tion center, thereby offering strong support for the recombina-tion center model (2).CTCF was a strong candidate for regulating the DP thymo-

cyte-specific chromatin hub because it has been shown to facili-tate DNA looping and contacts in other instances and becausewe found it to bind Eα, the TEAp, and many V gene segmentpromoters (Fig. 9). Indeed, the Eα–TEAp interaction was par-tially diminished in Ctcf conditional-knockout DP thymocytes,suggesting that CTCF binding at these two sites may contributeto their interaction. In addition, loss of CTCF reduced inter-actions between Eα and 3′ V gene segments and between theTEAp and 3′ V gene segments. Reductions in these various DNAcontacts likely explain impaired Tcra gene transcription and re-combination in CTCF-deficient DP thymocytes. Notably, CTCFbinding to Eα and to the TEAp is not developmentally regulated,whereas the Eα–TEAp interaction and TEA transcription clearlyare. We propose that enhancer- and promoter-bound transac-tivators other than CTCF provide developmental-stage specificityand that they cooperate with bound CTCF to mediate these de-velopmentally regulated events. Thus, the inefficient Eα–TEApinteraction in DN thymocytes may be mediated primarily by Eα-bound CTCF, with efficient Eα–TEAp interaction in DP thymo-cytes dependent not only on CTCF but also on additional Eα–bound transactivators induced at the DN-to-DP transition (47).Viewed in this way, CTCF may prime the TEAp to be targetedby Eα when Eα is activated in DP thymocytes. CTCF may primethe promoters of 3′ V gene segments similarly. However, some 3′V segment promoters display induced and Eα-dependent CTCFbinding in DP thymocytes, suggesting that CTCF recruitment andstabilization may depend on enhancer–promoter interaction aswell.CTCF and cohesin have been shown to colocalize and function

coordinately at many genomic sites (32–34). Because the re-duction of Eα–TEAp interaction and TEA expression in CTCF-deficient DP thymocytes was similar to the phenotype of cohesinsubunit Rad21-deficient DP thymocytes, it appears that CTCFand cohesin regulate the Tcra/Tcrd locus through the samepathway (30). Notably, Cd4-Cre–triggered deletion of Rad21caused a defect only in secondary Tcra recombination, whereasLck-Cre–triggered deletion of Ctcf caused a defect in primaryTcra recombination. This difference likely reflects the fact that

the Cd4-Cre–mediated deletion of Rad21 occurs too late toimpact primary Tcra recombination events (30). Nevertheless,the two results, taken together, argue that CTCF and cohesin arerequired continuously throughout the course of primary andsecondary Tcra recombination events in DP thymocytes.Although CTCF was shown previously to be needed for com-

plete Igh locus contraction (25), we did not observe impairedTcra/Tcrd locus 3′ end contraction in Ctcf-knockout DP thymo-cytes (Fig. 9). Moreover, we previously observed no loss of 3′ endcontraction on Eα-deleted Tcra/Tcrd locus alleles (35). Thus,neither CTCF nor Eα is required for 3′end contraction, and themechanisms of Tcra/Tcrd locus contraction remain unknown. Wespeculate, however, that 3′end contraction provides an initiallayer of organization that is essential to allow CTCF and Eα tomediate the DNA contacts needed for the activation, synapsis,and recombination of 3′ Vα and 5′Jα gene segments. This conceptwould explain why Eα-dependent DNA contacts and germ-linetranscription are detectable at the 3′ but not the 5′ end of thelocus, even though CTCF binds to both. It also would explainwhy the Eα–TEAp interaction is not detectable in B cells, even atlow levels, although CTCF binds to both elements in these cells.Although the ability of Eα to interact with upstream CTCF-

bound promoters was compromised in CTCF-deficient DP thy-mocytes, interactions with Tcrd gene segments that lack CTCF-binding sites were elevated (Fig. 9). As a result, Tcrd genesunderwent elevated transcription and recombination in CTCF-deficient as compared with WT DP thymocytes. We concludethat CTCF is not essential for long-distance contacts by Eα.Rather, CTCF biases these contacts to discrete sites in the locusand in this manner suppresses aberrant interactions.Finally, we note that the unique distribution of CTCF sites at

the Tcra/Tcrd locus may dictate a fundamentally different rolefor CTCF in the Tcra/Tcrd locus than in the Igh and Igk loci. AtIgh and Igk, CTCF sites are generally intergenic except for thosein the proximal one-third of the VH array, which are downstreamof VH RSSs; moreover, CTCF sites are not associated with themajor Igh and Igk enhancer elements (25, 26, 48–50). At theseloci, CTCF promotes long-distance interactions between CTCF-binding elements (24–26), but these looping interactions appearprimarily to restrict or insulate the activities of the major enhancerelements. As examples, CTCF-binding regions situated betweenVH and DHJH and between Vκ and Jκ segments appear to blockthe enhancers from activating relatively proximal V segments,thus promoting the formation of Igh and Igk repertoires in whichnatural biases to proximal V segments are suppressed (24, 26,50). Our data suggest that CTCF binding at the TEA promoterinhibits Eα from activating Tcrd gene segments immediately up-stream; thus, by analogy to Igh and Igk, the TEAp CTCF site

Fig. 9. An Eα- and CTCF-dependent chromatin hub within the contracted 3′ portion of the Tcra/Tcrd locus. In WT DP thymocytes, Eα and CTCF cooperate tonucleate interactions (thick lines) among V promoters and the TEAp within the contracted region. Eα-deleted alleles remain contracted, but long-distanceDNA contacts that compose the hub are disrupted. In CTCF-deficient DP thymocytes, Eα interactions with some V and Jα segments are reduced (thin lines),whereas interactions with Dδ and Jδ segments are increased (thin lines), leading to reduced Tcra rearrangement but increased Tcrd rearrangement in DPthymocytes.


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could be considered an insulator that limits Tcrd gene activationin DP thymocytes. However, unlike Igh and Igk, CTCF sites atthe Tcra/Tcrd locus mark Eα and many locus-promoter elements.In this scenario, the ability of CTCF to promote long-distanceinteractions, rather than insulating promoters from enhancers,appears to direct Eα to CTCF-marked promoters. Viewed in thislight, the Tcrd activation phenotype reflects not an insulationdefect but, more directly, a targeting defect. We suggest thatCTCF may influence enhancer–promoter communication at an-tigen receptor loci through predominant insulating or targetingmechanisms, with the distribution of CTCF-binding sites beingthe critical determinant.

Materials and MethodsMice. Eα

−/− mice (37), TEAp−/− mice (41), TEA-T mice (42), and Ctcf f/f mice (44)have been described previously. To induce Ctcf deletion in early DN thy-mocytes, Ctcf f/f mice were bred with Lck-Cre mice kindly provided by JeffreyRathmell (Duke University, Durham NC). Mice carrying modified Tcra/Tcrdand Ctcf alleles also were bred with Rag2−/− mice (38) for analysis on arecombinase-deficient background. Eα

−/−Rag2−/−, TEAp−/−Rag2−/−, TEA-TRag2−/−, and Ctcff/f Lck-Cre mice were of mixed genetic background butcarried the 129 Tcra/Tcrd locus. Ctcff/f Lck-Cre Rag2−/− mice were either ona mixed background with a 129 Tcra/Tcrd locus or on a pure C57BL/6 back-ground. C57BL/6 background Rag1−/− and Rag1−/− Tcrb transgene mice wereused for ChIP-seq. All mice were used in accordance with protocols approvedby the Duke University Animal Care and Use Committee.

Cell Collection. DN thymocytes were obtained from 3-wk-old Rag2−/−, Eα−/−

Rag2−/−, TEAp−/− Rag2−/−, and TEA-T Rag2−/− mice. DP thymocytes wereobtained from these mice 10 d after a single i.p. injection of 150 μg anti-CD3antibody (145-2C11; BioLegend). DP thymocytes were sorted from Ctcf f/f

Lck-Cre+ mice or Ctcf f/f Rag2−/− Lck-Cre+ mice or their Lck-Cre− littermates7 d after a single i.p. injection of 75 μg anti-CD3 antibody, using anti–CD71-PE (RI7217), anti–CD4-PE/Cy7 (GK1.5), and anti–CD8-APC/Cy7 (53-6.7). Be-cause Ctcf deletion induces LacZ expression, thymocytes were incubatedwith LacZ substrate fluorescein di β-D-galactopyranoside (FDG) from a Fluo-Reporter lacZ flow cytometry kit (Invitrogen) before sorting to identify LacZ+

cells. Splenic B lymphocytes were sorted from WT 129 mice by using anti–B220-PE/Cy7 (RA3-6B2) together with anti–CD3-PE5 (145-2C11), anti–CD4-PE5 (GK1.5), and anti–CD8-PE5 (53-6.7) to exclude T cells. All antibodies wereobtained from BioLegend.

3C. The 3C assays were performed as described previously with slight mod-ifications (51). In brief, 10 million cells were cross-linked in 8 mL RPMI con-taining 10% FBS (vol/vol) and 2% paraformaldehyde (vol/vol) for 10 min onice, followed by the addition of glycine to 0.125 M to stop the cross-linkingreaction. After washing with 1× PBS, cells were lysed in 10 mM Tris (pH8.0),10 mM NaCl, and 0.2% (vol/vol) Nonidet P-40 for 10 min on ice. Nuclei werepelleted by centrifugation and resuspended with 1.1× restriction enzymebuffer (NEBuffer 2; New England BioLabs) containing 0.3% (wt/vol) SDS.After 1 h of incubation at 37 °C, Triton-X was added to a final concentrationof 2% (vol/vol) for an additional hour of incubation at 37 °C to neutralizethe SDS. Chromatin then was digested by the addition of 200 U HindIII (NewEngland BioLabs) for overnight incubation at 37 °C, followed by a secondaddition of 200 U HindIII for an additional 4 h. Digestion efficiency averaged97%. Digestion was stopped by the addition of SDS to 0.8% (wt/vol) andheat inactivation at 68 °C for 10 min. Digested chromatin was purified fromcellular proteins by centrifugation for 16 h at 35,000 rpm through 8 M ureain a Beckman SW40Ti rotor at 10 °C. The cross-linked chromatin pellet wasresuspended in 2 mL of 30 mM Tris·HCl (pH 7.4) and 10 mM MgCl2 and wasdialyzed against the same buffer overnight to remove urea. Purified chro-

matin then was diluted by the addition of 5 mL of the same buffer followedby DTT to 10 mM and ATP to 1 mM. The chromatin then was ligated by theaddition of 200 U T4 ligase (New England BioLabs) for overnight incubationat 16 °C, followed by the addition of another 200 U of T4 ligase for 4 hadditional incubation. Ligated DNA was collected after overnight incubationat 65 °C with 10 μg/mL proteinase K and purification by phenol/chloroformextraction and ethanol precipitation. 3C products were quantified by Taq-man-based real-time qPCR assays using a LightCycler 480 probe master kit(Roche) and a LightCycler 480 Real-Time PCR system (Roche). The followingPCR program was used: 95 °C for 10 min, followed by 48 cycles of 95 °C for10 s and 65 °C for 30 s. All PCR reactions were run in duplicate. The se-quences of probes and PCR primers are shown in Table S1. An unbiasedpool of 3C products generated by digestion and religation of equally mixedbMQ-440L6 and bMQ-206H21 BACs was used to generate standard curves.bMQ-440L6 spans proximal Vα/δ segments from TRAV19 to downstream ofTRDV2-2, whereas bMQ-206H21 spans from Jδ2 to downstream of Dad1.Samples were normalized by setting the 3C signal between Eα and its 3′neighbor fragment to one.

Germ-Line Transcription and Tcra Recombination. RNA was isolated usingTRIzol reagent (Invitrogen) and was converted to cDNA using SuperScript III(Invitrogen) and random hexamers according to the manufacturer’s in-structions. Genomic DNA was isolated from sorted DP thymocytes by stan-dard procedures. Tcra transcripts and rearranged DNA were quantified byreal-time PCR using a QuantiFast SYBR Green PCR kit (Qiagen). All PCRreactions were run in duplicate using the following amplification program:95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s and 62 °C for 30 s.Primers for transcript analysis are shown in Table S2. Samples were nor-malized to signals for Actb. Primers for rearrangement analysis are shownin Table S3. Samples were normalized to signals for B2m.

ChIP. Chromatin to be used for ChIP of acetylated histone H3 was preparedand immunoprecipitated using rabbit antiserum to acetylated H3 histone(06-599; Millipore) or control rabbit IgG (ab-105-c; R&D Systems) as previouslydescribed (52). Chromatin to be used for ChIP of CTCF was prepared byformaldehyde cross-linking as described (53) and was immunoprecipitatedusing rabbit antiserum to CTCF (07-729; Millipore) or control rabbit IgG (ab-105-c; R&D Systems). In both cases, samples were analyzed by real-time qPCRusing the QuantiFast SYBR Green PCR kit. All PCR reactions were run induplicate using the following amplification program: 95 °C for 5 min, fol-lowed by 45 cycles of 95 °C for 10 s and 62 °C for 30 s. PCR primers for anti-AcH3 ChIP are shown in Table S4. PCR signals were normalized first to theamount of input DNA and then to acetylation of Actb. MageA2 served asa negative control. Primers for CTCF ChIP are shown in Table S5. Signals werenormalized first to input DNA and then to c-Myc for a positive control.

ChIP-Seq. CTCF ChIP-seq was performed as described in ref. 52.

3D-FISH. 3D-FISH was performed using BAC clones RP24-334B8 and RP23-10K20 as described in ref. 35.

LM-qPCR. Genomic DNA was prepared from sorted DP thymocytes, and linkerligation was performed as described in refs. 54 and 55. Ligated signal endswere detected by Taqman-based real-time qPCR assays. PCR reactions wereperformed using the program described for 3C analysis. Primers and probesare shown in Table S6.

ACKNOWLEDGMENTS.We thank Chih-Wen Ou-Yang and Yu-tsung Chen fortechnical advice and Nancy Martin of Duke Cancer Institute Flow Core Facil-ity for help with cell sorting. This work was supported by National Institutesof Health Grants R37 GM41052 (to M.S.K.) and RO1 AI082918 (to A.J.F.). A.T.was supported in part by National Institutes of Health Grant UL1 RR025774.

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