New roles for DNA cytosine modification, eRNA,anchors, and superanchors in developing Bcell progenitorsChristopher Bennera,1,2, Takeshi Isodab,1, and Cornelis Murreb,2

aThe Integrative Genomics and Bioinformatics Core, The Salk Institute for Biological Studies, La Jolla, CA 92037; and bDepartment of Molecular Biology,University of California at San Diego, La Jolla, CA 92093

Edited by Frederick W. Alt, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Howard Hughes MedicalInstitute, Boston, MA, and approved September 1, 2015 (received for review July 1, 2015)

B-cell fate is orchestrated by a series of well-characterized de-velopmental regulators. Here, we found that the onset of B-celldevelopment was accompanied by large-scale changes in DNA cy-tosine modifications associated with promoters, enhancers, andanchors. These changes were tightly linked to alterations in trans-cription factor occupancy and nascent RNA (eRNA) transcription. Wefound that the prepro-B to the pro–B-cell transition was associatedwith a global exchange of DNA cytosine modifications for poly-comb-mediated repression at CpG islands. Hypomethylated regionswere found exclusively in the active/permissive compartment ofthe nucleus and were predominantly associated with regulatoryelements or anchors that orchestrate the folding patterns of thegenome. We identified superanchors, characterized by clusters ofhypomethylated CCCTC-binding factor (CTCF)-bound elements, whichwere predominantly located at boundaries that define topologicalassociated domains. A particularly prominent hypomethylatedsuperanchor was positioned down-stream of the Ig heavy chain(Igh) locus. Analysis of global formaldehyde–cross-linking studiesindicated that the Igh locus superanchor interacts with the VH

region repertoire across vast genomic distances. We proposethat the Igh locus superanchor sequesters the VH and DHJH regionsinto a spatial confined geometric environment to promote rapidfirst-passage times. Collectively, these studies demonstrate how,in developing B cells, DNA cytosine modifications associated withregulatory and architectural elements affect patterns of geneexpression, folding patterns of the genome, and antigen recep-tor assembly.

DNA modification | superanchor | nuclear architecture | immunoglobulinheavy chain locus | superinsulator

The onset of B-cell development is initiated in the fetal liver oradult bone marrow at the common lymphoid progenitor cell

(CLP) cell stage (1, 2). Specification to the B-cell lineage is es-tablished by a spectrum of transcriptional regulators that actcollaboratively to prime and ultimately activate a B-lineage–specific program of gene expression (3, 4). Conspicuous amongthe activators that establish B-cell identity are the E2A, EBF1,and FOXO1 proteins (5–8). In CLPs, the E2A proteins inducethe expression of FOXO1, which, in turn, activates EBF1 tran-scription (6). EBF1 and FOXO1 subsequently act in a regulatoryfeedback loop to induce an active enhancer repertoire and ac-tivate a B-lineage specific program of gene expression (9, 10).It is now well established that the chromatin fiber is marked

by DNA methylation (11–13). Methylation of cytosines primarilyoccurs at CpG residues, although methylation in CHG and CHHnucleotides has also been observed (14). The vast majority of CpGnucleotides are methylated. Unmethylated CpG residues and CpGislands are closely associated with transcriptionally active promoters.Other demethylated regions, found distal to gene promoters, tend tobe cell-type specific, with changes in DNA methylation state oftencorrelating with alterations in patterns of gene expression (15).Unlike many other epigenetic modifications, mCpG has a well-

understood maintenance mechanism that copies methylation statesduring the cell cycle mediated by DNMT1 (16). Recent studieshave shown that patterns of DNA methylation are dynamic,with specific enzymes involved in de novo DNA methylation(DNMT3) and demethylation (TET1-3) (17). TET proteins ox-idize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine(5fC), and 5-carboxylcytosine (5caC) (18–20).Here, we describe how DNA cytosine modifications associ-

ated with regulatory and architectural elements affects patternsof gene expression, folding patterns of the genome, and antigenreceptor assembly.

ResultsDNA Methylation Landscape in B Cells. To determine whether al-terations in DNA cytosine modifications are associated with theonset of B-cell development, pro-B cells were isolated fromRAG1-deficient mice and cultured in the presence of IL-7 andSCF. Purified DNA was treated with sodium bisulfite by usingstandard procedures. Bisulfite sequencing does not distinguish5mC from 5hmC and C from 5fC and 5caC (20, 21). Hence, wewill refer to unconverted cytosines as mC (includes 5hmC/5mC),whereas converted cytosines will be referred to as C (includes 5fC/5caC/C). Consistent with previous observations, most CpGs acrossthe pro–B-cell genome were heavily methylated (81.8% on aver-age). Profiles of mCpG% at genes revealed hypomethylated

Significance

B cells are destined to produce a wide spectrum of antibodiesor immunoglobulins in response to the invading pathogens.Here, we found that the onset of B-cell development wasaccompanied by large-scale changes in DNA cytosine modifi-cations associated with DNA elements that control gene ex-pression and the folding patterns of genomes. We identifynovel DNA elements that function as superanchors and/orsuperinsulators. A prominent super-anchor is located in theimmunoglobulin heavy chain locus where it acts to facilitatethe interactions among variable, diversity, and joining DNAsegments. We propose that changes in DNA cytosine modifi-cations at regulatory DNA elements orchestrate a B cell-spe-cific transcription signature and genome structure to enablethe production of a diverse antibody repertoire.

Author contributions: C.B., T.I., and C.M. designed research; C.B. and T.I. performed re-search; C.B. analyzed data; and C.B. and C.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequence reported in this paper has been deposited in the GenBankdatabase (accession no. GSE72670).1C.B. and T.I. contributed equally to this work.2To whom correspondence may be addressed. Email: murre@biomail.ucsd.edu orcbenner@salk.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1512995112/-/DCSupplemental.

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promoter regions and elevated levels of methylation at genebodies (Fig. S1A).We next performed an unbiased search for hypomethylated re-

gions across the pro–B-cell genome, finding 43,363 regions with anaverage mCpG% less than 30%. The majority of hypomethylatedregions mapped to promoter-distal regions (Fig. S1B). Clustering ofthe hypomethylated domains revealed distinct groups that wereidentified based on the deposition of H3K4me3 (promoters),H3K4me1/2 (enhancers), H3K27me3 (polycomb bodies), andCCCTC-binding factor (CTCF) occupancy (Fig. 1 A and B and Fig.S1D). We found that the cistromes for all relevant transcriptionfactors were hypomethylated (Fig. S1C). Factors closely associatedwith promoter binding (i.e., Yy1) showed wider windows of hypo-methylation, whereas factors typically binding at enhancer elements(i.e., E2A) displayed a narrower pattern of decreased methyl-ation (Fig. S1C). CTCF occupancy was closely associated withnucleosome-sized fluctuations in the mCpG/CpG ratios (Fig. 1C).Using Global Run-On sequencing data from pro-B cells

(GRO-Seq), we assessed the differences in methylation profiles atintergenic E2A-bound sites associated with transcriptionally activeregions producing nascent RNA (eRNA) versus E2A-bound sitesthat were transcriptionally inactive (Fig. 1D). We found that E2Aoccupancy was associated with hypomethylation at bound sites,regardless of transcriptional activity (Fig. 1D and Fig. S1E).However, E2A bound sites at transcriptionally active enhancersshowed broader regions of hypomethylated DNA (Fig. 1D andFig. S1E). A similar pattern of mCpG depletion was observed

when considering gene transcription start sites with unidirec-tional versus bidirectional transcription. Hypomethylation waspredominately associated with promoters and genomic regionspositioned immediately downstream of active transcription, whichstrongly correlates with levels of H3K4me3 (Fig. 1E and Fig. S1F).Taken together, these data suggest a mechanistic link betweennoncoding RNA transcription and modulating DNA cytosinemodifications associated with enhancer regions.

Global Changes in Enhancer Methylation During B-Cell Development.To determine whether the pre–pro-B to pro–B-cell transition isaccompanied by changes in DNA methylation, DNA was extractedfrom E2A-deficient prepro-B cells. The overall pattern of methyl-ation was similar as described for pro-B cells (80.7% mCpG over-all), including depletion of mCpG at transcriptional start sites andincreased levels of DNA cytosine modifications associated withgene bodies (Fig. S2A). More than 14,000 genomic regions wereidentified that displayed large-scale changes in DNA cytosine mod-ifications, termed DMRs (differential methylated regions) (Fig.S3A). A large fraction of these changes could be segregated into twogroups that exchanged mCpG for either deposition of H3K4me2or H3K27me3 (Fig. S3A). In contrast, a mere 1,299 genomic re-gions displayed the reverse pattern (Fig. S3A). Prepro-B–specificDMRs were predominantly associated with prepro-B–specificH3K4me2 levels (Fig. S3A). Many of these changes in patterns ofmethylation occurred at the promoters of prominent B-lineagegenes (Fig. S2 B and C).

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Fig. 1. Global patterns of hypomethylation acrossregulatory elements in the pro–B-cell genome. (A) Un-biased hierarchical clustering of hypomethylatedgenomic regions with respect to mCpG%, histonemodification ChIP-Seq, DNase hypersensitivity, andCTCF ChIP-Seq in pro-B cells. Active promoters werecharacterized by enrichment for H3K4me3. En-hancer regions were assigned by deposition ofH3K4me1/2. Polycomb bodies were identified bythe deposition of H3K27me3. (B) Hypomethylatedgenomic regions associated with the Pax5 locus.Hypomethylated regions were observed across thePax5 promoter, enhancer, and CTCF binding sites.(C ) CpG methylation metaprofile associated withall CTCF-bound sites in pro-B cells revealing nucle-osomal fluctuations. (D) Mechanistic link involvingeRNA transcription and DNA hypomethylation.Differences in methylation profiles at intergenic E2Apeaks associated with transcriptionally active regions(>5 GRO-Seq reads per locus) versus intergenic E2A-bound sites that were not linked with eRNA tran-scription (<2 GRO-Seq reads per locus). Both sets ofbinding sites were hypomethylated at the bindingsites, but transcriptionally active E2A enhancers dis-played broader domains of hypomethylated DNA.The + and – symbols discriminate between nascentRNA transcription originating from the + and –

strand. (E) CpG methylation profile at active unidi-rectional and bidirectional promoters. Directionalityof promoters was determined by strand-specificGRO-Seq reads found upstream and downstream ofthe transcription start site (Fig. S1E).

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To identify regulatory signals in DMRs associated with thedevelopmental progression of B cells in an unbiased manner, weapplied HOMER, a de novo motif finding algorithm (Fig. S3B)(22). This analysis revealed transcription factor motifs closelylinked with a B-lineage–specific transcription signature, includingPU.1, EBF1, and E2A (Fig. S3B). MEF2, AP-1, RUNX, and PU.1binding sites were associated with genomic regions that acquiredmCpG (Fig. S3B) (23). Consistent with this analysis, we found thatE2A, EBF1, and PU.1 bound sites were highly enriched in ge-nomic regions that altered their DNA cytosine modification status(Fig. S3C). Analysis of intergenic DMRs specific to either cell typerevealed robust, bidirectional nascent RNA production indicativeof active eRNA transcription specific for each cell type (Fig. S3D).To further explore the relationship between transcription

factor binding and DNA cytosine modifications, we examinedthe distribution mCpG across genomic regions associated withE2A bound sites. We found that nearly half of the genomic re-gions bound by E2A in pro-B cells were hypermethylated(5hmC/5mC) in prepro-B cells (Fig. S2D). Notably, in pro-Bcells, hypomethylated genomic regions that displayed E47binding were associated with elevated levels of H3K4me2 (Fig.S2E). To determine how E2A binding modulates the epigeneticstate, we analyzed E2A cistromes derived from prepro-B cellstransduced with virus-expressing E47-ER (23). One hour aftertreatment with tamoxifen, E2A binding was associated withmore than 40,000 sites (Fig. S2F) (23). We found that more thanhalf of genomic regions associated with E2A occupancy werehypermethylated in prepro-B cells (Fig. S2G). Six hours aftertreatment with tamoxifen, E2A occupancy across these highlymethylated regions was closely associated with robust depositionof H3K4me1 (Fig. S2F) (23). Taken together, these data indicatethat commitment to the B-cell fate is closely associated withthe demethylation of a B-lineage–specific enhancer repertoireand that before activating a B-lineage–specific enhancer reper-toire, E47 is capable of binding to genomic regions that overallare hypermethylated.

Demethylated CpG Islands Are Predominantly Recruited to PolycombBodies. In contrast to the relatively balanced gain and loss ofDNA methylation at enhancers during the prepro-B to pro–B-cell transition, cytosine demethylation at regions that becomeassociated with H3K27me3 is specific to pro-B cells (Fig. S3A).The H3K27me3 histone modification is closely associated withpolycomb-mediated repression, representing an alternative mecha-nism to DNA methylation to silence gene expression. We foundthat more than 70% of pro-B DMRs that displayed H3K27me3deposition during the prepro-B to pro–B-cell transition were asso-ciated with CpG islands (Fig. S3E). Analysis of the entire spectrumof CpG Islands revealed a genome-wide pattern of cytosine de-methylation in pro-B cells. CpG islands that were inactive in prepro-B cells were methylated, but the methylation was lost in pro-B cellsand replaced with the acquisition of H3K4me2 (active), H3K27me3(repressed), or both (bivalent) (Fig. S3F, group II). Interestingly,two groups of CpG islands remained methylated in developingB-cell progenitors (groups III and IV) (Fig. S3F). Group III wasfound in the gene bodies of highly expressed genes because it wasclosely associated with H3K36me3, indicative of active transcriptionand possibly reflecting the presence of 5hmC (Fig. S3F). Group IVCpG islands also remained methylated (5hmC/5mC) during theprepro-B to pro–B-cell transition (Fig. S3F). Notably, group IVCpG islands were located at promoters that were highly enrichedfor genes associated with meiosis and reproduction, which mayrequire mCpG-mediated silencing rather than being localized inpolycomb bodies (Fig. S2H). Collectively, these data indicate thatduring the prepro-B to pro–B-cell transition, demethylated CpGislands are primarily recruited to polycomb bodies.

DNA Cytosine Modifications Across Heterochromatic CompartmentsAre Not Well Maintained. To determine how DNA methylationrelates to nuclear architecture, we examined the distribution ofmCpG across the pro–B-cell interactome (23). We found that the

vast majority of hypomethylated regions (>80%) were associatedwith the transcriptionally permissive compartment (Fig. S4A).To assess whether DNA cytosine modifications were maintainedduring developmental progression, we plotted the average per-centages of mCpG. We found that on average, the proportion ofmCpGs for genomic regions associated with the transcriptionallypermissive compartment was similar for prepro-B and pro-Bcells (Fig. S4B). However, we found lower mCpG rates in genomicregions associated with the heterochromatic compartment (Fig. S4B)(23). The difference in mCpG rates was derived from higher vari-ability in mCpG rates at individual CpG dinucleotides throughoutthe repressive compartment in prepro-B cells, rather than arisingfrom coordinately demethylated regions (Fig. S5A). A striking ex-ample of this was found at the Igκ locus (Fig. S5B). The proportionsof mCpGs across genomic regions spanning the Igκ locus appearedto fluctuate to a much higher degree in prepro-B cells where thelocus was associated with the heterochromatic compartment (Fig.S5A). This analysis indicates that the heterochromatic compartmentin prepro-B cells is associated with substantial allelic and/or indi-vidual cell variation across the population, whereas genomic regionsacross the euchromatic compartment are well maintained as it re-lates to the degree of cytosine modifications.

Identifying Superanchors. On a genic scale (100 kb to 1 Mb), wefound that hypomethylated genomic regions were significantlyenriched at putative anchors (Fig. S4C) (24). Consistent with theseobservations, anchors participating in multiple long-range geno-mic interactions, for example transcriptionally active hubs, weremore likely to be associated with hypomethylated than methylatedgenomic regions (Fig. S4D). Likewise, we found that putative cell-type specific anchors were highly enriched for genomic regionsthat displayed significant levels of demethylation (Fig. S4 E andF). Analysis of boundaries that flank densely interconnected as-sociated domains revealed enrichment for hypomethylated regionsand CTCF occupancy. In contrast, transcriptional active regionsdefined by the deposition of H3K4me2 or E2A occupancy werepredominantly found inside topological-associated domains (Fig.2A). We found that CTCF bound sites at boundaries containedmotifs in specific orientations, consistent with previous findingsdemonstrating that CTCF motif orientation dictates the directionof looping (Fig. 2B) (25). Furthermore, we found that boundariesassociated with topological-associated domains often revealed clus-ters of CTCF-binding sites (Fig. 2 C and D). Borrowing from theconcept that clusters of enhancers form superenhancers (26), weused the same computational framework to identify clusters ofCTCF sites, termed hereafter as “superanchors” or “superinsulators”(Fig. 2C) (Datasets S1–S3). We identified 645 superanchors in pro-Bcells. For example, we found a prominent superanchor positioned4 kb upstream of the Foxo1 locus (Fig. 2D). The Foxo1 superanchor,spanning 27 kb, is composed of eight CTCF-binding sites bound inboth pro-B and prepro-B cells and positioned on a boundary seg-regating two topological-associated domains (Fig. 2D). In prepro-Bcells, the upstream topological-associated domain harboringMaml3 was transcriptionally active, but the downstream topo-logical-associated domain encompassing Foxo1 is insulated fromthat activity and transcriptionally inactive (Fig. 2D). We found thatupon establishing B-cell identity, the Foxo1 promoter underwentdemethylation and enhancers associated with the Foxo1 locus wereactivated as characterized by the deposition of H3K4me2 (Fig. 2D).Global analysis of superanchors compared with “typical anchors”

showed that they are more enriched at topological-associateddomain boundaries, whereas superenhancers were found pre-dominantly inside topological-associated domains (Fig. 2E). Con-sistent with their placement at topological-associated domainboundaries, the distribution of CTCF binding within superanchorsrevealed that the CTCF motifs found at the 5′ and 3′ ends of thesuperanchors were oriented to form interactions either upstreamor downstream, respectively (Fig. 2F). Unlike superenhancers,which are usually cell type-specific, superanchors appeared to belargely invariant between the two cell types (Datasets S1–S3).More than 70% of superanchors identified in pro-B cells were also

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identified in prepro-B cells (Fig. S6A). Levels of CTCF bindingwithin superanchors were similar between the two cell types,consistent with the relatively invariant structure of topological-associated domains across different cell types (Fig. S6B) (27). Onenotable exception was the superanchor associated with thex-linked Firre locus (Fig. S6C), which displayed substantially moreCTCF binding and was associated with DNA hypomethylation infemale prepro-B cells compared with male pro-B cells. In othercases where superanchors were associated with developmentalprogression, developmental stage-specific CTCF occupancy bind-ing was accompanied by DNA demethylation and specific patterns

of gene expression (Fig. 2G and Fig. S6D). Collectively, theseobservations indicate a novel architectural element: superanchoror superinsulator consisting of clusters of CTCF sites.

The Ig Heavy Chain Locus Superanchor. Conspicuous among theidentified ensemble of superanchors was an unusual configurationof 10 densely bound, hypomethylated CTCF motifs that displayedthe same convergent orientation within an 8.5-kb genomic regionlocated 3′ of the Ig heavy chain (Igh) locus (Fig. 3 A and B) (28).CTCF-bound sites located across the Igh locus were hypomethy-lated and often found at a fixed distance from VH regions, whereas

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Fig. 2. Superanchors are closely associated with hypomethylated CTCF-binding sites at boundaries flanking topological associated domains. (A) Hypo-methylated regions are frequently associated with boundary elements that define topological-associated domains. Metaplot of hypomethylated regions andChIP-Seq peaks density across topological-associated domains. (B) Distribution of CTCF motifs based on matching strand at the 5′ and 3′ edges of topological-associated domains. Primary orientation of CTCF motif at each boundary is displayed above each plot. (C) Super enhancer-style plot used to identifysuperanchors depicting the ranked signal for CTCF at putative CTCF clusters. Superanchors and typical anchors were identified in the same manner thatsuperenhancers and typical enhancers are identified, the only difference being that superanchors were found by using CTCF occupancy. As a result, typicalanchors are associated with single CTCF peaks found in the genome. The cutoff for superanchors was defined where the slope of the graph equals one. Levelsof representative superanchors indicated. (D) Superanchor that separates the Malm3 and Foxo1 loci with levels of CTCF, DNA methylation, H3K4me2, andnascent RNA levels indicated. Note the cluster of CTCF bound sites across the putative superinsulator. (E) Distribution of superanchors versus typical anchorsand superenhancers versus typical enhancers across topological domains. Levels are normalized to the total number of elements in each category to comparethe relative levels for each feature. (F) Distribution of CTCF motifs and their orientation along superanchor elements. (G) Differentially bound superanchor atthe Thbd locus with differential CTCF recruitment, DNA methylation, and histone modifications.

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predicted CTCF motifs found with the opposite orientation withinthe Igh locus were hypermethylated and often found near pseudo-VHregions (Fig. 3 A and B and Fig. S7A). The only exception to thistrend was the IGCR1 element, found immediately adjacent to DHregions. The IGCR1 contains two CTCF sites with opposing orien-tations, one of which is oriented to interact with the Igh superanchorand the other pairing with one of the VH regions, and disrupts V(D)Jrecombination when knocked out (28). We note that a similar trendwas observed for the TCRα locus (Fig. S7A).The orientation of VH region-associated CTCF motifs suggests

that they all form interactions with the Igh superanchor or theIGCR1 control region (28). To explore this possibility, we examinedthe pro–B-cell interactome by using meta Hi–C interactions identi-fied in B-cell progenitors (24, 29). This analysis revealed substantialenrichment for a spectrum of long-range genomic interactions,spanning vast genomic distances involving the VH region repertoireand the Igh superanchor as well as weak enrichment for genomicinteractions involving distal V region gene segments and the IGCR1control region (Fig. 3 A and C and Fig. S7 B–D).

DiscussionB-lineage development is initiated by the combined activities oflineage-specific transcriptional regulators that act in concert to alterthe epigenetic landscape of B-lineage regulatory elements. Here wefound that specification of the B-cell fate was closely associated withlarge-scale changes in DNA cytosine modifications across a widespectrum of regulatory elements. How are such sweeping changesacross the epigenetic landscape established? The induction of a Blineage-specific program of gene expression requires the activationof an enhancer repertoire. The activation of such enhancer reper-toires is established by the combined activities of B lineage-specificregulators. Although in prepro-B cells hypermethylated CpGswere associated with B-lineage specific enhancers, the majority oftranscription factor binding sites lack CpG residues. Consistentwith these observations, we found that in prepro-B cells, forcedE2A expression readily binds to a hypermethylated enhancerrepertoire. We suggest that in progenitor cells, E2A by itself or inconjunction with other transcription factors promotes demethyla-tion across an enhancer repertoire. Although detailed mechanistic

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Fig. 3. Characterization of the Ig heavy chain (Igh) locus superanchor. (A) Schematic of the Igh locus depicting the locations of predicted CTCF motifs, boundCTCF motifs, DNA methylation, and ChIP-Seq signal across the locus. The normalized Hi-C interaction frequencies are shown above the locus. (B) Zoomed inrepresentation of the Igh superanchor, IGCR1 region, and representative VH and pseudo VH regions. (C) Model depicting looping patterns involving thesuperanchor and an ensemble of variable regions associated with the Igh locus.

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insight is lacking, it has been proposed that demethylation of highlytranscribed genes is accomplished by recruitment of TET proteinsby the RNA polymerase II complex (30). Here we find that eRNAtranscription is closely associated with the demethylation of aB lineage-specific enhancer repertoire. Thus, we propose that inB-cell progenitors, B lineage-specific transcription factors bindto a spectrum of hypermethylated enhancers to activate eRNAtranscription, which, in turn, leads to demethylation, convertinga poised into an active enhancer repertoire capable of formingloops with other regulatory DNA elements.We found considerable noise in the degree of methylation across

the heterochromatic compartment in multipotent progenitors. Whydoes variation in DNA cytosine modification to such a degree exist?We speculate that noise in DNA methylation permits the branchingout of different hematopoietic cell lineages from common pro-genitor cells. For example, it is well established that in multipotentprogenitor cells, the EBF1 locus is localized in the heterochromaticcompartment (24, 31). However, nuclear localization of the EBF1locus to the lamina is not absolute but variable, and we would like tosuggest that the decision of multipotent progenitors to differentiateinto a distinct cell lineage is controlled by changes in DNAmethylation at sites that control the nuclear positioning of keydevelopmental regulators.It is now well established the Igκ VJ rearrangements and allelic

exclusion are controlled at least in part by DNA cytosine modifica-tions (11, 32–34). Here, we find that the relocation of the Igκ ge-nomic region upon establishing B-cell fate is closely allied withincreased levels of CpG methylation. Why are elevated levels ofDNA cytosine modifications associated with the Igκ locus in com-mitted pro-B cells? We suggest that the increase in DNA cytosinemodifications ensures that Igκ VJ locus rearrangement is not pre-maturely initiated. As cells differentiate into small pre-B cells, theIgκ locus would then undergo allele-specific demethylation as de-monstrated in previous studies (11, 31, 32).Our data indicates that the Igh superanchor serves as a

platform to assemble variable regions separated by vast ge-nomic distances within close spatial proximity of the DHJH

elements. How does such an elaborate and intricate foldingpattern relate to the process of VDJ recombination? Recentstudies have indicated that spatial confinement is the domi-nant parameter that controls the frequencies of genomic en-counters, and we would like to propose that the Igh locussuperanchor spatially constrains subsets of VH elements andDHJH gene segments to promote rapid pairing of antigenreceptor coding elements (33, 35).

Experimental ProceduresPreviously published sequencing data used in this study: GSE40173 (Hi-C,GRO-seq, H3K4me2, H3K36me3, CTCF), GSE40173 (H3K4me1/3, H3K9ac, E2A,EBF, Foxo1), GSE21512 (PU.1), GSE35519 (Hi-C), GSE44288 (Med1), GSE38046(Pax5, DNase-Seq), GSE43008 (YY1), GSE53595 (Ikaros, Irf4), GSE21614(Smad3), and GSE45377 (Runx1).

All ChIP-Seq datawere reanalyzed from FASTQ files bymapping reads to themm9 genome using Bowtie2, and all downstream processing using HOMER.DNase-Seq data from GSE38046 was treated as ChIP-Seq data. Superanchorswere identified by using HOMER as described (22), which mimics the definitionof superenhancers described (26). Briefly, peaks were initially found in CTCFChIP-Seq data by using input as background. Peaks within 12.5 kb were thenmerged into putative superanchor regions. The combined CTCF signal fromthese regions was then normalized to input and ranked. The region where therank and signal normalized curve achieved a slope of 1 was used to determinethe cutoff for superanchors. Pro-B Hi-C data were pooled from two studies toboost the total coverage (GSE35519, GSE40173) (24, 36). Hi-C reads weretrimmed at HindIII sites and mapped to the mm9 genome by using Bowtie2.Interaction normalization, matrix creation, PCA analysis of compartments, andsignificant interaction identification at 10-kb resolution were performed byusing HOMER as described (24). Topological associated domain identificationat 10-kb resolution was also performed by using HOMER in the manner de-scribed (27) (SI Experimental Procedures).

ACKNOWLEDGMENTS. We thank Vivek Chandra and Yin Lin for help ingenerating the libraries. T.I. is the recipient of a research fellowship from theUehara Memorial Foundation. This work was supported by the NationalInstitutes of Health (C.M.).

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