1196 VOLUME 13 NUMBER 12 DECEMBER 2012 nature immunology

The genome is not merely organized as a linear structure but has a dis-tinct three-dimensional configuration. It must fold into an elaborate and coherent pattern to allow genomic elements to find each other with the appropriate frequencies. How the chromatin fiber is organ-ized into higher-order topologies remains to be determined. Distinct folding arrangements for chromosome topologies have been proposed that involve helical, radial or combined loop-helical folding of the genome1,2. Imaging studies with electron microscopy have suggested that chromosomes consist of clusters of loops separated by linkers3,4. The results of measurements of spatial distance and formaldehyde crosslinking approaches in conjunction with modeling strategies are consistent with the organization of the genome into chromatin glob-ules consisting of bundles of loops, related to that originally proposed by the multiloop subcompartment model5–8.

During developmental progression, genes frequently change their nuclear ‘neighborhoods’9,10. For example, after commitment to the B cell lineage, the immunoglobulin heavy-chain locus (Igh) moves from the nuclear periphery to more centrally located domains11–14. After productive rearrangement of the Igh locus, the nonproductive Igh allele moves to the transcriptionally repressive compartment to interact with the immunoglobulin κ-chain locus (Igk)15. Similarly, the Igk locus relocates during B cell development. In small pre-B cells, one of the Igk alleles becomes associated with the repressive compart-ment to favor rearrangement of the Igk allele in the transcriptionally permissive compartment16.

Chromosome-capture studies have provided insight into the folding patterns of genomes on a global scale17–23. Such studies have shown that genomes are organized into compartments that are transcription-ally repressive or permissive17,19. The transcriptionally permissive compartments, in turn, are folded into distinct domains organized by intradomain interactions that involve the architectural protein CTCF18,21. Here we used genome-wide strategies to characterize chromatin compartments and domains during B cell development. We identified distinct classes of anchors that acted on scales of differ-ent lengths. Whereas occupancy by CTCF was associated mainly with intradomain interactions, the histone acetyltransferase p300 and the transcription factors E2A or PU.1 were involved in both intra- and interdomain interactions. Intradomain and interdomain interactions that involved p300, E2A, Pax5 and PU.1 were developmentally regu-lated, but those that involved CTCF were not. We found that a large ensemble of genes relocated during developmental progression from the multipotent stage to the committed pro-B cell stage. Prominent among those was Ebf1, which encodes a key developmental regulator that orchestrates the B cell fate. The switching of genes between com-partments was closely linked with changes in transcription signatures. Specifically, we found that the Ebf1 locus was tightly associated with the nuclear lamina in multipotent progenitors but relocated away from the lamina in committed pro-B cells. We suggest that the sequestration of key developmental regulators underpins the mechanism by which the multipotent progenitor cell stage is enforced. Furthermore, we propose

1Department of Molecular Biology, University of California San Diego, La Jolla, California, USA. 2Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA. 3Integrative Genomics and Bioinformatics Core, Salk Institute for Biological Studies, La Jolla, California, USA. 4These authors contributed equally to this work. Correspondence should be addressed to C.Be. (cbenner@salk.edu), C.K.G. (cglass@ucsd.edu) or C.M. (murre@biomail.ucsd.edu).

Received 6 July; accepted 27 August; published online 14 October 2012; doi:10.1038/ni.2432

Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fateYin C Lin1,4, Christopher Benner2–4, Robert Mansson1, Sven Heinz2, Kazuko Miyazaki1, Masaki Miyazaki1, Vivek Chandra1, Claudia Bossen1, Christopher K Glass2 & Cornelis Murre1

The genome is folded into domains located in compartments that are either transcriptionally inert or transcriptionally permissive. Here we used genome-wide strategies to characterize domains during B cell development. Structured interaction matrix analysis showed that occupancy by the architectural protein CTCF was associated mainly with intradomain interactions, whereas sites bound by the histone acetyltransferase p300 or the transcription factors E2A or PU.1 were associated with intra- and interdomain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitor cells, the transcriptionally inactive locus encoding early B cell factor (Ebf1) was sequestered at the nuclear lamina, which thereby preserved their multipotency. After development into the pro-B cell stage, Ebf1 and other genes switched compartments to establish new intra- and interdomain interactions associated with a B lineage–specific transcription signature.

A rt i c l e snp

g©

201

2 N

atur

e A

mer

ica,

Inc.

All

right

s re

serv

ed.

nature immunology VOLUME 13 NUMBER 12 DECEMBER 2012 1197

that the relocation of chromatin globules between the transcription-ally permissive and repressive compartments and global changes in intra- as well as interdomain interactions mediated by active enhancer repertoires underlie the mechanism by which a B lineage–specific pro-gram of gene expression is established.

RESULTSHighly conserved folding patterns of B cell genomesPublished data have demonstrated that measurements of spatial dis-tance across the Igh locus are consistent with the topology predicted by the multiloop subcompartment model5–7. To determine whether at a global scale the architecture of the pro-B cell genome is organ-ized similarly, we used the ‘Hi-C’ high-throughput chromosome-conformation capture method17,19. We fixed pro-B cells deficient in the recombinase RAG-1 with formaldehyde alone or with formal-dehyde and ethylene glycol bis(succinimidylsuccinate), digested the DNA with HindIII, filled in the gaps with biotinylated nucleotide, ligated the DNA at a high dilution and analyzed the products by high-throughput DNA sequencing. From the filtered interaction data, we generated genome-wide interaction matrices for each chromosome (Fig. 1a, Supplementary Table 1 and Supplementary Fig. 1a–c; filter-ing and statistical analyses, Supplementary Methods). This analysis identified clusters of interacting genomic elements across the entire length of chromosomes in pro-B cells (Fig. 1a and Supplementary Fig. 2). Most genomic interactions were local (<3 megabases (Mb)), as reported before17 (Fig. 1a). To identify patterns in the interac-tion matrix, we applied principal-component analysis to each chro-mosome17 (Supplementary Fig. 1a). This analysis confirmed that chromosome 11 segregated into compartments that were transcrip-tionally permissive or inert (Supplementary Fig. 1a). The coordi-nates for each region along the first principal component (PC1 values) were highly reproducible across distinct Hi-C replicate experiments, with positive values reflecting transcriptionally active compartments and negative values indicating repressive or inert compartments (Supplementary Fig. 1d), and they were conserved in the genomes of

mouse pro-B cells and syntenic regions derived from mature human cells of the B lineage17,23 (Fig. 1b and Supplementary Fig. 1a). The ‘interactomes’ (the entire sets of molecular interactions in cells) of cells fixed with ethylene glycol bis(succinimidylsuccinate), Abelson virus–transformed pro-B cells, embryonic stem cells and RAG-1- deficient pro-B cells were also similar22,23 (Supplementary Fig. 1d–f). To compare the degree of sequence conservation in transcriptionally active and repressive compartments, as determined by PC1 values, we segregated genomic regions on the basis of scores obtained with the PhastCons program24 for identifying evolutionarily conserved elements in a 38-organism alignment that assesses the probability that DNA sequences are under selective pressure (Fig. 1c). We found that regions with high scores were present mainly in transcriptionally per-missive compartments rather than the heterochromatic environment (Fig. 1c). To determine domain size across the pro-B cell genome, we computed the average size of continuous regions with positive PC1 values. We found that the pro-B cell genome was organized mainly as 0.5- to 3-Mb chromatin globules (Table 1).

The organization of the genome into distinct chromatin globules or domains raised the question of whether domains were associated with lineage-specific programs of gene expression. To explore this possibility, we computed, for 96 mouse tissues, the average pairwise correlation coefficient of genes in each active domain composed of a minimum of ten genes with gene-expression values derived from the BioGPS gene-annotation portal. We then compared those values with the expression patterns of genes dispersed across different chro-matin domains. We found that genes located in the same domain had a greater tendency to be coordinately expressed than did genes located in different domains (Fig. 1d). To determine how lineage-specific patterns of gene expression relate to the location of genes in a transcriptionally repressive or permissive compartment, we quanti-fied the distinct cell types that expressed the ensemble of annotated genes as a function of PC1 values. We found that genes located in inert compartments had a more cell type–restricted pattern of expres-sion than did genes located in permissive compartments, with a more

Figure 1 The folding pattern of the pro-B cell genome. (a) Strategy for Hi-C data normalization from expected interaction frequencies for a genome-wide contact matrix showing intrachromosomal interactions involving chromosome 11 (Chr 11). Colors indicate the ratio of observed interaction frequency to expected interaction frequency (obs/exp): blue, lower than expected; red, higher than expected. (b) Genome-wide comparison of PC1 values for mouse chromosome 11 of pro-B cells and syntenic regions derived from mature human cells of the B lineage, with 50-kb windows; color intensity corresponds to density. (c) Association of regions with high and low PhastCons scores (above plots) with transcriptionally permissive and/or euchromatic compartments (high PC1 values) and inert and/or heterochromatic compartments (low PC1 values). (d) Distribution of pairwise correlations of gene-expression values for 96 mouse tissues, calculated by comparison of genes (at least 10 per active domain) within the same domain (intradomain) or in different domains (interdomain). P < 1 × 10−7 (Mann–Whitney test). (e) Distribution of the number of tissues with detectable expression of each annotated gene (BioGPS; GEO accession code, GSE10246) in the transcriptionally repressive compartment (PC1 < −25), in regions with intermediate PC1 values (−25 < PC1 < 25) or in the transcriptionally permissive compartment (PC1 > 25). Small horizontal bold lines (d,e) indicate median. Data are from one experiment.

b100

100

r2 = 0.74

150

50

50

0

0

PC1 mouse pro-B cell

PC

1 hu

man

mat

ure

B c

ell

–50

–50

–100

–100

d0.10

0.05

0

–0.05

–0.10

Intra

dom

ains

Inte

rdom

ains

Rando

mize

d

Rando

mize

d

Exp

ress

ion

corr

elat

ion

Chr 11

a

0 Mb

30 Mb

60 Mb

90 Mb

120 Mb

10–1

10–2

10–3

10–4

10–5

10–6

10–7

103 104 105 106

Interaction distance Expected interactions

Normalized matrixRaw interaction matrix

Ave

rage

rat

e

107 108 109

e

80

60

40

Cel

l typ

esex

pres

sing

eac

h ge

ne

20

0

<–25PC1:

–25 to 25> 25

c > 0.15(r2 = 0.71)

0.10–0.15(r2 = 0.72)

Hum

an P

C1

Mouse PC1

Interaction (obs/exp)

8 4 2 11/

21/

41/

8

0.05–0.10(r2 = 0.71)

< 0.05(r2 = 0.64)

A rt i c l e snp

g©

201

2 N

atur

e A

mer

ica,

Inc.

All

right

s re

serv

ed.

1198 VOLUME 13 NUMBER 12 DECEMBER 2012 nature immunology

A rt i c l e s

ubiquitous pattern of gene expression (Fig. 1e). These results, along with published observations17–23, indicated that the folding patterns of genomes of different cell types and species were highly conserved, were organized as 0.5- to 3-Mb domains and were composed of clus-ters of genes with a tendency to be coordinately transcribed.

Recruitment of promoters to enhancers modulate eRNA abundanceTo determine whether and how epigenetic marks pair at genomic features, we computed interactions between loci (P < 0.05) with a window of 10 kilobases (kb). We identified a total of 64,101 of such interactions in pro-B cells (false-discovery rate, <10%). We examined the interacting DNA elements for enrichment for epige-netic marks of promoter activity (histone H3 trimethylated at Lys4 (H3K4me3), dimethylated at Lys4 (H3K4me2) or acetylated at Lys9

and Lys14 (H3K9-K14ac)), enhancer activity (H3K4me2 and histone H3 monomethylated at Lys4 (H3K4me1)), elongation and/or splic-ing (H3K36me3) and repression (H3K27me3)25 (Fig. 2a). We found considerable enrichment for the entire spectrum of epigenetic marks across loop-attachment regions (Fig. 2b). Notably, we found con-siderable depletion of loop-attachment regions with deposition of H3K27me3 for interactions involving active promoter and enhancer elements (Fig. 2b), which indicated that islands of deposition of H3K27me3 (silencing) and H3K4me2 (activation) were spatially segregated across the pro-B cell genome25.

To determine how genomic interactions between regulatory ele-ments related to nascent transcription at promoter and enhancer regions, we plotted the density of nascent RNA, as determined by glo-bal nuclear run-on sequencing (GRO-Seq), as a function of genomic distance from the transcription start site and sites of occupancy by E2A at the promoter and distal regions (Fig. 2c). Transcription start sites tethered with putative enhancer elements showed substantial enhancement for both sense and antisense transcription (Fig. 2c, left). To determine how nascent transcription initiated at enhancer elements (‘eRNA’) related to looping to transcription start sites, we plotted the abundance of nascent transcripts at intergenic putative enhancer regions bound by E2A (and with deposition of H3K4me2; Fig. 2c, right). The abundance of eRNA was much greater at E2A-bound regions that interacted with promoters than at those that did not (Fig. 2c). However, the degree of E2A occupancy affected the abundance of nascent transcripts only modestly (Fig. 2c,d). Consistent with those observations, we found that whereas enhancer elements in the pro-B cell genome were closely associated with B cell–specific interaction endpoints, enhancer regions identified in embryonic stem cells were not (Fig. 2e). These data indicated that the abundance of eRNA was greater at enhancers that interacted with

Table 1 Domain size of the pro-B cell genome and the MLS modelDomain size Domains Active region

≥5 Mb 37 25.0%3–5 Mb 51 18.4%1–3 Mb 138 22.5%500 kb–1 Mb 177 11.7%<500 kb 22.4%

Domain size Domains Inert region

≥5 Mb 38 17.7%3–5 Mb 76 19.2%1–3 Mb 311 36.5%500 kb–1 Mb 256 12.2%<500 kb 14.3%

Distribution of domain size across the pro-B cell genome, as defined by continuous regions of positive PC1 values across the genome, for the transcriptionally permissive compartment (top) and repressive compartment (bottom). Data are representative of one experiment.

e

b

H3K4me1

H3K27me3

H3K36me3H3ac

Frequency(obs/exp)

0.25–0.25

H3K4me3

H3K4me2

0.6 CTCF peaksTSSPro-B cell H3K4me2enh (>50 kb)ES cell H3K4me2enh (>50 kb)

Sig

nific

ant i

nter

actio

nen

dpoi

nts

(per

pea

k pe

r 10

kb)

0.5

0.4

0.3

0.2

0.1

0–30 –20

Distance from peaks and TSS (kb)

–10 0 10 20 30

8a

7

6

5

4

3Enr

ichm

ent

2

1

0

H3K

4me3

H3K

4me2

H3K

4me1

H3K

36m

e3H

3K27

me3

Pro

mot

ers

TT

SE

xons

Intr

ons

Gen

e de

sert

s

H3K

9-K

14ac

d

60

40

20Nor

mal

ized

E2A

rea

ds (

per

peak

)

0–

Interactionwith TSS

+

c No enh int (+)No enh int (–)Enh int (+)Enh int (–)

GR

O-S

eq r

ead

(per

bp

per

peak

)

0.0180.0160.0140.0120.0100.0080.0060.0040.002

0.020

0

–1,0

00–5

00

Distance from TSS (bp)

500

1,00

00

0.0040

GR

O-S

eq r

ead

(per

bp

per

peak

)

Distance from intergenicE2A peak (bp)

–1,0

00–5

00 500

1,00

00

0.0035

0.0030

0.0025

0.0020

0.0015

0.0010

0.0005

0

No TSS int (+)No TSS int (–)TSS int (+)TSS int (–)

Figure 2 The transcriptionally permissive compartment is spatially segregated into islands of H3K27me3 and H3K4me2. (a) Enrichment for epigenetic marks and genomic annotations such as promoters, transcription start sites (TSS), exons, introns and ‘gene deserts’ (>100 kb from a gene in the RefSeq database) at the end points of significant interactions (false-discover rate, <10%) relative to those at random genomic positions (10-kb window). (b) Frequency with which epigenetic marks are associated across loop-attachment points (node size) and the significance and reproducibility of tethering, in terms of P values (line thickness); color (red-blue) represents the log ratio of observed frequency relative to expected frequency (association strength). H3ac, acetylated histone H3. (c) Density of nascent RNA (determined by GRO-Seq) as a function of genomic distance from the transcription start site, for promoters that show looping toward putative enhancers defined by promoter-distal H3K4me2 peaks (left), and as a function of genomic distance from E2A-binding sites located in intergenic H3K4me2 peaks (putative enhancer regions) for enhancers (right), including sense (+) and antisense (−) transcription initiated from putative enhancer regions (Enh int; left) or the transcription start site (TSS int; right), as well as nascent transcripts initiated from promoters in the absence of enhancer interactions (No enh int; left) or interactions with transcription start site (No TSS int; right). (d) Distribution of the normalized number of E2A ‘reads’ per peak (obtained by chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq)) at peaks either interacting with (+) or lacking interaction with (−) nearby TSSs. (e) Distribution of loop-attachment points as a function of genomic distance from transcription start sites and transcription factor–binding sites (peaks). ES, embryonic stem. Data are representative of one experiment.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature immunology VOLUME 13 NUMBER 12 DECEMBER 2012 1199

A rt i c l e s

promoter regions, which suggested that the transcription of eRNA is initiated, at least in part, by looping of polymerase II from promoter elements to enhancer elements.

Putative anchors that act at different length scalesTo identify potential anchors associated with the pro-B cell inter-actome, we focused our analysis on active enhancers and factors known to be involved in early B cell development. Loop-attachment regions showed substantial enrichment for active enhancers marked by p300, as well as factors such as E2A, PU.1, EBF1, CTCF and Rad21 (Fig. 3a). However, we found significant depletion of interactions linking CTCF and lineage-specific transcription factors (E2A, PU.1, EBF1 and Foxo1) relative to interactions among CTCF or lineage- specific factors themselves (Fig. 3b). Hence, two distinct classes of factors were closely associated with loop-attachment regions. Lineage-restricted factors paired at regulatory elements with them-selves or with other lineage-specific transcription factors, whereas CTCF-bound sites associated mainly with themselves.

The range of detected significant interactions (false-discovery rate, <10%) with endpoints spanning windows of 10 kb was lim-ited to approximately 3 Mb, given the sequencing depth (Fig. 1a). We found interactions mainly within chromatin domains rather than between domains because of the greater Hi-C ‘read’ coverage for shorter genomic distances. To characterize interdomain inter-actions, at which Hi-C read pairs were sparse, we developed the structured interaction matrix analysis (SIMA) strategy. We boosted sensitivity by pooling sites of factor occupancy within domains and analyzed their collective Hi-C read counts (Fig. 3c). We then compared those values with peak positions randomized within the same domains and calculated P values associated with enrichment (Fig. 3c). For domain comparisons with a P value of less than 0.05, we computed the ratio of the value from observed peak positions to the average of randomized peak positions plotted across the interaction matrix (Fig. 3d). By SIMA we found notable differences between interactions involving CTCF or E2A (Fig. 3d and Supplementary Fig. 3). Whereas we found enrichment for CTCF interactions across regions involved in intradomain interactions, enrichment for E2A occupancy was not restricted to looping within domains but instead seemed to be associated with both intra- and interdomain interactions (Fig. 3d). Thus, SIMA identified the presence of distinct classes of anchors associated with either intradomain interactions or intra-and interdomain interactions.

Sequestration of Ebf1 at the nuclear laminaTo determine whether developmental progression from the pre-pro-B cell stage to the pro-B cell stage was associated with changes in long-range genomic interactions, we generated interactomes of pre-pro-B cells through the use of Hi-C and compared those with the interactomes of pro-B cells. The interaction matrices of pre-pro-B cells and pro-B cells were similar (Fig. 4a). However, a small fraction of loci differed in PC1 values (Fig. 4a and Supplementary Fig. 4a). To further evaluate these differences, we generated and directly compared Pearson corre-lation matrices (Fig. 4b,c). This analysis identified a subset of loci with PC1 values that differed in pre-pro-B cells and pro-B cells (Fig. 4b, Supplementary Fig. 4b,c and Supplementary Table 2). Conspicuous among those loci were the Ebf1, Foxo1 and the light-chain loci Igk and Igl, which repositioned from the repressive compartment to the permissive compartment (Supplementary Fig. 4 and Supplementary Table 2).

To verify the changes in compartmentalization at the single-cell level, we used three-dimensional fluorescence in situ hybridization in conjunction with epifluorescence microscopy. We generated fluo-rescence-labeled probes to detect the domain containing Ebf1 as well as genomic regions adjacent to the Ebf1 locus (Fig. 4d). Consistent with the Hi-C analysis, the Ebf1 domain in pre-pro-B cells was closely associated with the neighboring region located in the repressive compartment, whereas in pro-B cells the Ebf1 locus interacted more frequently with genomic elements located in the transcriptionally permissive compartment (Fig. 4d).

To investigate in greater detail how the Ebf1 locus was associated with the transcriptionally repressive environment, we examined the position of the Ebf1 locus relative to the nuclear lamina. For this, we fixed pre-pro-B cells and pro-B cells with formaldehyde, stained the cells for lamina and analyzed them. Notably, in pre-pro-B cells, the Ebf1 locus was closely associated with the nuclear lamina, whereas in committed RAG-1-deficient pro-B cells, it was located mainly away from the nuclear lamina (Fig. 5a,b).

To determine whether developmental regulators associated with alternative cell lineages were also sequestered in the transcription-ally repressive compartment, we determined PC1 values for loci encoding such regulators (Gata1, Gfi1, Tcf7, Cebpa, Cebpb, Bcl11b and Id2). Most of these loci were located in the transcriptionally permissive compartment in both multipotent progenitors and pro-B cells. Notably, however, we found that in both cell types, the Bcl11b locus was associated with the transcriptionally repressive compart-ment (Fig. 5c). Thus, these observations indicated that in multipotent

b

Foxo1

PU.1

EBF1

E2A

Rad21

p300

c-Myc

CTCF–0.5 0.5

Frequency(obs/exp)

8a

7

6

5

4

Enr

ichm

ent

3

2

1

0

c-M

ycR

ad21

p300

Fox

o1E

2AC

TC

FE

BF

1P

U.1

cPC1 E2A

1.6

High-resolution

E2A

Randomized

1.41.21.00.80.60.40.2

00

0.5

Interactions per matrix/expected interactions

1.0

1.5

2.0

2.5

3.0

3.5

**

d

CTCF E2A

CTCF E2A1/2 21 1/4 41Interactions(obs/exp)

Interactions(obs/exp)Figure 3 Two distinct classes of anchors establish the pro-B cell interactome. (a) Enrichment for occupancy by E2A, PU.1, CTCF,

Rad21, EBF1, c-Myc and p300 at loop-attachment regions. (b) Frequency with which putative anchors are associated with loop-attachment points (node size) and significance and reproducibility of tethering (as in Fig. 2b, as is color). (c) SIMA of the interaction of binding sites within domains (pooled) across the domains to binding sites in other domains (left and middle), followed by comparison of those values with peak positions randomized 10,000 times and calculation of P values associated with enrichment for interactions (middle and right). (d) Enrichment of interactions at sites with occupancy by CTCF or E2A, assessed for compartmental interactions across the transcriptionally permissive compartment. Data are from one experiment.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1200 VOLUME 13 NUMBER 12 DECEMBER 2012 nature immunology

A rt i c l e s

progenitor cells, the Ebf1 and Bcl11b loci, which encode key regulators that promote the B cell fate and T cell fate, respectively, were posi-tioned in the transcriptionally repressive compartment.

Nuclear repositioning, epigenetics and gene expressionTo determine whether the spatial repositioning of loci related to dif-ferences in programs of gene expression, we plotted the ratio of the abundance of nascent RNA in pro-B cells to that in pre-pro-B cells against PC1 values (Fig. 6a). Indeed, loci that switched from a tran-scriptionally repressive compartment to a permissive compartment had a greater abundance of nascent RNA, whereas loci that relocated from a transcriptionally permissive to a repressive compartment had a lower abundance of in nascent transcripts (Fig. 6a). Globally, the abundance of RNA encoded by 1,534 annotated genes was greater in pro-B cells than in pre-pro-B cells, whereas the abundance of RNA encoded by 1,623 annotated genes was less in pro-B cells than in pre-pro-B cells (Fig. 6b). Of the genes with a greater transcript abundance in pro-B cells, 240 (16%) were in regions that switched from a repres-sive environment to a permissive environment, whereas 87 (5%) of the genes whose transcript abundance was lower in pro-B cells were in compartments that changed from transcriptionally permissive com-partments to repressive compartments (Fig. 6b and Supplementary Tables 2 and 3). Regions associated with the transcription of noncod-ing RNA, microRNA and eRNA showed similar patterns (data not shown). As predicted, we found significant enrichment for occupancy by E2A, EBF1 and Foxo1 across domains that were actively tran-scribed in pro-B cells (P < 1 × 10–10; Supplementary Fig. 5a).

However, the entire spectrum of genes that switched from the repressive compartment to the permissive compartment did not have more nascent transcription (Fig. 6b). Instead, a substantial fraction of genes showed no or minimal changes in the abundance of nascent RNA (Fig. 6b). To examine this group of genes in greater detail, we

compared the deposition of H3K27me3 to the abundance of nascent transcripts for genes that switched from the repressive compartment to the permissive compartment. Notably, we found enrichment for the abundance of H3K27me3 across genes that made such a switch but whose nascent transcript amounts were equivalent in pre-pro-B cells and pro-B cells (Supplementary Fig. 5b). Thus, repositioning of loci during developmental progression from the transcription-ally repressive compartment to the permissive compartment was accompanied either by activation of gene expression or silencing by deposition of H3K27me3.

Repositioning of genes during ontogenyThe findings described above indicated large-scale changes in the compartmentalization of genes during B cell development. Do such changes also occur during ontogeny? To address this question, we compared the PC1 values of the interactomes of pro-B cells and embryonic stem cells (Supplementary Fig. 6a). Indeed, we found a subset of loci that seemed to have relocated from a transcription-ally permissive chromatin environment in embryonic stem cells to a repressive environment in pro-B cells (Supplementary Fig. 6a).

Published studies have indicated that transcriptionally active domains have a high density of short interspersed elements (SINEs), which represent short DNA sequences that originate from reverse-transcribed small nuclear RNA. The enrichment for SINEs across transcriptionally active regions23,26 prompted us to investigate the presence of SINEs in both embryonic stem cells and pro-B cells. Consistent with the published observations23,26, we found that tran-scriptionally active regions had a relatively high density of SINEs (Supplementary Fig. 6b). In contrast, long interspersed elements, which are clusters of actively transcribed genomic repeat elements, had a reverse pattern, in that the repressive compartment showed sub-stantial enrichment for these elements (Supplementary Fig. 6b).

150

a b c

150

100

100

50

50

0

0

PC1 pre-pro-B cells

PC

1 pr

o-B

cel

ls

Pre-pro-B cell PC1Chr 11: Chr 11 (Mb): 4641

PC151 5644000000 45000000

1 Mb46000000

Pro-B cell PC1Different

correlation valuesPre-pro-B cell GRO-Seq

Pro-B cell GRO-Seq

Pre-pro-B cell H3K4me2

Pro-B cell H3K4me2

Pre-pro-B cell

PC1

Pro-B cell

Differentcorrelation values

Pro-B cell E2ARefSeq genes

Ebf1

–50

–50

–100

–100

Pro-B cellPre-pro-B cell

100

3′ EBF toinactive domain

3′ EBF Inactive (I)

2.24

44.9

445

.14

47.3

8

Genomic distance (×106 bp)

47.5

750

.01

50.2

1

2.44

Active (A)

80604020

Cum

ulat

ive

freq

uenc

y

00 0.5 1.0 1.5

Distance (µm)2.0

100

3′ EBF toactive domain

80604020

Cum

ulat

ive

freq

uenc

y

00 0.5 1.0 1.5

Distance (µm)2.0

Pro-B cellPre-pro-B cell

d Allele 1 Allele 2 Allele

1

2

2

1

Pro-B cell

Pre-pro-Bcell

Figure 4 Switching between transcriptionally repressive and permissive nuclear compartments during B lineage specification. (a) PC1 values of pre-pro-B cells and pro-B cells, defined at intervals of 25 kb; signals away from the diagonal indicate genomic regions that switch nuclear environments during the transition between those stages. (b) Ebf1 locus, showing PC1 values of chromosome 11 for pre-pro-B and pro-B cells in a region surrounding this locus (top two rows), as well as interexperimental correlation (Different correlation values) and read densities for nascent RNA (GRO-Seq) and H3K4me2 and E2A ChIP-Seq (bottom five rows). (c) Correlation matrices derived from chromosome 11 for pre-pro-B cells (top) and pro-B cells (middle) by correlation of the normalized interaction ratios for each 25-kb interval against those of all other regions, and differences in correlation values, calculated by comparison of the two correlation matrices at each locus (bottom). Yellow box indicates the genomic region containing Ebf1. (d) Three-dimensional fluorescence in situ hybridization (top) of nuclei from pre-pro-B cells and pro-B cells with fluorescence-labeled bacterial artificial chromosomes that span three distinct regions across chromosome 11 (middle; colors match images above); top right, digitally enlarged images of the domains adjacent to the Ebf1 locus. Nuclei were visualized by staining with the DNA-intercalating dye DAPI (blue). Middle, genomic organization of the Ebf1 locus, including location of probes for the Ebf1 domain (3′ EBF), the adjacent constitutively inert domain (Inactive (I)) and a distal constitutively permissive domain (Active (A)). Bottom, distribution of spatial distances between the probes, measured for 102 and 106 alleles in pre-pro-B cells and pro-B cells, respectively. Original magnification, ×100 (top left); enlargement, (top right). Data are representative of one experiment (a–c) or are pooled from two experiments (d).

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature immunology VOLUME 13 NUMBER 12 DECEMBER 2012 1201

A rt i c l e s

We next sought to determine why a subset of the pro-B cell genome with high SINE density was located in a transcriptionally repressive compartment. To address this, we considered the possibility that SINE-rich regions located in the transcriptionally repressive chroma-tin compartment were in fact located in transcriptionally permissive compartments during earlier stages of ontogeny. Consistent with our prediction, we found that SINE-rich regions located in a repressive chromatin compartment in pro-B cells tended to show considerable enrichment for H3K4me2 in embryonic stem cells (Supplementary Fig. 6c,d). These data indicated that during embryogenesis, a subset of chromatin domains switched from a transcriptionally permissive compartment to a repressive compartment. Thus, in pro-B cells, most SINE-rich domains were located in a transcriptionally permissive compartment, but a fraction of the SINE-rich domains located in the transcriptionally repressive compartment represented domains that switched from a transcriptionally permissive nuclear compartment to a transcriptionally repressive nuclear compartment during an earlier stage in ontogeny.

Identifying developmentally regulated anchorsThe data described above identified distinct classes of factors that were associated with intra- and/or interdomain interactions. To determine whether these interactions were developmentally regu-lated, we used SIMA to assess enrichment for putative anchors across loop-attachment regions in multipotent pre-pro-B cells and pro-B cells. We found that pre-pro-B cells and pro-B cells did not differ significantly in their pattern of genomic interactions associated with CTCF occupancy (Fig. 6c). However, these cells

did differ significantly in their patterns of intra- and interdomain interactions associated with E2A occupancy. As expected, intra- and interdomain interactions associated with E2A occupancy in pro-B cells were absent from E2A-deficient multipotent progenitor cells (Fig. 6c). Thus, the developmental progression from the pre-pro-B cell stage to the pro-B cell stage was associated with large-scale changes in loop-attachment regions associated with E2A occupancy but not with CTCF occupancy.

To identify additional potential anchors associated with the pro-B cell interactome, we used SIMA to assess occupancy by PU.1, Pax5 and Foxo1 (refs. 26–30; Supplementary Fig. 5c). We used occupancy

a

cPre-pro-B cell PC1

83.860

0

1

–10

4930465M20RikBcl11b Setd3

CcnkCcdc85c

Bcl11b

–59.43

–78.44

50.17Pro-B cell PC1

Differentcorrelation value

b

Pre-p

ro-B

cell

Pro-B

cell

100

> 0.5 µm≤ 0.5 µm

90

80

70

60

50

Dis

tanc

ce (

%)

40

30

20

10

0

Pre-pro-B cell Pro-B cellFigure 5 The Ebf1 locus is closely associated with the nuclear lamina in pre-pro-B cells. (a) Immunofluorescence in situ hybridization of nuclei from pre-pro-B cells and pro-B cells with fluorescence-labeled bacterial artificial chromosome containing the Ebf1 domain (green; as Fig. 4d), antibody to lamin (orange; nuclear lamina) and DAPI (nuclei), presented as ‘digitally magnified’ images. Original magnification, ×100. (b) Fraction of spatial distances (<500 nm) separating the Ebf1 domain from the nuclear lamina in pre-pro-B cells (112 measurements) and pro-B cells (102 measurements). (c) PC1 values for the Bcl11b locus in pre-pro-B cells and pro-B cells. Data are pooled from two experiments (a,b) or are from one experiment (c).

10

5

0

GR

O-S

eq r

eads

log 2

(pro

-B c

ell/

pre-

pro-

B c

ell)

–5

–10

–150

–100 –5

0

PC1 pro-B cell –PC1 pre-pro-B cell

a

0 50 100

150

E2A

SOX2

dESC Pre-pro-B cell Pro-B cell

1/4 1 4Interactions(obs/exp)

6

–10 –8 –6 –2 0 2 4 6–4

Pre-pro-B cellGRO-Seq (log2 r.p.k.m.)

Pro

-B c

ell

GR

O-S

eq (

log 2

r.p.

k.m

.)

bPre-pro-B cell switchedPre-B cell switched

420

–2–4–6–8

–101/4

1/2 2CTCF

E2A

1

1 4Interactions(obs/exp)

E2A

CTCF

Pre-pro-B cell

c

Pro-B cell

Figure 6 Repositioning of loci during the developmental progression from a transcriptionally repressive to a permissive compartment is accompanied with activation of gene expression or silencing by deposition of H3K27me3. (a) Difference in nascent RNA read density (log2; GRO-Seq) versus the difference in PC1 values for pre-pro-B cells and pro-B cells, for each 25-kb interval of the genome; black line indicates moving average values for 100 regions. (b) The r.p.k.m. values (reads per kilobase of gene model per million reads) for RefSeq genes in pre-pro-B cells versus those in pro-B cells. Red, switch from repressive to permissive compartment in pro-B cells; blue, switch from permissive compartment in pre-pro-B cell to inert compartment in pro-B cells. (c) SIMA of E2A- and CTCF-binding sites in pre-pro-B cell (left) and pro-B cell (right) domains (pooled reads) for interactions across the transcriptionally permissive environment to binding sites in other compartments, compared with randomized peak positions; P values associated with enrichment for paired interactions were calculated (red and blue as in Fig. 1a). (d) SIMA of enrichment for E2A and SOX2 at loop-attachment points in embryonic stem cells (ESC), pre-pro-B and pro-B cells, with E2A-binding sites identified in the pro-B cell genome and SOX2 occupancy derived from embryonic stem cells. Data are from one experiment.

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1202 VOLUME 13 NUMBER 12 DECEMBER 2012 nature immunology

A rt i c l e s

by p300 to assess enhancers involved in intra- and interdomain inter-actions (Supplementary Fig. 6c). This analysis showed that overall, the spectrum of loop-attachment regions showing enrichment for enhancers as well as occupancy by Pax5, PU.1 and Foxo1 was dis-tinct from that for CTCF but was similar, albeit not identical, to that for E2A (Supplementary Fig. 5c). The active enhancer repertoire, as marked by p300 occupancy, also seemed to be associated with both intra- and interdomain interactions (Supplementary Fig. 6c).

To determine how occupancy by transcription factors associ-ated with loop-attachment regions changed during embryogenesis, we used SIMA in conjunction with occupancy of the E2A and SOX2 transcription factors28 in embryonic stem cells, pre-pro-B cells and pro-B cells (Fig. 6d). As expected, loop-attachment regions in embryonic stem cells did not show enrichment for the E2A-associated binding sites identified in the pro-B cell genome (Fig. 6d). As pre-dicted, we observed the reverse pattern for Sox2 occupancy ‘gated on’ binding sites detected in embryonic stem cells (Fig. 6d). In sum, these data indicated that loop-attachment regions associated with lineage- specific transcription factors were developmentally regulated.

Spatial clustering of k-chain variable regionsTo determine whether changes in nuclear location were associated with changes in chromatin topology, we compared the interactomes of Ebf1 and Igk in pre-pro-B cells and pro-B cells. We found a wider spec-trum of genomic interactions across the Ebf1 locus in pro-B cells than in pre-pro-B cells (Supplementary Fig. 7), indicative of widespread changes in topology. Looping across the Ebf1 locus involved mainly promoter-promoter interactions and enhancer-enhancer interactions, as well as enhancer-promoter interactions (Supplementary Fig. 7).

We also observed notable changes in genomic interactions across the Igk locus during developmental progression (Fig. 7a). We found looping mainly adjacent to the Igk locus in pre-pro-B cells, whereas in pro-B cells, we observed an intricate and elaborate pat-tern of interactions involving both small and large loops (Fig. 7a). Conspicuous were interactions involving the Igk intronic enhancer and genomic elements across the Igk locus (Fig. 7a). Many of these interactions were closely associated with E2A occupancy (Fig. 7a). To examine genomic interactions in greater detail, we applied SIMA for transcription factors, epigenetic marks, and joining and variable regions (Fig. 7b). Notably, κ-chain variable regions (Vκ regions) used in the Igk repertoire seemed to cluster in pro-B cells but not in pre-pro-B cells. In contrast, pseudo-variable regions did not gather (Fig. 7b). The clustering of Vκ elements indicated close spatial prox-imity that permitted the entire repertoire of functional Vκ regions, separated by large genomic distances, to encounter κ-chain joining (Jκ) elements with similar probabilities (Fig. 7c).

DISCUSSIONGlobal studies using formaldehyde crosslinking approaches have pro-vided insight into the folding patterns of genomes17–23. Such studies have shown that genomes are organized into compartments that are transcriptionally repressive and transcriptionally permissive17. The compartments themselves are organized into domains that are struc-tured by intradomain interactions, mediated mainly by CTCF21,23. We developed the SIMA strategy and used it here to identify addi-tional factors associated with loop-attachment regions. Consistent with published observations21,23, we found that occupancy by CTCF was associated mainly with genomic interactions within chromatin globules. We also identified additional putative anchors, such as E2A, PU.1, EBF1 and Pax5, that seemed to bind ‘collaboratively’ across loop-attachment regions. Thus, different classes of putative anchors established the intradomain interactome. SIMA also allowed us to detect interdomain interactions. Interdomain interactions are medi-ated mainly by enhancer-enhancer interactions that involve transcrip-tionally active domains, such as transcription factories, as they are closely associated with p300-bound sites31–33. We noted that distinct interdomain interactions were rather infrequent, which indicated that these genomic interactions occurred only in a small fraction of the population of cells.

The aforementioned data indicated that at least two distinct classes of anchors establish the pro-B cell interactome, as follows: first, archi-tectural proteins such as CTCF acting inside domains; and second, transcriptional regulators such as E2A and PU.1 acting within and between chromatin domains. How do these two classes, CTCF versus enhancer-binding proteins, differ? The biochemical activities and regu-latory functions of CTCF and of E2A and PU.1 are distinct. Whereas there is not enrichment for CTCF-bound sites across transcriptionally active regions, occupancy by E2A and PU.1 is associated with active enhancer repertoires29,30. Furthermore, tethers associated with CTCF occupancy were not developmentally regulated, whereas the spectrum of loop-attachment regions, enriched for E2A- and PU.1-bound sites,

a

c

GRO-SeqE2ACTCFH3K4me2

70.770.8

70.9

71.071.1

70.670.570.4

70.370.2

70.1

70.0

69.9

69.8

69.7

69.6

69.5

69.4

69.3

69.2

69.1 Chr 6

69.0

68.968.8

68.768.6

68.5

68.4

68.3

68.2

68.1

68.0

67.967.8

67.767.667

.567.467

.367.2

67.1

67.0

70.770.8

70.9

71.071.1

70.670.570.4

70.3

70.2

70.1

70.0

69.9

69.8

69.7

69.6

69.5

69.4

69.3

69.2

69.1 Chr 6

69.0

68.968.8

68.768.6

68.5

68.4

68.3

68.2

68.1

68.0

67.967.8

67.767.667

.567.467

.367.2

67.1

67.0

J

Pseudo VVκE2A

bPU.1

Eiκ

Pseudo VCTCF

H3K4me2

Vκ

PU.1

Eiκ

Pseudo V

CTCFE2A

H3K4me2

Vκ

1.00–0.5

Figure 7 Switching nuclear compartments is closely associated with remodeling of chromatin architecture. (a) Genomic regions, including the Igk locus, with significant intrachromosomal interactions (P < 0.05 (binomial test)) in pre-pro-B cells (top) and pro-B cells (bottom): red, deposition of H3K4me2; green, CTCF occupancy; blue, E2A occupancy; orange, GRO-Seq read density; green bar, intronic enhancer (Eiκ)-Jκ region; black bars, pseudo-V segments; red bars, functional V segments. Small vertical bars along the inner perimeter indicate the genomic location of the Igk intronic enhancer. Numbers around margin indicate genomic position (in Mb). (b) SIMA of the significance and reproducibility of tethering between various genomic regions; line thickness is proportional to the log P value of association (color, as in Fig. 2b). (c) Model of the Igk locus, with a subset of Vκ segments anchored in close spatial proximity, surrounding an inner cavity containing the Jκ elements. Data are from one experiment.np

g©

201

2 N

atur

e A

mer

ica,

Inc.

All

right

s re

serv

ed.

nature immunology VOLUME 13 NUMBER 12 DECEMBER 2012 1203

A rt i c l e s

differed extensively in the pre-pro-B cell stage and the pro-B cell stage. Across what genomic regions do the E2A- and PU.1-bound contacts originate? As the sites occupied by p300 overlapped most of the E2A- and PU.1-binding sites, we suggest that this spectrum of genomic inter-actions involves mainly enhancer elements34. Do any such factors have a direct role in establishing genome topology, or is there a hierarchy of primary and secondary participants? We would like to consider the possibility that the E2A proteins act as the main participants. E2A pro-teins have been particularly well studied for their roles in the assembly of antigen receptors35,36. Enforced expression of the E2A isoform E47 readily promotes the assembly of genes encoding antigen receptors in embryonic kidney cells37,38. A substantial fraction of the Vκ regions are located in close genomic proximity to E2A-binding sites39. Here we observed that in the bulk population, functional Vκ regions seemed to cluster, but pseudo-Vκ repertoires did not. Such clustering of functional Vκ elements indicated close spatial proximity. As E2A occupancy was closely associated with a subset of V regions, we propose that binding by E2A directly promotes the assembly of Vκ regions. Such a configura-tion would permit the entire Igk V-region repertoire, separated by vast genomic distances, to encounter Jκ elements with similar probabilities, which would provide an equal ‘playing field’ for the V-region repertoire. We suggest that, similarly, the E2A proteins act directly to mediate enhancer-enhancer interactions as well as enhancer-promoter interac-tions inside chromatin domains as well as between domains. Ultimately, biochemical approaches will be needed to determine whether indeed the E2A proteins function directly as anchors.

The observations described here have also shown that in multipo-tent progenitor cells, the Ebf1 locus was sequestered at the nuclear lamina. Is the sequestration of key developmental regulators at the nuclear lamina a general principle that governs developmentally spe-cific gene expression? Notably, after closer inspection, we found that in E2A-deficient multipotent progenitor cells, Bcl11b, which encodes a key regulator that promotes commitment to the T cell lineage, was also located in the heterochromatic environment40–42. Why are the Ebf1 and Bcl11b loci located in the heterochromatic environment in progenitor cells? We suggest that the nuclear lamina serves to ensure efficient silencing. Thus, being located in a transcriptionally repres-sive compartment would not readily lead to aberrant activation of gene expression, as the entire neighborhood is predominantly in a silent state. In contrast, transcriptionally inactive genes located across the transcriptionally permissive compartment might be subject to aberrant activation—for example, by the aberrant looping of nearby located active enhancers. Furthermore, we suggest that genes encod-ing key developmental regulators, such as Ebf1 and Bcl11b, need to be efficiently silenced in multipotent progenitor cells, as inappropriate activation of such genes may lead to premature differentiation. It is now well established that the E2A proteins act indirectly and directly to induce Ebf1 expression30,43,44. However, during early hematopoiesis, E2A expression is already high in multipotent hematopoietic progeni-tors, and why E2A does not activate Ebf1 expression prematurely at the multipotent progenitor cell stage has remained an open question45. The data described here have provided a mechanism for this. The Ebf1 locus in multipotent progenitor cells was sequestered away from tran-scription factors and was tightly associated with the nuclear lamina, which prevented premature activation and developmental progres-sion toward the B cell lineage. Similarly, the Bcl11b locus might be sequestered in the heterochromatic compartment in hematopoietic progenitor cells to suppress premature developmental progression toward the T cell lineage. Hence, we propose that the positioning of key developmental regulators at the nuclear lamina in progenitor cells is a general principle for ensuring efficient silencing.

The critical question to be addressed now is how the Ebf1 and Bcl11b loci are sequestered in the heterochromatic environment and how they are released at the appropriate developmental stage. Studies have identi-fied Th-POK as a factor that mediates the sequestration of the genes at the nuclear lamina46. It is conceivable that Th-POK might similarly be involved in recruiting the Ebf1 or Bcl11b locus to the nuclear lam-ina. However, as expression of Ebf1 and Bcl11b leads to the induction of either a B lineage–specific or T lineage–specific program of gene expression, it seems more likely that they are sequestered in the lamina by distinct tethers. The identification of such tethers and how they are regulated during hematopoiesis deserves further scrutiny.

METHODSMethods and any associated references are available in the online version of the paper.

Accession codes. GEO: sequencing data, GSE40173.

Note: Supplementary information is available in the online version of the paper.

ACKnowLedGMentSWe thank G. Hardiman, C. Ludka, L. Edsall, S. Kuan, C. Espinoza, U. Wagner, J. Sprague and Z. Ye for help with Solexa DNA sequencing; J. Dixon for help during the initial phase of Hi-C analysis; B. Wold for suggesting fixation with ethylene glycol bis(succinimidylsuccinate); B. Ren for access to the Illumina Hi-Seq; and members of the Murre laboratory for comments on the manuscript. Supported by the American Recovery and Reinvestment Act (ARRA PHS 3RO1AI082850 to Y.C.L.), the European Molecular Biology Organization (C.Bo.), the Swiss National Science Foundation (C.Bo.), the University of California San Diego, Cancer Center (P30 CA23100) and the US National Institutes of Health (AI082850A and AI00880 to C.K.G. and C.M.).

AUtHoR ContRIBUtIonSR.M. and S.H. contributed equally to this work. Y.C.L., R.M. and S.H. designed and did most of the experiments; S.H. did Gro-Seq analysis; C.Be. did the bioinformatics analysis, analyzed data, suggested new approaches and developed SIMA; M.M., K.M. and V.C. did chromatin immunoprecipitation followed by deep sequencing; C.Bo. provided insight into the topology of the Igk locus; Y.C.L., C.Be. and C.M. wrote the manuscript with contributions from C.K.G., R.M. and S.H.; and C.K.G. and C.M. supervised the study.

CoMPetInG FInAnCIAL InteReStSThe authors declare no competing financial interests.

Published online at http://www.nature.com/doifinder/10.1038/ni.2432. reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Sedat, J. & Manuelidis, L. A direct approach to the structure of eukaryotic chromosomes. Cold Spring Harb. Symp. Quant. Biol. 42, 331–350 (1977).

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

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

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

5. Munkel, C. & Langowski, J. Chromosome structure described by a polymer model. Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 575B, 5888–5896 (1998).

6. Jhunjhunwala, S. et al. The 3D-structure of the immunoglobulin heavy chain locus: Implications for long-range genomic interactions. Cell 133, 265–279 (2008).

7. Bau., D. et al. The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules. Nat. Struct. Mol. Biol. 18, 107–114 (2011).

8. Guo, C. et al. Two forms of loops generate the chromatin conformation of the immunoglobulin heavy chain locus. Cell 147, 332–343 (2011).

9. Peric-Hupkes, D. et al. Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation. Mol. Cell 38, 603–613 (2010).

10. Schneider, R. & Grosschedl, R. Dynamics and interplay of nuclear architecture, genome organization and gene expression. Genes Dev. 21, 3027–3043 (2007).

11. Brown, K.E., Baxtger, J., Graf, D., Merkenschlager, M. & Fisher, A.G. Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. Mol. Cell 3, 207–217 (1999).

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1204 VOLUME 13 NUMBER 12 DECEMBER 2012 nature immunology

12. Kosak, S.T. et al. Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science 296, 158–162 (2002).

13. Fuxa, M. et al. Pax5 induces V-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev. 18, 411–422 (2004).

14. Roldán, E. et al. Locus ‘decontraction’ and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat. Immunol. 6, 31–41 (2005).

15. Hewitt, S.L., Chaumeil, J. & Skok, J.A. Chromosome dynamics and the regulation of V(D)J recombination. Immunol. Rev. 237, 43–54 (2010).

16. Goldmit, M. et al. Epigenetic ontogeny of the IgK locus during B cell development. Nat. Immunol. 6, 198–203 (2005).

17. Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).

18. Fullwood, M.J. et al. An oestrogen-receptor-α-bound human chromatin interactome. Nature 462, 58–64 (2009).

19. Kalhor, R., Tjong, H., Jayathilaka, N., Alber, F. & Chen, L. Genome architectures revealed by tethered chromosome conformation capture and population-based modeling. Nat. Biotechnol. 30, 90–98 (2011).

20. Sexton, T. et al. Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148, 458–472 (2012).

21. Li, G. et al. Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell 148, 84–98 (2012).

22. Zhang, Y. et al. Spatial organization of the mouse genome and its role in recurrent chromosomal translocations. Cell 148, 908–921 (2012).

23. Dixon, J.R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).

24. Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034–1050 (2005).

25. Dawson, M.A. & Kouzarides, T. Cancer epigenetics: from mechanism to therapy. Cell 150, 12–27 (2012).

26. Schmidt, D. et al. Waves of retrotransposon expansion remodel genome organization and CTCF binding in multiple mammalian lineages. Cell 148, 335–348 (2012).

27. Ochiai, K. et al. A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation. Nat. Immunol. 13, 300–307 (2012).

28. Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).

29. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

30. Lin, Y.C. et al. A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate. Nat. Immunol. 11, 635–643 (2010).

31. Iborra, F.J., Pombo, A., Jackson, D.A. & Cook, P.R. Active RNA polymerase are localized within discrete transcription factories in human nuclei. J. Cell Sci. 109, 142–2436 (1996).

32. Edelman, L.B. & Fraser, F. Transcription factories: genetic programming in three dimensions. Curr. Opin. Gen. Dev. 22, 110–114 (2012).

33. Corcoran, A.E. The epigenetic role of non-coding RNA transcription and nuclear organization in immunoglobulin repertoire generation. Semin. Immunol. 6, 353–361 (2010).

34. Simonis, M. et al. Nuclear organization of active and inactive chromatin domains uncovered by chromosome capture-on-chip (4C). Nat. Genet. 38, 1348–1354 (2006).

35. Jhunjhunwala, S., van Zelm, M.C., Peak, M. & Murre, C. Chromatin architecture and the generation of antigen receptor diversity. Cell 138, 435–448 (2009).

36. Inlay, M.A., Tina, H., Lin, T. & Xu, Y. Important roles for E protein binding sites within the immunoglobulin kappa chain intronic enhancer in activating Vκ to Jκ rearrangement. J. Exp. Med. 200, 1205–1211 (2004).

37. Romanow, W.J. et al. E2A and EBF act in synergy with the V(D)J recombinase to generate a diverse immunoglobulin repertoire in non-lymphoid cells. Mol. Cell 5, 343–353 (2000).

38. Sakamoto, S. et al. E2A and CBP/p300 act in synergy to promote chromatin accessibility of the immunoglobulin κ locus. J. Immunol. 188, 5547–5560 (2012).

39. Bossen, C., Mansson, R. & Murre, C. Chromatin topology and the regulation of antigen receptor assembly. Annu. Rev. Immunol. 30, 337–356 (2012).

40. Li, L., Leid, M. & Rothenberg, E.V. An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b. Science 329, 89–93 (2010).

41. Ikawa, T. et al. An essential developmental checkpoint for production of the T cell lineage. Science 329, 93–96 (2010).

42. Li, P. et al. Reprogamming of T cells to natural killer-like cells upon Bcl11b deletion. Science 329, 85–89 (2010).

43. Welinder, E. et al. E2A and HEB act in concert to induce the expression of FOXO1 in the common lymphoid progenitor. Proc. Natl. Acad. Sci. USA 108, 17402–17407 (2011).

44. Seet, C.S., Brumbaugh, R.L. & Kee, B.L. Early B cell factor promotes B lymphopoiesis with reduced interleukin 7 responsiveness in the absence of E2A. J. Exp. Med. 199, 1689–1700 (2004).

45. Semerad, C.L., Mercer, E.M., Inlay, M.A., Weissman, I.L. & Murre, C. E2A proteins maintain the hematopoietic stem cell pool and promote the maturation of myelolymphoid and myeloerythroid progenitors. Proc. Natl. Acad. Sci. USA 106, 1930–1935 (2009).

46. Zullo, J.M. et al. DNA sequence-dependent compartmentalization and silencing of chromatin at the nuclear lamina. Cell 149, 1474–1487 (2012).

A rt i c l e snp

g©

201

2 N

atur

e A

mer

ica,

Inc.

All

right

s re

serv

ed.

nature immunologydoi:10.1038/ni.2432

ONLINE METHODSMice. Animal studies were approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. All mice were main-tained on the C57BL/6 background.

Cell culture. E2A-deficient hematopoietic progenitor cells were isolated and grown in the presence of IL-7, stem-cell factor and the hematopoietic growth factor Flt3L as described47. RAG-1-deficient pro-B cells were grown for 7 d in OptiMEM with 10% FCS, 2% glutamine, penicillin and streptomycin, and 50 µM β-mercaptoethanol in the presence of IL-7 and stem-cell factor as described48.

ChIP-Seq. Chromatin was immunoprecipitated as described30 (details, Supplementary Methods). Antibodies used in these experiments were as follows: antibody to (anti-) H3K4me2 (07-030; Millipore), anti-H3K27me3 (07-449; Millipore), anti-CTCF (07-729; Millipore), anti-Rad21 (ab992; Abcam), anti-H3K36me3 (ab9050; Abcam), anti-c-Myc (sc-764x; Santa Cruz Biotechnology) and anti-p300 (sc-585; Santa Cruz Biotechnology) antibody.

Imaging. Fluorescent in situ hybridization and image acquisition were done as described48. The bacterial artificial chromosome probes were RP23-118P17, RP23-286D14 and RP24-74E7 from the BACPAC Resource Center at Children’s Hospital Oakland Research Institute. Three-dimensional fluo-rescent images were acquired on a deconvolution microscope (Deltavision) with a 100× objective. Optical sections (z stacks) 0.2 µm apart were obtained in the DAPI, FITC, Red and Cy5 channels. Distances separating probes

were measured with the center of mass as the focal point. The center of mass was determined by SoftWorx software package. Chromatic aberration was corrected by measurement of TetraSpeck microspheres (size, 0.2 µm) as described6. Immunofluorescent in situ hybridization and image acquisi-tion were done as described48 with the following changes. The probe used for Ebf1 domain (RP23-118P17) was labeled with Alexa Fluor 488–5-dUTP. The nuclear lamina was stained first with primary antibodies to lamin A and B (sc-6214 and sc-6217; Santa Cruz Biotechnology), followed by secondary staining with donkey antibody to goat IgG conjugated to Alexa Fluor 594 (A11058; Invitrogen). Fluorescent images were acquired on a deconvolution microscope (Deltavision) with a 100× objective. Optical sections (z stacks) 0.2 µm apart were obtained in the DAPI, FITC and Red channels. Distances between the nuclear lamina and the Ebf1 locus were measured in two dimen-sions with SoftWorx software package.

Hi-C analysis. Computational data were analyzed with software developed in-house. Hi-C analysis was integrated into HOMER, a general Next-gen sequenc-ing analysis suite (http://biowhat.ucsd.edu/homer/). Specialized visualization of HOMER analysis files was accomplished with Java Tree View, Circos soft-ware and Cytoscape software (details, Supplementary Methods).

47. Ikawa, T., Kawamoto, H., Wright, L.Y. & Murre, C. Long-term cultured E2A-deficient hematopoietic progenitor cells are pluripotent. Immunity 20, 349–360 (2004).

48. Sayegh, C.E., Jhunjhunwala, S., Riblet, R. & Murre, C. Visualization of looping involving the immunoglobulin heavy-chain locus in developing B cells. Genes Dev. 22, 322–327 (2005).

npg

© 2

012

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.