Two Forms of Loops Generate
the Chromatin Conformation of the
Immunoglobulin Heavy-Chain Gene Locus
Changying Guo,1,4Tatiana Gerasimova,1,4Haiping Hao,2Irina Ivanova,1Tirtha Chakraborty,1,5Roza Selimyan,1
Eugene M. Oltz,3and Ranjan Sen1,*
1Gene Regulation Section, Laboratory of Molecular Biology and Immunology, National Institute on Aging, 251 Bayview Boulevard, Baltimore,
MD 21224, USA
2JHMI Deep Sequencing & Microarray Core, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
3Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA
4These authors contributed equally to this work
5Present address: Immune Disease Institute, Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston,
MA 02115, USA
The immunoglobulin heavy-chain (IgH) gene locus
undergoes radial repositioning within the nucleus
and locus contraction in preparation for gene recom-
bination. We demonstrate that IgH locus conforma-
tioninvolves twolevelsofchromosomal compaction.
At the first level, the locus folds into several multi-
looped domains. One such domain at the 30end of
the locus requires an enhancer, Em; two other do-
mains at the 50end are Em independent. At the
second level, these domains are brought into spatial
proximity byEm-dependent interactions withspecific
sites within the VHregion. Em is also required for
radial repositioning of IgH alleles, indicating its es-
sential role in large-scale chromosomal movements
in developing lymphocytes. Our observations pro-
vide a comprehensive view of the conformation of
IgH alleles in pro-B cells and the mechanisms by
which it is established.
Radial positioning of loci within the nucleus and chromosome
conformation have recently gained prominence as mechanisms
for developmentally regulated gene expression (Kadauke and
Blobel, 2009; Takizawa et al., 2008). This interest rides on the
foundation of pioneering studies that examined global chro-
mosome structure and folding within the nucleus (Gasser and
Laemmli, 1987; Paulson and Laemmli, 1977). Of particular
note, the concept of chromosomal loops arose from a com-
bination of biochemical and direct visualization studies (Cook
et al., 1976). Based on the observation that loops were tethered
at their base to the nuclear scaffold, Laemmli and colleagues
proposed a rosette-like configuration for chromosomes (Mars-
den and Laemmli, 1979). Chromosomal loops are also the
central feature of computational models of chromosome
structure that account for chromosome conformation by
varying the size and numbers of loops associated with chromo-
somal domains (Knoch et al., 2000; Sachs et al., 1995). The
extent to which these features apply to developmentally regu-
lated loci and the mechanisms by which these structures
are generated are critical for understanding gene regulatory
Antigen receptor genes of B and T lymphocytes are assem-
bled from gene segments that are spread over several mega-
bases of the genome (Krangel, 2009; Perlot and Alt, 2008). The
immunoglobulin heavy-chain (IgH) locus in the mouse consists
of 150 variable (VH) gene segments, 8–12 diversity (DH) gene
segments, and 4 joining (JH) gene segments (Johnston et al.,
2006; Retter et al., 2007). Two rearrangement steps assemble
functional IgH genes during B cell development. First, a DH
gene segment recombines with a JHgene segment to form
a DJHjunction; this is followed by VHrecombination to the DJH
junction to generate V(D)J recombined alleles.
Prior to initiation of DNA rearrangements, the IgH locus
undergoes two forms of chromosome movements. First, radial
repositioning moves the locus away from the nuclear periphery
to a more central location (Kosak et al., 2002). This step does
not occur in progenitors that lack the transcription factor E2A
(Sayegh et al., 2005), in which B cell differentiation is blocked
at a very early stage. Second, locus contraction brings the two
ends of the IgH locus into physical proximity (Kosak et al.,
2002; Sayegh et al., 2005). These movements are independently
regulated because locus contraction, but not radial reposition-
ing, is abolished in B cell progenitors that lack the transcription
Busslinger and colleagues proposed that Pax5 mediates locus
contraction via a conserved sequence element that they named
teen PAIRs, of which seven bind Pax5 in pro-B cells, are spread
332 Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc.
whether YY1 is mechanistically connected to the Pax5/PAIR
Jhunjhunwala et al. (Jhunjhunwala et al., 2008) developed
a model for IgH locus conformation in its germline (prerearrange-
ment) state. They measured spatial distances between different
points throughout the IgH locus using 3D-FISH and trilateration.
The data was used to mathematically compute the conformation
of the genomic region. They found that, in transcription factor
E2A-deficient pre-pro-B cells, IgH locus conformation fit best
within the framework of the computational major loop subcom-
partment (MLS) model. In further differentiated pro-B cells, how-
ever, the conformation is more compact and deviates signifi-
cantly from the MLS model. A central feature of the state in
pro-B cells is that the distal VHgenes (labeled J558 and 3609
in Figure 1A) and proximal VHgenes (labeled 7183 in Figure 1A)
are positioned at comparable spatial distance from the DH-JH
part of the IgH locus. The molecular mechanisms by which these
changes are brought about are not clear.
The tissue-specific enhancer Em (Figure 1A) regulates both
steps of IgH locus recombination (Afshar et al., 2006; Perlot
et al., 2005). We previously showed that deletion of the 220
nucleotide Em core results in a partially active locus in precursor
B cells (Chakraborty et al., 2009). Em-deleted alleles lack acety-
lated histones H3 and H4, but other activation-specific epige-
netic marks, such as H3K4me2 or tissue-specific loss of
H3K9me2, are clearly evident. Based on these observations,
we proposed that full activation of the IgH locus requires Em-
independent and Em-dependent steps. Here, we demonstrate
that the conformation of the IgH locus is generated by Em-
dependent and Em-independent chromatin loops. One set of
Em-dependent interactions defines a domain that encompasses
the 30262 kb of the locus. A second set of Em-dependent inter-
actions brings parts of the VH locus close to the DH gene
segments. All Em-interacting sequences bind the transcription
factor YY1, indicating a role for this factor in establishing Em-
dependent loops. We also found evidence for Em-independent
looping between CTCF-bound sites in the IgH locus. Further-
more, Em-deleted alleles did not undergo radial repositioning,
indicating that Em-independent forms of locus activation can
occur at the nuclear periphery. Our observations provide a com-
prehensive view of the conformational state of the IgH locus in
pro-B cells and the mechanisms by which it is established.
Em Regulates Radial Positioning and IgH Locus
To understand the relationship between cis-regulatory se-
quences and epigenetic changes at the IgH locus, we deter-
mined radial positioning of IgH alleles with defined deletions
(Figure 1A). P?E+alleles (that delete only a promoter, PQ52,
associated with DQ52) and P?E?alleles (that delete both Em
and PQ52) have been previously described (Afshar et al.,
2006). Both of these alleles were analyzed in a recombinase-
deficient context to maintain the locus in unrearranged state.
JHT alleles lack a 3.5 kb region starting at the 50end of the
P?deletion and extending to the 30end of the E?deletion
(Gu et al., 1993). These alleles were assayed in recombinase-
sufficient cells because the absence of all JHgene segments
precludes any rearrangement of these alleles. We isolated
primary pro-B cells from the bone marrow by positive selection
using anti-CD19-coupled magnetic beads and used the cells
tion (FISH) studies.
We used bacterial artificial chromosome (BAC) probes that
mark the 50and 30ends of the IgH locus to study IgH radial posi-
tioning and locus contraction. WT IgH alleles were located away
averaged in 1C). Loss of PQ52 (P?E+alleles) did not affect radial
positioning in either pro-B or pro-T cells. However, P?E?alleles
were located closer to the nuclear periphery in pro-B cells
compared to WT or P?E+alleles (Figures 1B and 1C and Fig-
ure S1 available online). Indeed, the location of P?E?alleles in
pro-B cells was similar to that of WT or P?E+alleles in primary
pro-T cells (Figure 1B, bottom). These observations indicate
that Em is necessary for radial repositioning of IgH alleles in
primary pro-B cells.
genotypes. We found that P?E?and JHT alleles did not undergo
locus contraction in pro-B cells, as visualized by the lack of over-
lap of FISH signals (Figure 1B and averaged in 1D). Instead, the
average distance between the two probes in P?E?and JHT pro-
B cells was similar to that seen in pro-T cells of each genotype or
in non-B lineage cells from the bone marrow of WT mice (Fig-
ure 1D). This effect was specific to loss of Em, as P?E+alleles
underwent normal locus contraction. Additionally, IgH alleles
deleted only for Em also did not contract (Figures 3D and 3E).
Thus, Em is essential for locus contraction; in contrast, PQ52
does not contribute to either radial positioning or locus contrac-
tion of IgH alleles.
Em-Dependent Locus Contraction
To understand the basis for Em-dependent locus contraction, we
locus that were in close proximity to Em. For this, crosslinked
chromatin from a RAG-deficient pro-B cell line, D345, was
digested with Nla III or Mse I, ligated, and then amplified using
anchor primers from the test region (Figure 2A). Sequences
ligated between the anchor primers were identified by hybridiza-
tion to mouse genomic tiling 2.0R E arrays that contained mouse
chromosome 12; as a control, we used sonicated genomic DNA
from the same cells. Array data were analyzed using CisGenome
(Ji et al., 2008).
We found that the 30regulatory region (30RR) of the IgH locus
was prominently represented in sequences amplified with Em
anchor primers (Figure 2B, arrow 1 and Figure S2A). The 30RR
comprises a cluster of eight DHSs distributed over 30 kb; five
of these (HS3a, b, 1, 2, and 4) are found only in activated mature
B cells, whereas HS5–7 are present in pro-B cell lines (Garrett
et al., 2005). The HS1,2 region has been previously shown to
be close to Em in mature splenic B cells and a myeloma cell
line (Ju et al., 2007; Wuerffel et al., 2007). Sequences that we
amplified within Em anchors corresponded to the HS5 region
(Figure 2C). Thus, Em-30RR association occurs in the earliest B
cell progenitors prior to initiation of V(D)J recombination. We
Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc. 333
also identified sequences just 50of DFL16.1 (50DFL) (Figures 2B
and 2C, arrow 2, and S2A) and a region close to the 50end of the
proximal VH7183 gene family (Figures 2B and 2C, arrow 3, and
S2A) in Em-anchored 4C assays. HS5, 50DFL, and 507183 are
located ?206 kb, ?57 kb and ?400 kb from Em, respectively,
suggesting that these regions are brought into proximity of Em
by chromosome looping.
We also identified a sequence located toward the 30end of the
VHJ558 genes ?1 Mb from Em (Figures 2B and 2C, arrow 4, and
S2A), but the signal intensity was much lower. Em association
with the 30RR,50DFL,and 507183was alsodetected in 4Cassays
using a different restriction enzyme, Mse I (Figure 2B, bottom
line). The inability to detect 30558 sequence using Mse I may
be because the sites for this restriction enzyme are not appropri-
atelyjuxtaposed in crosslinked chromatin. Finally, wecarried out
4Cwithanchorprimerslocated withinthenewlydetected 507183
region. We detected prominent interactions with Em and the
30RR, thereby strengthening the idea that these regions were in
spatial proximity in pro-B cells (Figure S2B). We conclude that
Em interactions form a domain that contains all DHand JHgene
segments, as well as exons that encode all Ig isotypes. In addi-
Figure 1. Nuclear Positioning and Locus
Contraction of IgH Alleles with cis-Regula-
tory Sequence Deletions
(A) Top line is a schematic representation of
the murine IgH locus. Approximate distances
between regions of interest are derived from the
sequence of the locus in C57BL6 mouse strain
(Johnston et al., 2006).The telomere (black circle)-
proximal variable region (VH) spans ?2.5 Mb and
contains 150 VHsegments. Gene segments cor-
responding to J558 and 3609 families are largely
interspersed at the 50end. The 7183 family lies at
the 30end of the cluster. The 50-most and 30-most
diversity (DH) gene segments, DFL16.1 and DQ52,
are indicated; between them are variable numbers
of DSP gene segments depending on the mouse
strain. JHgene segments are depicted as black
vertical lines. Two cis-regulatory elements dis-
cussed here, PQ52 and Em, are indicated as
ovals and are marked by tissue-specific DNase
I-hypersensitive sites (red arrows). The region
containing exons of various IgH isotypes spans
another 200 kb and is followed by a cluster of
DNase I-hypersensitive sites that comprise the
30regulatory region (30RR). The next three lines
show IgH alleles that carry deletions of specific
regulatory sequences (shown by dotted lines) as
indicated; IgH genotype notations used in the text
are noted on the right. Red and green lines below
the WT allele show the position of BAC probes
used in FISH analyses.
(B) Two-color FISH using bone marrow pro-B cells
(a–d) and thymocytes (e–h) that carry wild-type
(WT) or mutated IgH alleles as indicated. BAC
probes are indicated in (A), and blue color marks
nuclear DNA with DAPI. A representative nucleus
from each genotype is shown.
(C) Radial positioning of WT and mutated IgH
alleles was determined by measuring the distance
of red and green FISH signals from the nuclear
boundary in ?200 nuclei from pro-B cells and
thymocytes of the indicated IgH genotypes. y axis
shows the distance between FISH signals and the
nuclear periphery divided by the nuclear radius.
Error bars represent the SD between nuclei. The
percentage of IgH alleles close to the nuclear
periphery in each genotype is shown in Figure S1.
(D) Locus contraction of WT and mutated IgH
alleles was estimated by measuring the distance
between red and green FISH signals in ?200
nuclei. y axis shows the distance between FISH
signals divided by the nuclear radius. B lineage-depleted bone marrow cells from RAG2-deficient mice were used as non-B controls. Error bars represent the SD
between nuclei. Measurements were made with three independent cell preparations each obtained from five to six mice of the indicated genotypes.
334 Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc.
30558; these interactions are possible sources of Em-dependent
locus contraction. Neither of the VH-associated Em-interacting
sequences corresponds to PAIR elements.
To substantiate the interactions detected by 4C, we carried
out quantitative 3C analyses. Using Em as anchor (Figure 3A),
we detected prominent interactions with 50DFL, the 30RR
(labeled HS1,2 and HS5), and 507183. The interaction with
30558 was weaker but significantly higher than regions A–D,
which served as negative controls. Conversely, using 30558 as
the anchor (Figure 3B), we detected interactions with 507183,
50DFL, Em, and the 30RR, thereby confirming spatial proximity
of these widely separated parts of the IgH locus. Fragment B,
located 34 kb from 30558, scored strongly with the 30558 anchor,
but not with Em anchor. Proximity between the 30558 anchor and
fragment B could be one possible explanation for this; alterna-
tively, 30558 could be involved in more than one kind of loop.
We also carried out 3C studies with anchors located at 507183
and the 30RR (Figure S3A and S3B) and confirmed reciprocal
interactions between all five interacting sequences identified
by 4C analyses.
Though it is difficult to directly compare 3C results using
different anchor primers (and associated Taqman probes), we
noticed that the relative association frequency between different
parts of the locus varied with the anchor used. For example, the
113.5 114.5 115.5116.5Mb
Site 1 (40 kb)Site 2 (150 kb)
Site 3 (40 kb)Site 4 (50 kb)
7183.18.35Q52.11.34 J558.2.88J558.1.85VH10.1.86 3609.1.85J606.5.83
Figure 2. Em-Mediated Long-Range Chromatin Interactions in the IgH Locus
(A) Schematic representation of the unrearranged IgH locus oriented with the DH-Cm region on the right and VHregion on the left. The region surrounding Em is
expanded below to show the positions of restriction enzyme sites (N = Nla III; X = Xba I; E = EcoR I) and bait primers (black triangles) used in 4C assays.
(B) 4C was carried out as described in the Experimental Procedures using D345, a recombinase-deficient Abelson virus transformed pro-B cell line of C57BL6
origin. Position of bait primers close to Em is marked by the asterisk. Genomic sequences that were ligated between the Nla III sites (top) or Mse I sites (bottom)
were amplified by PCR using the bait primers and hybridized to Affymetrix Genomic tiling 2.0R E array. Hybridization signal intensity was compared to input DNA
in CisGenome and fold enrichment (y axis) calculated as described in Experimental Procedures. Numbered arrows mark enriched regions. 1 corresponds to the
HS5region of the 30RR,2corresponds tosequences50of DFL16.1(50DFL in text), 3corresponds tosequence referred toas 507183inthe text, and 4corresponds
to sequence referred to as 30558 in the text. One of two independent experiments with each restriction enzyme is shown; high-resolution 4C data is shown in
Figure S2A. 4C analysis with 507183 anchor is shown in Figure S2B.
further high resolution 4C data is shown in Figure S2A.
Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc. 335
Em anchor detected interaction with 50DFL and HS5 more effec-
tively than with 507183 or 30558. Conversely, the 30558 anchor
detected 507183 more effectively than 50DFL, Em, or HS5. Our
working hypothesis is that these selectivities represent preferen-
tial associations in pro-B cells. One set of prominent interactions
involves Em, 50DFL, and HS5 that leads to a 206 kb domain at the
30end of the IgH locus. Another set of interactions, exemplified
by 30558 to 507183, occurs within the VHpart of the locus. Inter-
domain interactions represented by Em to 30558 or 507183, or by
HS5 to 30558 or 507183, are relatively less efficient and may
occur, for example, in a smaller proportion of cells. Such con-
tacts may get ‘‘fixed’’ during crosslinking to be revealed in the
3C or 4C assays.
To determine the role of Em in establishing locus conformation,
we carried out quantitative 3C analyses using chromatin pre-
pared from primary bone marrow pro-B cells carrying WT,
P?E+and P?E?IgH alleles. An Em anchor readily amplified
sequences 50of DFL16.1 and within the 30RR on WT alleles (Fig-
ure 3C, blue bars labeled 50DFL and HS5). The signal to HS1,2
likely represents the lower part of a peak centered around
HS5. Additionally, we detected Em interactions with 507183 and
30558 regions within the VHlocus on WT alleles. Two regions,
labeled c116 and c32, that flank 507183 served as negative
controls. All Em associations were substantially reduced in
P?E?pro-B cells, but not in P?E+pro-B cells, demonstrating
bars). Loops within the b-globin locus were comparable in all
three cell preparations (Figure S3C). We only detected Em inter-
action with 50DFL and the 30RR in CD4?CD8-thymocytes (Fig-
ure 3C, light blue bars), indicating that Em associations with the
VHlocus were B lineage specific. We conclude that Em is essen-
tial to establish chromatin loops to 50DFL, 30RR, and specific
sites in the VHlocus. The absence of Em-dependent loops to
these VHsites (507183 and 30558) in pro-B cells may be the basis
for the lack of locus contraction of P?E?alleles noted in FISH
analyses (Figure 1).
To further confirm the presence of Em-dependent loops, we
carried out high-resolution 3D-FISH analyses on primary pro-B
cells from RAG2?/?and P?E?(RAG2?/?) mice using 10 kb
probes. Previously described probes h4 and h11 (Jhunjhunwala
et al., 2008) were used to validate our results; these probes mark
sequences close to Em and toward the 50end of the VHJ558 gene
family, respectively (Figure 4A). We also generated new probes
corresponding to looping sites identified by our 4C assays (Fig-
ure 4A). To visualize Em-dependent interactions, we used probe
h4 with probes h11, 507183, and 30558. Each probe combination
gave closely juxtaposed signals in WT and P?E+pro-B cells, but
not in P?E?pro-B cells (Figure 4B). After quantitation of interp-
robe distance, we found that compaction of P?E?alleles was
reduced by 1.4- to 1.8-fold for h4-h11, as well as for each new
Figure 3. 3C Analyses of Em-Interacting
Quantitative 3C analyses in D345 pro-B cells using
different anchors (gray) as indicated. Taqman
probes for detection of amplicons were located
close to the anchor primers. Data with Em (A) and
30558 (B) anchor primers are shown (additional 3C
studies with 507183 and HS5 anchors are in Fig-
ures S3A and S3B). Association frequency (y axis)
between two primers was normalized to long-
range 30HS1 interaction in the b-globin locus; gray
arrows mark the site of anchor primers in the bar
graphs. Data shown are the average of three in-
dependent 3C experiments, with error bars rep-
resenting the standard error of mean between
(C) Quantitative 3C analyses using primary bone
marrow pro-B cells and thymocytes that carry IgH
alleles of the indicated genotypes (all cells were
obtained from RAG2-deficient background). Co-
ordinates of the IgH locus in the 129 strain
(Simpson et al., 1997) are shown on the top line
with positions of the relevant sequences identified
by 4C. 3C assays were carried out using anchor
primer in combination with primers located near
HS1,2, HS5, 50DFL, 507183, and 30558; the c116
and c32 sequences served as negative controls.
Amplification products were detected using a
Taqman probe located close to the Em anchor
two primers was normalized to long-range 30HS1
interaction in the b-globin locus (Figure S3C). Data
shown are the average of three independent
3C experiments in each genotype; error bars re-
present the standard error of mean between
336 Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc.
pairwise probe combination (Table S1). We also determined the
proportion of IgH alleles in which the two FISH signals were
separated by different distances. For all probe combinations,
the separation between FISH signals was skewed toward
greater separation on P?E?alleles (Figure 4C).
Finally, we used two probe sets for FISH analyses in pro-B cell
cells, the separation distance between probes was increased in
?80% of Em?alleles (Figures 4D and 4E and Table S1). Thus,
physical proximity of the VHregion to the DH-Cm region requires
Em. Interestingly, FISH analysis also showed decontraction of
the interaction between DFL16.1 and the 30RR on P?E?alleles
(Figures 4F and 4G and Table S1). Taken together, these obser-
vations demonstrate the presence of several Em-dependent
loops in the IgH locus.
Em-Associated Looping Sites Bind YY1
ThebasisforlackofIgHlocuscontraction inYY1-deficient pro-B
cells (Liu et al., 2007) is not known. We reasoned that Em-bound
YY1 (Park and Atchison, 1991) may interact with other YY1-
bound sequences to induce locus contraction. We therefore
analyzed YY1 binding to key Em-associated looping sites by
chromatin immunoprecipitation. We detected YY1 binding to
sequences 3 kb 50of DFL16.1 and in HS5-7 of the 30RR that
are involved in Em-50DFL and Em-30RR loops (Figure 5A, blue
bars in the section marked D-J-Cm). These sites also bound
CTCF (Featherstone et al., 2010; Garrett et al., 2005), though
Em itself did not (Figure 5A, red bars). Immunofluorescence
studies demonstrated that YY1 and CTCF also colocalized at
a subset of nuclear sites (Figure S4). These observations are
consistent with Em-50DFL and Em-30RR loops being mediated
% IgH alleles
% IgH alleles
% IgH alleles
Figure 4. Visualization of Em-Dependent
(A) The unrearranged IgH locus as represented in
Figure 2A showing the location of six 10 kb probes
used for FISH.
(B) Three color 3D-FISH were carried out with
bone marrow pro-B cells of the indicated IgH
genotypes cells in a RAG2-deficient background.
Short probes labeled with Alexa Fluor 594 (red)
and 488 (green) and BAC RP23-201H14 labeled
withAlexa Fluor 697(blue) werehybridizedtofixed
pro-B cells. Signals were visualized by epifluor-
escence microscopy, and distances between
probes were determined as described (Jhunjhun-
wala et al., 2008) and shown in Table S1. Probe
combinations were: a, d, and g h4, red and h11,
green; b, e, and h h4, red and 507183, green; c, f,
and i h4, green and 30558, red. Red line represents
(C) Quantitation of FISH data shown in (B).
Distances between red and green 3D FISH signals
in (A) were divided into five categories (<0.2, 0.2–
0.5, 0.5–0.8, 0.8–1.0, and >1.0 mm) for 60–90
nuclei. The percentage of IgH alleles in each
category was determined (y axis) for each IgH
genotype (x axis) and is represented in different
colors. Probe combinations are shown above the
bars.Pro-Bcells were purified from fivetosix mice
of each genotype.
(D) Three-color 3D-FISH with RAG2-deficient
pro-B cell lines carrying WT and Em-deleted IgH
alleles. Probe combinations were: a and c h4, red;
h11, green; b and d h4, green and 30558, red.
(E) Quantitation of the FISH data shown in (D), as
described in (C).
(F) Three-color 3D-FISH with bone marrow pro-B
cells of the indicated IgH genotypes cells obtained
from RAG2-deficient background. Probe combi-
d DFL, red and 30RR, green. Pro-B cells were
purified from five to six mice of each genotype.
(G) Quantitation of FISH data shown in (F), as
described in (C).
Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc. 337
by homotypic YY1 interactions or by Em-bound YY1 interacting
with CTCF-bound 50DFL or 30RR.
We also detected YY1 binding near 507183 and 30558, but not
at several other sites in the VHlocus (Figure 5A). However, YY1
binding to the 507183 and 30558 was lower than that at the
30RR or 50DFL. Sites that did not bind YY1 were located within
different VHgene families at the 50, middle, and 30ends of the
VHlocus. Thus, 507183 and 30558 are distinctly different from
other parts of the VHlocus with regard to YY1 binding, which
may be why they are preferred sites of interaction with Em. We
cannot rule out the presence of other YY1-binding sites in
regions that were not queried by our primer sets. To determine
whether Em regulates YY1 binding to looping sites, we assayed
YY1 binding on Em-deficient alleles. We found that YY1 bound
normally to all sites, other than Em, on Em-deficient alleles (Fig-
ure 5B). We conclude that Em does not regulate YY1 binding to
Figure 5. Interaction of YY1 with Em-Interacting
(A) Chromatin immunoprecipitations were carried out with
anti-YY1 and anti-CTCF antibodies using D345 pro-B
cells. ChIP primersfrom the 30region of the IgH locus used
are shown on the top line. VHprimers assay gene families
noted above the bar graph. The relative abundance of
specific amplicons in the immunoprecipitate compared to
input DNA is shown on the y axis. y axis scales differ for
YY1 ChIP (left) and CTCF ChIP (right). RPL30 is a positive
control for YY1 binding (Liu et al., 2007). HS5–7 corre-
spond to amplicons located within these DNase I-hyper-
sensitive sites in the 30RR, DFL(-3), and DFL(-6) amplicons
are located 3 and 6 kb 50of DFL16.1; amplicons near
507183 and 30558 are indicated. Data shown are the
average of three independent ChIP experiments with each
antibody; error bars represent the SD between experi-
ments. See also Figure S4.
(B) Chromatin immunoprecipitation was carried out with
anti-YY1 antibody using RAG2-deficient pro-B cell lines
carrying WT and Em-IgH alleles. Amplicons are as noted
in part A. Error bars represent the SD between three
other parts of the locus; rather, it provides
a YY1-binding site that other YY1-bound parts
of the locus can interact with.
IgH Locus Loops that Involve CTCF
The transcription factor CTCF has been impli-
cated in chromosome looping at several loci
(Gerasimova et al., 2007; Phillips and Corces,
2009; Wallace and Felsenfeld, 2007). More
than60 CTCF-binding siteshave been identified
in the germline IgHlocus (Degner et al.,2009), of
which the majority are located within the VH
domain. To determine whether CTCF is involved
in looping of the VHregion, we carried out 4C
assays using chromatin immunoprecipitated
with anti-CTCF antibodies (ChIP-loop). The an-
chor locations VH3 and VH10 were chosen as
representative sites within the proximal and distal VHgenes,
respectively (Figure 6A).
VH3 anchorprimers identified several regions within 140 kb,as
well as sequences 50of DFL16.1 located ?250 kb away (Fig-
ure 6B, red trace, arrow DFL(?3)). Because CTCF binds
50DFL16.1 sequences, we infer that a CTCF-containing loop
brings DFL16.1 into the proximity of the VH7183 gene family.
VH10 anchor primers identified four major interacting sites span-
ning ?500 kb (Figure 6B, blue trace, arrows 1–4). One of these
sites (VH10-3) corresponds to a previously identified CTCF-
binding site (VH8 in Degner et al., 2009). To obtain independent
evidence that these sequences were involved in chromosome
looping, we carried out regular 4C using VH3 and VH10 anchor
primers. In each case, we noted interactions with sites identified
in the ChIP-loop assay (Figure 6C, indicated by arrows), as
well as sites that were not detected in the ChIP-loop assay.
338 Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc.
The latter could be due to looping factors other than CTCF or
sites bound weakly by CTCF.
We further tested whether the newly identified VH3- and VH10-
interacting regions bound CTCF. We found that most VH3 inter-
acting sequences bound CTCF at high levels (Figure 6D, VH1-3
and VH3-[1, 3, 4]). In contrast, only VH10 and VH10-3 bound
CTCF efficiently (Figure 6D), suggesting that loops to VH10-1, 2
and VH10-4 may contain other factors. CTCF binding to these
h4 DFLV3 V10-3V10 201H14
% IgH alleles
Figure 6. CTCF-Containing Loops in the IgH Locus
(A) A schematic of the IgH locus as described in Figure 2A.
J558/3609, S107, and 7183 refer to VHgene families.
Triangles show positions of oppositely oriented primers
labeled VH3 and VH10 used in ChIP-loop and 4C assays.
The nearest VH gene segment to these primers is
(B) ChIP-loop 4C assays were performed using D345 pro-
B cells. Crosslinked chromatin was immunoprecipitated
with anti-CTCF antibodies, followed by digestion of the
associated chromatin with Nla III. After re-ligation, the
DNA was amplified with VH3 or VH10 primers. Sequences
amplified within VH3 primers (red trace) or VH10 primers
(blue trace) were hybridized to Affymetrix chromosome 12
tiling arrays and quantitated as described in Experimental
Procedures. Asterisk indicates position of anchor primers;
labeled arrows indicate positions of sequences identified
in the assay. Data shown are representative of two inde-
pendent experiments with each anchor primer.
(C) Conventional 4C assay using Nla III restriction enzyme
was carried out using VH3 (red trace) or VH10 (blue trace)
anchor primers. Asterisks mark the position of anchor
primers; arrows indicate interacting regions shared
between ChIP-loop and 4C arrays.
(D) CTCF binding to sites identified by anti-CTCF ChIP-
loop assays. Crosslinked chromatin from D345 cells was
immunoprecipitated with anti-CTCF or anti-Rad21 anti-
bodies. Coprecipitated genomic DNA was amplified with
primers close to regions identified in ChIP-loop and 4C
assays. VH1, 2, 3, VH3-1 to VH3-5 lie in the cluster of in-
teracting sequencesidentifiedwithVH3primers. VH10-1to
VH10-4 correspond to interacting sequences identified
with VH10 primers. VH3 and VH10 amplicons are close to
the corresponding anchor primers used in 4C assays.
CTCF binding to the 30DNase I-hypersensitive site (30HS1)
in the b globin locus was used as the positive control.
CTCF binding within the IgH locus is Em independent
(Figure S5A). Error bars represent the SD between three
(E) Top line shows the location of five short probes used in
3D FISH (see also Figure S5B). Three-color 3D-FISH was
carried out using bone marrow pro-B cells with wild-type
and P?E?IgH alleles on a RAG2-deficient background.
Probes were labeled as follows: a and c, DFL, red and V3,
green; b and d, V10-3, red and V10, green. Distances
between probes were determined as described in Fig-
ure 4C and shown in Table S1. (Right) Quantitation of FISH
of 60–90 nuclei from two independent cell preparations.
3C analysis of V3-DFL loops in WT and Em?pro-B cells is
shown in Figures S5C and S5D.
sites did not require Em (Figure S5A). Our
biochemical data are consistent with separate
loops from VH10 to each of VH10-(1–4), leading
to 500 kb, 450 kb, 287 kb, and 113 kb loops, respectively. Alter-
natively, the four interacting sites may coalesce to form a ‘‘hub’’
from which four loops of 113 kb, 170 kb, 180 kb, and 20 kb
radiate. Additional studies are needed to distinguish between
To independently verify proximity between these sequences
and to determine whether they required Em, we used 3D-FISH
to measure spatial distances between DFL16.1 and VH3 and
Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc. 339
between VH10-3 and VH10. New probes corresponding to loop-
ingsitesidentified byourChIP-loop 4Cassayswereprepared by
amplification of appropriate BAC templates (Figure 6E, top line,
labeled DFL, V3, V10-3, and V10). DFL/V3 and V10/V10-3 probe
pairs resulted in virtually superimposed FISH signals in pri-
mary pro-B cells with WT or P?E?IgH alleles (Figure 6E and
Table S1). In contrast, V10-3/h4 probe proximity was disrupted
on P?E?alleles (Figure S5B). Quantitation of the distribution of
interprobe distances on WT and P?E?alleles (Figure 6E) or locus
compaction (Table S1) revealed no difference between WT and
P?E?alleles. Because the comparably sized DFL-30RR loop
undergoes easily discernible locus decontraction on P?E?
alleles, we conclude that CTCF-involving loops, such as those
dent. We further confirmed Em independence of the DFL-V3 loop
by 3C assays in cells containing WT or Em?IgH alleles (Figures
S5C and S5D).
The mechanisms by which coordinated chromosomal move-
scriptional regulation. Such movements remove genes from the
‘‘repressive’’ environment of the nuclear periphery or bring
together clusters of genes in transcription factories. Beyond
nuclear location, conformational changes within a locus permit
interactions betweenregulatorysequences orhelpdemark inde-
pendently regulated chromatin domains. The IgH locus under-
goes several forms of chromosomal movements that ensure
developmental stage- and lineage-specific DNA recombination
and transcription. Here, we demonstrate that IgH locus confor-
mation is generated in two steps. The first step generates multi-
looped domains whose sizes range from 200 to 400 kb. The
second step brings these domains together to spatially juxta-
pose the 50and 30ends of the locus and thereby generate a fully
compacted state. A cis-regulatory element, Em, participates in
both steps of locus compaction. The functional implications of
each chromatin domain are discussed below.
Based on a combination of 3C, 4C, and FISH studies, we
propose that Em nucleates a domain that extends from a few
kb 50of DFL16.1 to the 30RR. This domain contains all of the
DH and JH gene segments and constant region exons. We
propose a three-loop configuration for this domain. The smallest
2.8 kb loop between Em and PQ52 contains the JH gene
segments and DQ52 (Figure 7C, green). Em-PQ52 interaction is
indicated by the observed Em dependence of PQ52 transcription
and DNase I hypersensitivity. This minidomain is marked by ex-
tremely high levels of H3/H4ac and H3K4me3, high DNase I
sensitivity (Chakraborty et al., 2007; Chakraborty et al., 2009;
Mae ¨s et al., 2001), and RAG1/2 binding (Ji et al., 2010). We
suggest that the likely function of this domain is to recruit RAG
proteins to the IgH locus to initiate recombination.
A somewhat larger loop of about 57 kb is generated by 50DFL/
Em interaction. The majority of DHgene segments are seques-
tered within this minidomain (Figure 7C, smaller red loop labeled
DSPs), which is marked by heterochromatic H3K9me2, except
near DFL16.1. We propose that DHrearrangements are initiated
within this chromatin domain by RAG proteins bound to the
JH-associated recombination center. The most readily available
DHgene segments in the proposed chromatin configuration are
DFL16.1 and DQ52, which are localized at the base of loops
tethered to Em. Thus, these gene segments recombine preferen-
tially, thereby providing a mechanistic basis for the overrepre-
sentation of DFL16.1 and DQ52 in V(D)J recombined alleles of
B lymphocytes (Subrahmanyam and Sen, 2010). Activation of
DSP gene segments for recombination might involve transient
association of Em with a DSP-associated promoter (Chakraborty
etal., 2007) and consequent recruitment of that genesegment to
The largest (206 kb) loop in this region is created by Em/30RR
interactions (Figure 7C, labeled Cg3-Ca). Its epigenetic features
are similar to the intermediate loop in that active histone modifi-
cations only occur at the base of the loop at Em and 30RR. The
function of this domain, particularly for IgH gene assembly by
recombination, is not clear. It is not our intention to imply that
a stable three-loop structure is present in all pro-B cells. Rather,
we envisage a dynamic structure in which loops between these
interaction sites form and break continuously.
Em-Independent Looping within the VHRegion
Using anti-CTCF ChIP-loop assays, we present evidence for
multiple cis-interactions in the 50region of the IgH locus that
contains VHgene segments. Importantly, these loops are Em
independent. Three interesting conclusions follow from these
observations. First, both sets of interactions identified by anti-
CTCF ChIP-loop extend over a few hundred kb. For example,
the interacting sites in the VH3 region are spread over ?250 kb,
and in the VH10 region, they are spread over ?500 kb. The
striking difference between the two regions is the high density
of ‘‘peaks’’ near VH3 and the relatively few ‘‘peaks’’ near VH10.
One possibility is that the proximal VHregion (near VH3) may
be folded into multiple (>6) 30–40 kb loops, whereas the distal
VHregion (near VH10) may be folded into three 100–150 kb loops
(Figure 7A). Alternatively, even the VH3 region may be folded into
two to three 100–120 kb loops, with the exact configuration of
loops being different from cell to cell (Figure 7B).
Second, CTCF-bound sites near VH10 do not interact with
CTCF-bound sites near VH3, or vice versa. We suggest that
this may be because VH3 and VH10 fall in different chromatin do-
mains that do not interact significantly with each other. Though
interdomain interaction is not evident by the assays that we
have used, it is important to note that both domains are brought
close to the DH/JHpart of the locus by interacting with Em. We
note that the VH10-associated domain lies completely within
the 50part of the IgH locus that contains the newly identified
PAIR elements (Ebert et al., 2011). However, neither VH10 itself
sites within PAIR elements.
Third, the presence of VH3-associated loops to DFL16.1
are brought into the vicinity of DFL16.1 in the absence of Em-
dependent large-scale locus contraction. We surmise that it is
these Em-independent loops that allow proximal VH recom-
bination to continue in the absence of locus contraction, for
340 Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc.
example, in Pax5- or YY1-deficient pro-B cells (Hesslein et al.,
2003; Liu et al., 2007). Furthermore, close examination of the
residual VHrecombination on Em-deleted alleles reveals prefer-
ential utilization of proximal VHgene segments (Perlot et al.,
2005). These rearrangements are readily explained by Em-
independent VH3 to DFL16.1 loops identified in this study.
Em-Mediated IgH Locus Contraction
The location and sizes of three domains described in the
preceding sections do not account for IgH locus contraction as
defined by FISH studies. We provide evidence for a second level
of compaction that occurs via Em interaction with specific parts
of the VHregion. We propose that these latter interactions bring
together the two ends of the IgH locus and account for the
phenomenon of locus contraction (Figure 7D). The role of Em in
able mechanism for the absence of VHrecombination on Em-
deleted alleles (Afshar et al., 2006; Klein et al., 1984; Perlot
et al., 2005; Sakai et al., 1999). Notably, Em-independent clus-
tering of VHgene segments in domains such as the one near
VH10 ensures that each Em-dependent interaction with the VH
region brings multiple VHgene segments close to the DH-Cm
part of the locus.
The Em-interacting regions, 507183 and 30558, are located
?400 kb and 1.0 Mb away from Em. Because both 507183 and
30558 bind YY1, we propose that locus contraction results from
interactions between Em-bound YY1 and YY1 bound to these
distal sites. Em-bound YY1 may also make heterotypic interac-
tions with CTCF bound close to 507183. Additionally, interaction
of Em with 507183 or 30558 may be increased by Em-independent
compaction of the VHdomain by CTCF or by inter-PAIR interac-
tions in the distal VHpart of the IgH locus. The interactions that
we identified also provide an explanation for the observed prox-
imity of distal VHgene segments to the very 30end of the IgH
locus. We suggest that Em interaction with 30558 and the 30RR
draws together the 50and 30ends of the IgH locus (Figure 7D).
Finally, our studies provide insight into the multistep process
of locus activation, particularly the distinction between Em-
dependent and Em-independent steps. For example, CTCF and
YY1 binding to the IgH locus is Em independent. Because Em-
deficient alleles are located at the nuclear periphery, we infer
that lymphoid-restricted binding of these factors can occur at
the nuclear periphery. The resulting structure, comprising of
VHregion loops, may be the basis for the conclusion from trilat-
mation in E2A-deficient pre-pro-B cells (Jhunjhunwala et al.,
Figure 7. Two Levels of Chromatin Compaction at
(A–D) Top line shows a schematic of the unrearranged IgH
locus. For description, see Figure 1 legend. Regulatory
sequences PQ52 and Em are indicated as ovals. Em-
dependent and Em-independent chromatin loops identi-
fiedin thisstudy are shown as redand blue colored curved
arrows, respectively. The first level of chromatin com-
paction involves the formation of multilooped domains
(middle). Three such domains were identified. Those in the
50VHpart of the locus near VH10 and VH3 (A and B,
respectively), are Em independent. The number of loops in
each domain is inferred from the number of interaction
sites, as discussed in the text. CTCF binding is indicated
by yellow ovals; possible role for factors other than CTCF
at VH10 is indicated by blue ovals. At the 30end an Em-
dependent domain (C) extends from sequences 50of
DFL16.1 (50DFL) to the 30RR. A proposed three-loop
configuration for this domain is discussed in the text. The
smallest loop that contains the four JHgene segments and
DQ52 is indicated in green because it has the highest
levels of activating histone modifications and binds RAG1
and RAG2 to form a recombination center (Ji et al., 2010).
The two other loops that contain DSP gene segments and
constant region exons (Cg3-Ca)(shown in red) are marked
with H3K9me2. Red ovals at the base of these loops
represent the possible role of YY1 protein in establishing
this domain. The second level of chromatin compaction
involves the interaction of Em with specific sites in the VH
region (D). We identified two such sites, 507183 and 30558
(top line); both of these interaction sites are outside of
VH10- and VH3-associated domains, suggesting that
the multilooped structure within each domain does not
change with these interactions. Rather, Em-30558 and Em-
507183 interactions bring these domains into the vicinity of
the DFL-30RR domain and, thereby, in physical proximity
to the RAG-rich recombination center. Linker regions
between identified domains are shown in gray.
Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc. 341
and 30558 would reconfigure the locus to deviate away from the
MLS model in pro-B cells. Finally, Em-dependent generation of
the 50DFL-30RR domain and associated RAG-rich recombination
center leads to the fully active state of the locus that is ready to
Mice and Cell Lines
JHT (Gu et al., 1993), P?E+and P?E?mice have been previously described
(Afshar et al., 2006). RAG2-deficient mice on 129 or C57BL6 background
were purchased from Taconic or maintained at the NIA animal facility. Abelson
virus transformed cell lines Em?contains a 220 bp deletion of Em and lacks
recombination-activating gene (RAG) 2 (Chakraborty et al., 2009). D345
pro-B cell line contains an inactive RAG1 allele in a C57BL6 background
(Ji et al., 2010) and was kindly provided by Dr. David Schatz (Yale University).
Pro-B cells were purified from the bone marrow by positive selection using
anti-CD19-coupled magnetic beads (Stem Cell Technologies, BC, Canada)
according to the manufacturer’s protocol. Thymocytes were prepared by
making single-cell suspensions of the thymus and filtration through nylon
mesh. All procedures were carried out at 4?C. Cell purity and viability were
assessed by flow cytometry.
ChIPs, real-time PCR, and data analysis were performed as described (Chak-
raborty et al., 2009). The following antibodies were used for ChIP: anti-Rad21
(Abcam ab992), anti-CTCF (Millipore 17-10044), and anti-YY1 (Santa Cruz
H414). Previously described primers for ChIP assays were from (Chakraborty
et al., 2007) and (Liu et al., 2007). New primers used for ChIP assays are noted
in Table S2.
Chromosome Conformation Capture Assay and Circular
Chromosome Conformation Capture Assay
Chromosome conformation capture(3C) assays were performed as described
(Wuerffel et al., 2007)using Hind III to digest crosslinked chromatin.3C ligation
products were measured by the Taqman quantitative PCR technology
(Hage `ge et al., 2007). We normalized 3C results between experiments using
the IgH-unrelated b-globin locus long-range interaction of 30HS1 (Splinter
et al., 2006). PCR control fragments for determination of primer efficiency of
each primer combination (Table S2) were generated using genomic DNA
from the regions of interest as described (Nativio et al., 2009) or BAC clones
covering the genomic segments under study.
For circular chromosome conformation capture (4C) assays, crosslinked
chromatin was digested with Mse I or Nla III followed by religation for
3 days. After reversal of crosslinking by incubation at 65?C overnight, genomic
DNA was purified and nested PCR carried with different anchor primer pairs
(Table S2) as described (Go ¨ndo ¨r et al., 2008). PCR products were fragmented,
labeled with biotin, and hybridized to the Affymetrix mouse GenChip Mouse
tiling 2.0 R E array according to the manufacturer’s specifications. Data
normalization and enrichedregion detection wasperformed using CisGenome
(Ji et al., 2008) with default parameters. Significantly enriched regions were
determined with one-tail t test statistics. Moving averages of normalized
log2 ratio between sample and input were calculated using the msProcess
Package of bioconductor (http://www.bioconductor.org) and plotted along
chromosomal coordinates (mm8) for visualization.
Partially sonicated formaldehyde-crosslinked chromatin was incubated over-
night at 4?C with anti-CTCF antibody complexes to Dynabeads (Invitrogen,
CA) at 4?C. DNA-Dynabead complexes were washed extensively with restric-
tion enzyme buffer followed by incubationwith Mse I or Nla III.Further work-up
was carried out as described for 4C.
Fluorescent In Situ Hybridization and Immunolocalization
Locus specific DNA probes for fluorescent in situ hybridization (FISH) were
prepared from BACs RP23-230L2 and RP23-458I14 (Invitrogen, CA). BAC
probes were labeled by nick translation using ChromaTide Alexa Fluor 568-4
dUTP (red) and ChromaTide Alexa Fluor 488-5 dUTP (green) (Invitrogen, CA)
(Sayegh et al., 2005).
with primers listed in Table S2. Probes h4 and h11, as well as BAC RP23-
201H14, were kindly provided by Dr. Cornelis Murre (UCSD). FISH with 10 kb
probes were performed as described (Jhunjhunwala et al., 2008) using a Nikon
T2000 microscope equipped with a 1003 lens and motorized 100 mm Piezo
Z-stage (Applied Scientific Instrumentation, OR). Depending on the size of
the nucleus 30–40 serial optical sections spaced by 0.2 mm were acquired.
The data sets were deconvolved using NIS-Elements software (Nikon, NY)
and optical sections merged to produce 3D images. The spatial distance
between probes was measured as described (Jhunjhunwala et al., 2008).
Supplemental Information includes two tables and five figures and can be
found with this article online at doi:10.1016/j.cell.2011.08.049.
We are indebted to Cornelis Murre and Suchit Jhunjhunwala for sharing and
troubleshooting the procedures for small probe FISH. We thank Drs. Dinah
Singer, Amy Kenter, Cornelis Murre, Fred Alt, and Sebastian Fugmann for
discussions throughout the work and critical appraisal of the manuscript.
This work was supported by the Intramural Research Program of the National
Institute on Aging (Baltimore, MD) and by NIH grant (P01 HL68744 and
CA100905) to E.M.O.
Received: November 11, 2010
Revised: April 26, 2011
Accepted: August 26, 2011
Published online: October 6, 2011
Afshar, R., Pierce, S., Bolland, D.J., Corcoran, A., and Oltz, E.M. (2006). Regu-
lation of IgH gene assembly:role of the intronic enhancer and 50DQ52 region in
targeting DHJH recombination. J. Immunol. 176, 2439–2447.
Chakraborty, T., Chowdhury, D., Keyes, A., Jani, A., Subrahmanyam, R.,
Ivanova, I., and Sen, R. (2007). Repeat organization and epigenetic regulation
of the DH-Cmu domain of the immunoglobulin heavy-chain gene locus. Mol.
Cell 27, 842–850.
Chakraborty, T., Perlot, T., Subrahmanyam, R., Jani, A., Goff, P.H., Zhang, Y.,
Ivanova, I., Alt, F.W., and Sen, R. (2009). A 220-nucleotide deletion of the
intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy
chain locus activation. J. Exp. Med. 206, 1019–1027.
Cook, P.R., Brazell, I.A., and Jost, E. (1976). Characterization of nuclear struc-
tures containing superhelical DNA. J. Cell Sci. 22, 303–324.
Degner, S.C., Wong, T.P., Jankevicius, G., and Feeney, A.J. (2009). Cutting
edge: developmental stage-specific recruitment of cohesin to CTCF
sites throughout immunoglobulin loci during B lymphocyte development.
J. Immunol. 182, 44–48.
Ebert, A., McManus, S., Tagoh, H., Medvedovic, J., Salvagiotto, G., Novatch-
V(H) gene cluster of the Igh locus contains distinct regulatory elements with
Pax5 transcription factor-dependent activity in pro-B cells. Immunity 34,
Featherstone, K., Wood, A.L., Bowen, A.J., and Corcoran, A.E. (2010). The
mouse immunoglobulin heavy chain V-D intergenic sequence contains insula-
tors that may regulate ordered V(D)J recombination. J. Biol. Chem. 285, 9327–
342 Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc.
Fuxa, M., Skok, J., Souabni, A., Salvagiotto, G., Roldan, E., and Busslinger, M. Download full-text
(2004). Pax5 induces V-to-DJ rearrangements and locus contraction of the
immunoglobulin heavy-chain gene. Genes Dev. 18, 411–422.
Garrett, F.E., Emelyanov, A.V., Sepulveda, M.A., Flanagan, P., Volpi, S., Li, F.,
Loukinov, D., Eckhardt, L.A., Lobanenkov, V.V., and Birshtein, B.K. (2005).
Chromatin architecture near a potential 30end of the igh locus involves
modular regulation of histone modifications during B-Cell development and
in vivo occupancy at CTCF sites. Mol. Cell. Biol. 25, 1511–1525.
Gasser, S.M., and Laemmli, U.K. (1987). A glimpse at chromosomal order.
Trends Genet. 3, 16–22.
Gerasimova, T.I., Lei, E.P., Bushey, A.M., and Corces, V.G. (2007). Coordi-
nated control of dCTCF and gypsy chromatin insulators in Drosophila. Mol.
Cell 28, 761–772.
Go ¨ndo ¨r, A., Rougier, C., and Ohlsson, R. (2008). High-resolution circular chro-
mosome conformation capture assay. Nat. Protoc. 3, 303–313.
Gu, H., Zou, Y.R., and Rajewsky, K. (1993). Independent control of immuno-
globulin switch recombination at individual switch regions evidenced through
Cre-loxP-mediated gene targeting. Cell 73, 1155–1164.
Hage `ge, H., Klous, P., Braem, C., Splinter, E., Dekker, J., Cathala, G., de Laat,
W., and Forne ´, T. (2007). Quantitative analysis of chromosome conformation
capture assays (3C-qPCR). Nat. Protoc. 2, 1722–1733.
Hesslein, D.G., Pflugh, D.L., Chowdhury, D., Bothwell, A.L., Sen, R., and
Schatz, D.G. (2003). Pax5 is required for recombination of transcribed, acety-
lated, 50IgH V gene segments. Genes Dev. 17, 37–42.
Jhunjhunwala, S., van Zelm, M.C., Peak, M.M., Cutchin, S., Riblet, R., van
Dongen, J.J., Grosveld, F.G., Knoch, T.A., and Murre, C. (2008). The 3D struc-
ture of the immunoglobulin heavy-chain locus: implications for long-range
genomic interactions. Cell 133, 265–279.
Ji, H., Jiang, H., Ma, W., Johnson, D.S., Myers, R.M., and Wong, W.H. (2008).
An integrated software system for analyzing ChIP-chip and ChIP-seq data.
Nat. Biotechnol. 26, 1293–1300.
Ji, Y., Resch, W., Corbett, E., Yamane, A., Casellas, R., and Schatz, D.G.
(2010). The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor
loci. Cell 141, 419–431.
Johnston, C.M., Wood, A.L., Bolland, D.J., and Corcoran, A.E. (2006).
Complete sequence assembly and characterization of the C57BL/6 mouse
Ig heavy chain V region. J. Immunol. 176, 4221–4234.
Ju, Z., Volpi, S.A., Hassan, R., Martinez, N., Giannini, S.L., Gold, T., and Birsh-
tein, B.K. (2007). Evidence for physical interaction between the immunoglob-
ulin heavy chain variable region and the 30regulatory region. J. Biol. Chem.
Kadauke, S., and Blobel, G.A. (2009). Chromatin loops in gene regulation. Bio-
chim. Biophys. Acta 1789, 17–25.
Klein, S., Sablitzky, F., and Radbruch, A. (1984). Deletion of the IgH enhancer
does not reduce immunoglobulin heavy chain production of a hybridoma IgD
class switch variant. EMBO J. 3, 2473–2476.
Knoch, T.A., Munkel, C., and Langowski, J., eds. (2000). Three-dimensional of
chromosome territories in human interphase nucleus (Heidelberg, Germany:
Kosak, S.T., Skok, J.A., Medina, K.L., Riblet, R., Le Beau, M.M., Fisher, A.G.,
during lymphocyte development. Science 296, 158–162.
Krangel, M.S. (2009). Mechanics of T cell receptor gene rearrangement. Curr.
Opin. Immunol. 21, 133–139.
Liu, H., Schmidt-Supprian, M., Shi, Y., Hobeika, E., Barteneva, N., Jumaa, H.,
Pelanda, R., Reth, M., Skok, J., Rajewsky, K., and Shi, Y. (2007). Yin Yang 1 is
a critical regulator of B-cell development. Genes Dev. 21, 1179–1189.
Mae ¨s, J., O’Neill, L.P.,Cavelier, P.,Turner,B.M., Rougeon, F.,and Goodhardt,
M. (2001). Chromatin remodeling at the Ig loci prior to V(D)J recombination. J.
Immunol. 167, 866–874.
Marsden, M.P., and Laemmli, U.K. (1979). Metaphase chromosome structure:
evidence for a radial loop model. Cell 17, 849–858.
Nativio, R., Wendt, K.S., Ito, Y., Huddleston, J.E., Uribe-Lewis, S., Woodfine,
K., Krueger, C., Reik, W., Peters, J.M., and Murrell, A. (2009). Cohesin is
required for higher-order chromatin conformation at the imprinted IGF2-H19
locus. PLoS Genet. 5, e1000739.
Park, K., and Atchison, M.L. (1991). Isolation of a candidate repressor/
activator, NF-E1 (YY-1, delta), that binds to the immunoglobulin kappa 30
enhancer and the immunoglobulin heavy-chain mu E1 site. Proc. Natl. Acad.
Sci. USA 88, 9804–9808.
Paulson, J.R., and Laemmli, U.K. (1977). The structure of histone-depleted
metaphase chromosomes. Cell 12, 817–828.
Perlot, T., and Alt, F.W. (2008). Cis-regulatory elements and epigenetic
changes control genomic rearrangements of the IgH locus. Adv. Immunol.
Perlot, T., Alt, F.W., Bassing, C.H., Suh, H., and Pinaud, E. (2005). Elucidation
of IgH intronic enhancer functions via germ-line deletion. Proc. Natl. Acad. Sci.
USA 102, 14362–14367.
Phillips, J.E., and Corces, V.G. (2009). CTCF: master weaver of the genome.
Cell 137, 1194–1211.
Retter, I., Chevillard, C., Scharfe, M., Conrad, A., Hafner, M., Im, T.H., Lude-
wig, M., Nordsiek, G., Severitt, S., Thies, S., et al. (2007). Sequence and char-
acterization of the Ig heavy chain constant and partial variable region of the
mouse strain 129S1. J. Immunol. 179, 2419–2427.
Sachs, R.K., van den Engh, G., Trask, B., Yokota, H., and Hearst, J.E. (1995).
A random-walk/giant-loop model for interphase chromosomes. Proc. Natl.
Acad. Sci. USA 92, 2710–2714.
Sakai, E., Bottaro, A., Davidson, L., Sleckman, B.P., and Alt, F.W. (1999).
Recombination and transcription of the endogenous Ig heavy chain locus is
effected by the Ig heavy chain intronic enhancer core region in the absence
of the matrix attachment regions. Proc. Natl. Acad. Sci. USA 96, 1526–1531.
Sayegh, C.E., Jhunjhunwala, S., Riblet, R., and Murre, C. (2005). Visualization
of looping involving the immunoglobulin heavy-chain locus in developing B
cells. Genes Dev. 19, 322–327.
Simpson, E.M., Linder, C.C., Sargent, E.E., Davisson, M.T., Mobraaten, L.E.,
and Sharp, J.J. (1997). Genetic variation among 129 substrains and its impor-
tance for targeted mutagenesis in mice. Nat. Genet. 16, 19–27.
Splinter, E., Heath, H., Kooren, J., Palstra, R.J., Klous, P., Grosveld, F., Galjart,
N., and de Laat, W. (2006). CTCF mediates long-range chromatin looping and
local histone modificationinthebeta-globinlocus.Genes Dev.20,2349–2354.
Subrahmanyam,R.,andSen, R.(2010).RAGs’ eyeview oftheimmunoglobulin
heavy chain gene locus. Semin. Immunol. 22, 337–345.
Takizawa, T., Meaburn, K.J., and Misteli, T. (2008). The meaning of gene posi-
tioning. Cell 135, 9–13.
Wallace, J.A., and Felsenfeld, G. (2007). We gather together: insulators and
genome organization. Curr. Opin. Genet. Dev. 17, 400–407.
Wuerffel, R., Wang, L., Grigera, F., Manis, J., Selsing, E., Perlot, T., Alt, F.W.,
Cogne, M., Pinaud, E., and Kenter, A.L. (2007). S-S synapsis during class
switch recombination is promoted by distantly located transcriptional
elements and activation-induced deaminase. Immunity 27, 711–722.
Cell 147, 332–343, October 14, 2011 ª2011 Elsevier Inc. 343