Download Recombination Mediators across Cell Cycle Stage by Regulating

IL-7 Functionally Segregates the Pro-B Cell
Stage by Regulating Transcription of
Recombination Mediators across Cell Cycle
This information is current as
of June 15, 2017.
Kristen Johnson, Julie Chaumeil, Mariann Micsinai, Joy M.
H. Wang, Laura B. Ramsey, Gisele V. Baracho, Robert C.
Rickert, Francesco Strino, Yuval Kluger, Michael A. Farrar
and Jane A. Skok
Supplementary
Material
http://www.jimmunol.org/content/suppl/2012/05/11/jimmunol.120036
8.DC1
Subscription
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Permissions
Email Alerts
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
J Immunol published online 11 May 2012
http://www.jimmunol.org/content/early/2012/05/11/jimmun
ol.1200368
Published May 11, 2012, doi:10.4049/jimmunol.1200368
The Journal of Immunology
IL-7 Functionally Segregates the Pro-B Cell Stage by
Regulating Transcription of Recombination Mediators across
Cell Cycle
Kristen Johnson,* Julie Chaumeil,* Mariann Micsinai,†,‡,x Joy M. H. Wang,*
Laura B. Ramsey,{ Gisele V. Baracho,‖ Robert C. Rickert,‖ Francesco Strino,x
Yuval Kluger,x Michael A. Farrar,{ and Jane A. Skok*
D
evelopmental progression involves a delicate balance
between differentiation, survival, and proliferation. The
juxtaposition of the differentiation and proliferation
programs is particularly important during lymphocyte development
because early developmental stages are punctuated by V(D)J recombination events, which enable diversification of Ag receptors
*Department of Pathology, New York University School of Medicine, New York, NY
10016; †New York University Center for Health Informatics and Bioinformatics, New
York, NY 10016; ‡New York University Cancer Institute, New York, NY 10016; xDepartment of Pathology, Yale Cancer Center, Yale University School of Medicine,
New Haven, CT 06520; {Department of Laboratory Medicine and Pathology, Center
for Immunology, University of Minnesota, Minneapolis, MN 55455; and ‖Program of
Inflammatory Disease Research, Sanford-Burnham Medical Research Institute, La
Jolla, CA 92037
Received for publication January 30, 2012. Accepted for publication April 13, 2012.
This work was supported by National Institutes of Health Grants K99GM088408-01
and R01GM086852 (to K.J.), a Wellcome Trust Project grant (to J.A.S.), and National Institutes of Health Grant R01GM086852 (to J.A.S.). J.A.S. is a Leukemia and
Lymphoma Society Scholar. This work was also supported by National Institutes of
Health Grant R01GM086852 (to J.C.), an Irvington Institute Fellowship program of
the Cancer Research Institute (to J.C.), and National Institutes of Health Grant
R01AI041649 (to R.C.R.). M.A.F. is a Leukemia and Lymphoma Society Scholar.
This work was also supported by National Science Foundation Grant Integrative
Graduate Education and Research Traineeship 0333389 (to M.M.) and an
American-Italian Cancer Foundation Postdoctoral Research fellowship (to F.S.). This
work was also supported by National Institutes of Health Research Grant CA-16359
from the National Cancer Institute (to Y.K.).
Address correspondence and reprint requests to Dr. Jane A. Skok, Department of
Pathology, New York University School of Medicine, 550 First Avenue, MSB 599,
New York, NY 10016. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BAC, bacterial artificial chromosome; ChIP-seq,
chromatin immunoprecipitation combined with deep sequencing; dUTP, deoxyuridine triphosphate; FISH, fluorescence in situ hybridization; Igh, Ig H chain; PCH,
pericentromeric heterochromatin; PRCC, putative regulator of RAG expression
through cell cycle.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200368
in B and T lineage cells (1). Because recombination involves the
repeated cutting and joining of widely separated gene segments,
the process must be tightly regulated to ensure that mistakes do
not occur. Indeed, deregulation of recombination can have serious
consequences resulting in aberrant chromosomal rearrangements
that give rise to leukemia and lymphoma.
RAG1 and RAG2 play an essential part in recombination, guiding
the process from the cleavage phase through to synapsis and repair.
First, RAG proteins recognize and cleave conserved recombination
signal sequence elements that flank individual Ag receptor gene
segments (2). Next, RAG stabilizes the postcleavage complex to
ensure that the four cleaved ends are held in place, which is critical
for their proper repair by classical nonhomologous end joining (3).
This repair pathway predominates during the G1 early S phase of
cell cycle (4) and is important for maintaining genome stability (5–
7). RAG cleavage is also directed to occur during the G1-G0 phase
of the cell cycle, which couples cleavage and repair to ensure that
replication does not occur over unrepaired breaks (8).
It has been known for some time that RAG2 is regulated across
cell cycle by a mechanism that involves phosphorylation-dependent
protein degradation during the G1-S transition (9–11). The importance of this restriction was recently shown in mice harboring
a targeted threonine-to-alanine mutation at position 490 on RAG2,
which prevents degradation at the appropriate stage of a cell cycle.
When RAG2T490A/T490A is expressed on a p53-deficient background, it gives rise to lymphoid tumors that contain translocations
involving Ag receptor genes (12). Somewhat surprisingly though,
death is not accelerated in these animals compared with p53deficient mice. Furthermore, the lymphoid tumors are phenotypically more mature, with a broader spectrum of translocations,
compared with lymphomas isolated from Core RAG2, p53 doubledeficient mice (13). This implies additional mechanisms of protection at the early stages of lymphocyte development.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Ag receptor diversity involves the introduction of DNA double-stranded breaks during lymphocyte development. To ensure fidelity,
cleavage is confined to the G0-G1 phase of the cell cycle. One established mechanism of regulation is through periodic degradation
of the RAG2 recombinase protein. However, there are additional levels of protection. In this paper, we show that cyclical changes
in the IL-7R signaling pathway functionally segregate pro-B cells according to cell cycle status. In consequence, the level of
a downstream effector of IL-7 signaling, phospho-STAT5, is inversely correlated with cell cycle expression of Rag, a key gene
involved in recombination. Higher levels of phopho-STAT5 in S-G2 correlate with decreased Rag expression and Rag relocalization
to pericentromeric heterochromatin. These cyclical changes in transcription and locus repositioning are ablated upon transformation with v-Abl, which renders STAT5 constitutively active across the cell cycle. We propose that this activity of the IL-7R/
STAT5 pathway plays a critical protective role in development, complementing regulation of RAG2 at the protein level, to ensure
that recombination does not occur during replication. Our data, suggesting that pro-B cells are not a single homogeneous
population, explain inconsistencies in the role of IL-7 signaling in regulating Igh recombination. The Journal of Immunology,
2012, 188: 000–000.
2
Materials and Methods
both Rag1 and Rag2 genes) was detected using the bacterial artificial
chromosome (BAC) RP23-313G3. The Igh locus was detected using the
two BACs CT7-34H6 and CT7-526A21, mapping the 39 constant and the
59 variable regions, respectively (22). BAC probes were directly labeled
by nick translation with deoxyuridine triphosphate (dUTP)-A594 or
dUTP-A488 (Invitrogen). The gamma satellite probe was prepared from
a plasmid containing eight copies of the gamma satellite repeat sequence
(23) and was directly labeled with dUTP-Cy5 or dUTP-A488 (GE
Healthcare).
Immuno-DNA FISH
Combined detection of H3S10ph and Rag or Igh loci was carried out on
cells adhered to poly-L-lysine–coated coverslips as described previously
(18). Briefly, cells were fixed with 2% paraformaldehyde/PBS for 10
min and permeabilized for 5 min with 0.4% Triton X-100/PBS on ice.
After 30 min blocking in 2.5% BSA, 10% normal goat serum, and 0.1%
Tween 20/PBS, H3S10ph staining was carried out using an Ab against
phosphorylated Ser10 of histone H3 (Millipore) diluted at 1:400 in
blocking solution for 1 h at room temperature. Cells were rinsed three
times in 0.2% BSA and 0.1% Tween 20/PBS and incubated for 1 h with
goat-anti-rabbit IgG Alexa 488 or 594 or 633 (Invitrogen). After three
rinses in 0.1% Tween 20/PBS, cells were postfixed in 3% paraformaldehyde/PBS for 10 min, permeabilized in 0.7% Triton X-100 in
0.1 M HCl for 15 min on ice, and incubated in 0.1 mg/ml RNase A for 30
min at 37˚C. Cells were then denatured with 1.9 M HCl for 30 min at
room temperature and rinsed with cold PBS. DNA probes were denatured for 5 min at 95˚C, preannealed for 45 min at 37˚C, and applied to
coverslips, which were sealed onto slides with rubber cement and incubated overnight at 37˚C. Cells were then rinsed three times 30 min
with 23 SSC at 37˚C, 23 SSC, and 13 SSC at room temperature. Cells
were mounted in ProLong Gold (Invitrogen) containing DAPI to counterstain total DNA.
Mice
Confocal microscopy and analysis
CaStat5 and Erag mice have been described previously (19, 20). Wildtype mice were littermate controls and/or when wild-type were used
alone were C57BL/6 mice (The Jackson Laboratory). Mice were housed
in specific pathogen-free conditions and were maintained and used in
accordance with the Institutional Animal Care and Use Committee
guidelines.
Cells were analyzed by confocal microscopy on a Leica Sp5 AOBS
(Acoustica Optical Beam Splitter) system. Optical sections separated by 0.3
mm were collected, and only cells with signals from both alleles were
analyzed using ImageJ software. Alleles were defined as associated with
PCH if BAC probe signals were overlapping or juxtaposed to the gamma
satellite signal. Individual alleles from the same cell are shown in different
confocal sections. Sample sizes were 100 cells minimum per experiment
(see supplementary tables for exact numbers), and experiments were repeated at least two to three times.
Cells and culture conditions
Short-term bone marrow cells were established by harvesting total bone
marrow and placed at a concentration of 1 3 106–2 3 106 cells/ml in T-25
flasks in 8 ml in Optimem media supplemented with 5% FCS and 5 ng/ml
IL-7. One-half of the media was replaced every 3–4 d for a total of 6–10 d,
with fresh media being added 1 d prior to analysis. This culture system
makes use of endogenous stromal cells present within the bone marrow
and produces a pro-B cell population that is .90% pure as measured by
CD19+/CD252 surface expression. For IL-7 dilution experiments, cultures
were established as above for 6–7 d, counted, washed, and replated for 36–
40 h at 2 3 106 cells/ml in 6-well dishes without stroma with 10, 5, 2.5, or
1 ng/ml or no IL-7 and then directly analyzed. When indicated, cells were
sorted prior to analysis on CD19+/CD252/IgM2 to ensure equivalent purity of pro-B cell populations were assessed.
The v-Abl lines were cultured without stromal cells or cytokine, in RPMI
1640 medium supplemented with 10%FBS.
The gating strategy of sorted cells is as follows: pro-B cells are CD19+/
c-kit+/CD252/IgM2, pre-B cells are CD19+/c-kit2/CD25+/IgM2 and DP
cells are Thy1.2+/CD192/CD4+/CD8+. Experiments were done directly
after sorting. Fetal liver pro-B cells were analyzed directly after sorting
and were directly compared with bone marrow pro-B that were similarly
sorted and directly analyzed.
RT-PCR
Total RNA was isolated with TRIzol (Invitrogen), and cDNA was made
using SuperScript II reverse transcriptase (Invitrogen). Quantitative PCR
was performed in triplicate with a SYBR green kit (Stratagene) using genespecific primers described previously (16). All samples are normalized
against b2-microglobulin.
Three-dimensional DNA fluorescence in situ hybridization
Cells were washed three times in PBS and then fixed onto poly-L-lysine–
coated slides for three-dimensional DNA–fluorescence in situ hybridization (FISH) analysis as described in detail (21). The Rag locus (containing
Flow cytometry
Surface staining, intracellular staining, and cell cycle staining using Hoechst
have all been described previously (16, 24). Abs specific for murine CD19
(1D3), IL-7R a-chain (A7R34), c-kit (2B8), CD25 (PC61), IgM (II/41),
and pY695 STAT5 were purchased from BD Pharmingen. Data were
collected with the LSR II and were analyzed with FCS Express (De Novo
Software).
ChIP-seq analysis
We obtained raw data sets of chromatin immunoprecipitation combined
with deep sequencing (ChIP-seq) of H3K27me3, STAT5, and total input
from recent experiments performed by the Clark laboratory (25). We
aligned ChIP-seq reads with Bowtie 0.12.7 software to the mm9 mouse
genome data, using the following command line option–best–all -m1 -n2
(26). To identify regions of increased sequence tag density obtained after
enrichment by ChIP with specific Abs relative to the measured background
along the genome (input chromatin), we used the Qeseq algorithm (27).
We analyzed the H3K27me3 using –s 150 setting, reflecting the experimental fragment size. For STAT5, the setting was –s 250. We visualized
the obtained peaks and the enrichment scores of the reads located within
the peaks using Integrated Genome Browser (http://bioviz.org/igb/).
Statistical analysis
The two-tailed Fisher’s exact test was used to analyze the significance of
association with PCH and association of gamma satellite. SD was used to
create errors bars for transcription analysis. In some cases, single representative experiments are shown to compare multiple experimental parameters within a single experiment. Replicate experimental data are
provided within supplementary tables to show reproducibility.
The statistical tests described above were applied to combined data from
repeated experiments. Data for individual experiments is displayed in
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Environmental signals govern lineage and stage-specific gene
programs, including the processes of proliferation and recombination. In this context, the IL-7 signaling pathway serves a pivotal
role. Not only is it required for survival and proliferation of early B
and T cell progenitors (14) but it is also involved in the negative
regulation of Rag expression (15, 16). Furthermore, phosphoSTAT5, a downstream signaling component of the IL-7 signaling
pathway, has been shown to enhance accessibility of the Ig H
chain (Igh) locus for rearrangement (17, 18), a stage at which IL-7
is purported to inhibit Rag expression (15). Thus, it remains unclear how IL-7 could coordinate these disparate activities at the
pro-B cell stage during Igh recombination.
In this study, we show that pro-B cells are in fact a heterogeneous
population that can be subdivided on the basis of IL-7R expression
and levels of phospho-STAT5. Expression of IL-7R/phosphoSTAT5 is found predominantly in the actively dividing population. As a consequence, Rag1 has a different transcriptional
profile within B cell subsets. Repositioning of the Rag locus to
repressive pericentromeric heterochromatin (PCH) occurs preferentially within cells at the G2 phase and correlates with increased
phospho-STAT5 levels. Our data reconcile the role of IL-7 in
positively regulating Igh accessibility and negatively regulating
Rag expression in pro-B cells. Importantly, we reveal an additional mechanism to enforce segregated recombination and proliferation in a developmental context.
IL-7R/STAT5 SUBDIVIDES THE PRO-B CELL COMPARTMENT
The Journal of Immunology
supplementary tables to show the low level of variation between
the repeats.
Results
Differential Rag1 transcription occurs within pro-B cells at
different phases of the cell cycle as a result of fluctuations in
IL-7 responsiveness
FIGURE 1. Differential Rag1 transcription occurs within pro-B cells at
different phases of the cell cycle as a result of fluctuations in IL-7 responsiveness. (A) Wild-type bone marrow pro-B
cells were analyzed for IL-7R expression
and cell cycle stage using flow cytometry.
CD19+/IgM2/c-kit+ pro-B cells were divided into G0-G1 and S-G2-M by DAPI
staining and back gated for either IL-7R
(blue) or an isotype (IG) control (red).
(B) Wild-type bone marrow pro-B cells
cultured short term with 5 ng/ml IL-7
in vitro were analyzed for phosphoSTAT5 levels and cell cycle stage using
flow cytometry. Cells were divided into
G0-G1 and S-G2-M by DAPI staining and
back gated for either phospho-STAT5
(blue) or an isotype (IG) control (red).
(C) Phospho-STAT5 levels were analyzed
across cell cycle stages within v-Abl–
transformed pro-B cells as described in
(B). (D, E) Graphs showing the level of
Rag1 and Im transcripts assessed by QPCR (lower panels) in sorted G0-G1 and
S-G2-M bone marrow pro-B cells cultured short term in the presence of IL-7
(5 ng/ml) (D) or v-Abl pro-B cells (E)
(upper panels). Transcripts were normalized against b2-microglobulin, and
the G0-G1 population set at 1. Each data
set is representative of three to four
experiments.
functional consequences. Activation of the STAT pathway begins
after IL-7R engagement, which allows activation of the STAT
protein via phosphorylation, dimerization, and subsequent translocation to the nucleus where transcription of target genes can be
directly controlled. Thus, we analyzed levels of activated STAT5
(phospho-STAT5) during G0-G1 and S-G2-M in pro-B cells. For
these experiments, we used ex vivo-derived wild-type pro-B cells,
cultured short term in the presence of 5 ng/ml IL-7. The use of
cultured cells was necessary because of technical limitations of
the assay including, 1) the inability to use multiparameter surface
and DAPI staining in conjunction with the methanol fixation step
that is required for intracellular staining with the phospho-STAT5
Ab, and 2) the transient nature of the downstream effects of IL-7
signaling: phospho-STAT5 levels fall off rapidly after removal of
cells from their IL-7–containing environment and are therefore
lost during cell sorting. In addition, the use of cultured cells
allowed us to obtain adequate cell numbers to analyze gene expression in concurrent experiments (see below). Intracellular
staining was performed on cultured ex vivo-derived pro-B cells
using an Ab to phospho-STAT5 or an isotype control. As shown in
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
To explain the seemingly contradictory roles of IL-7 in regulating
V(D)J recombination within pro-B cells we considered the possibility that pro-B cells are in fact a heterogeneous population
in terms of their responsiveness to IL-7 signaling. To test this
hypothesis we assessed surface IL-7R levels within pro-B cells
at different phases of the cell cycle (Supplemental Fig. A). Our
analyses indicate that a higher proportion of S-G2-M cells
expressed IL-7R as compared with G0-G1 pro-B cells (Fig. 1A).
These data demonstrate that pro-B cells are a heterogeneous
population and differentially express the IL-7R on their surface in
a fashion that correlates with cell cycle status.
Signaling components downstream of the IL-7R pathway must
also be differentially regulated during cell cycle for there to be
3
4
IL-7R/STAT5 SUBDIVIDES THE PRO-B CELL COMPARTMENT
The Rag locus is repositioned to PCH in G2 cells
Association of genes with PCH correlates with gene silencing (29).
Furthermore, repositioning of select loci within the nucleus, including Rag, has been shown to occur in cycling but not quiescent
cells (30). We considered the possibility that a similar mechanism
may function to silence Rag expression within a population of
actively dividing cells. To address this, we performed threedimensional immuno-FISH using an Ab to the phosphorylated
form of Ser10 on histone H3 (H3S10ph) in combination with a
BAC probe that spans both the Rag1 and Rag2 loci (RP23313G3) and a gamma satellite probe that hybridizes to PCH
(Fig. 2A). H3S10 phosphorylation begins in early G 2 at the
chromocenters, overlapping with PCH, and continually spreads
along the chromosome until prophase (31). By this method, we
were able to determine whether the Rag locus was preferentially
associated with repressive PCH during G2 when cells have committed to the process of cell division. Alleles were considered to
be associated with PCH if signals overlapped or were directly
adjacent to gamma satellite. Our analysis of wild-type pro-B cells,
cultured short term in IL-7, indicate that the Rag locus was
preferentially associated with PCH in G2 cells (Fig. 2, Supplemental Table I). This trend is not common to other loci that are
regulated by IL-7: as shown in Fig. 2B, association of Igh with
PCH did not change between cell cycle phases. Moreover, relocation of the Rag locus to PCH during G2 is not simply due to
FIGURE 2. The Rag locus is repositioned to PCH in G2 cells. (A) Threedimensional immuno-DNA FISH was performed on wild-type pro-B cells
(cultured short term in the presence of IL-7) and v-Abl–transformed pro-B
cell lines. Cells were separated into G2 or G0-G1-S on the basis of their
immunostaining pattern for H3S10ph (green). DNA FISH probes used
were gamma satellite (white) in conjunction with either Rag (red) or Igh
(data not shown). Both alleles were scored for association with repressive
PCH as determined by overlapping or juxtaposition of the locus-specific
and gamma satellite signals. Mitotic cells were excluded. A representative
example of a G0-G1-S and G2 cell is shown. The positions of the two Rag
alleles are shown in separate confocal sections. Scale bar, 1 mm. (B) Graph
showing the percentage of association of at least one Rag or Igh allele with
PCH in the total population and in G2 or G0-G1-S cells from both shortterm wild-type pro-B cell cultures or v-Abl B cell lines. Over 250 cells of
each cell type was counted. Data shown are from one representative experiment, with experiments repeated two to three times. Data from two
individual experiments is shown in Supplemental Table I to show the low
variability between experiments.
a progression through cell cycle, because repositioning did not
occur during G2 in v-Abl–transformed cells. These data demonstrate that the Rag locus is dynamically repositioned to PCH
during G2 in IL-7–responsive B cells, which correlates with reduced Rag transcription.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Fig. 1B, phospho-STAT5 was present in all S-G2-M cells and in
only a proportion of G0-G1 cells. This pattern mirrors the pattern
of IL-7R expression (Fig. 1A), indicating that both these signaling
components are dynamically regulated within pro-B cells.
We next asked whether differential levels of IL-7/phosphoSTAT5 in G0-G1 and S-G2-M impact Rag expression in proB cells. Ex vivo-derived wild-type pro-B cells were stained with
Hoechst and sorted according to cell cycle status prior to analyzing levels of Rag1 transcription. As shown in Fig. 1D, in G0-G1
cells, Rag1 transcripts were detected at a level that was 8- to 11fold higher compared with S-G2-M, whereas other transcripts such
as Im did not vary substantially in the same cells. These data
demonstrate that Rag transcription is specifically decreased in SG2-M in which IL-7R/phospho-STAT5 is present in the majority
of cells. This result is consistent with previous analyses showing
that IL-7 signaling represses Rag transcription (15, 16). In addition, these data are also consistent with V(D)J recombination
occurring in the G1 phase of the cell cycle (8) and highlight a
second level of control for restricting Rag expression to the G0-G1
compartment.
The above data links IL-7R/phospho-STAT5 expression with
cell cycle status and Rag transcription. However, it remains
possible that differential Rag transcription is a consequence of
a change in cell cycle status rather than a consequence of IL-7
signaling. To differentiate between these possibilities, we analyzed Rag1 transcription during cell cycle in v-Abl–transformed
pro-B cells. Transformation with v-Abl dissociates proliferation
from exogenous IL-7, resulting in constitutive activation of
downstream signaling components (28). Indeed, our data indicate
that in contrast to ex vivo-derived cells STAT5 remains phosphorylated in v-Abl–transformed cells during G0-G1 (Fig. 1C),
and Rag1 transcription is not altered to the same extent across the
cell cycle relative to untransformed cells (in v-Abl–transformed
cells, there is a ,2-fold difference between G0-G1 and S-G2-M
compared with an 8- to 11-fold difference in Rag1 transcription in
untransformed cells) (Fig. 1E). Thus, we conclude that differential Rag transcription is not simply a consequence of cell cycle
status.
The Journal of Immunology
Phospho-STAT5 levels inversely correlate with Rag expression
During ontogeny, B cell development begins in the fetal liver and
subsequently moves to the bone marrow. Interestingly, there is
a differential dependence on IL-7 signaling within these two anatomical locations, such that in the absence of the IL-7R, B cell
development is ablated in the adult bone marrow but only partially blocked in fetal liver (32, 33). Thus, we asked whether
differential dependence on the IL-7 signaling pathway within
these two physiological settings manifested itself in differential
levels of phospho-STAT5 in bone marrow and fetal liver-derived
pro-B cells. As shown in Fig. 3A, phospho-STAT5 is present at
lower levels in ex vivo-sorted fetal liver pro-B cells compared
with their bone marrow counterparts. Again, we compared the
phospho-STAT5 signals with an isotype control Ab because (as
shown in this study) the background signal can change as a result
of cell size. FACs analyses indicate that fetal liver pro-B cells also
have fewer cells in S-G2-M compared with their bone marrow
counterparts (Supplemental Fig. Bi). Importantly, reduced levels
of phospho-STAT5 in fetal liver pro-B (compared with bone
marrow pro-B cells) correlated with increased Rag expression
(Fig. 3A). Thus, reduced levels of phospho-STAT5 impact on the
cell cycle profile and levels of Rag expression in the two environments.
To extend these observations, we next asked whether reciprocal
alterations in phospho-STAT5 levels also influence Rag expression.
For this we analyzed Rag expression and cell cycle status in proB cells isolated from mice that express a constitutively active form
of STAT5b (caStat5). In these mice, although transgenic STAT5b
can be activated independent of IL-7 signals, it becomes hyperphosphorylated upon IL-7 stimulation and decays at a slower rate
(24). STAT5 is phosphorylated in both wild-type and caStat5 proB cells that have been cultured short term in the presence of IL-7.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 3. Phospho-STAT5 levels inversely correlate with Rag expression. (A)
Intracellular staining and FACS analysis of
phospho-STAT5 levels in pro-B cells
(CD19+/c-kit+) derived from bone marrow
and E16 fetal liver. Phospho-STAT5 (blue)
staining and the IG control (red) are shown
for each cell type (left panels). Q-PCR
analysis of the level of Rag1 transcripts in
bone marrow and fetal liver derived pro-B
cells is shown (right panel). Transcripts
were normalized against b2-microglobulin
and the bone marrow pro-B cell population set at 1. (B) Comparison of phospho-STAT5 levels in wild-type and
caStat5 pro-B cell (CD19+/CD252) shortterm cultures (left panels). Corresponding
Rag1 transcriptional analysis is also shown
(right panel). (C) Comparison of phosphoSTAT5 levels across cell cycle stages
within wild-type (top) or caStat5 (bottom)
pro-B cell cultures as described in Fig. 1B
(left panels). (D) Graph showing the percentage of association of the Rag locus
with PCH as determined by three-dimensional DNA FISH in sorted wild-type
or caStat5 pro-B short-term cultures. A
gamma satellite DNA probe was used in
conjunction with a probe that hybridizes to
the Rag locus. Two hundred cells of each
genotype were scored. Experimental variability is shown in Supplemental Table II.
Data are representative of three experiments. Data from two individual experiments are shown in Supplemental Table II
to show the low variability between experiments.
5
6
STAT5 binds downstream of the Rag1 locus in pro-B cells
The transcriptional regulation of the Rag genes is complex. The
two genes (Rag1 and Rag2) are closely linked and convergently
transcribed and share multiple regulatory elements within a 110kb region, including several lineage-specific enhancers (19, 34,
35). To date, Erag is the only defined B cell regulatory element
FIGURE 4. STAT5 binds downstream of the Rag1
locus in pro-B cells. (A) Q-PCR analysis of the level of
Rag1 transcripts in wild-type and Erag2/2 pro-B cells.
(B) Graph showing percentage association of the Rag
locus with PCH in wild-type and Erag2/2 pro-B cells.
Over 180 cells of each genotype were counted for the
pair. Data from two individual experiments is shown
in Supplemental Table III to show the low variability
between experiments. (C) ChIP-seq enrichment and
peaks of STAT5 and H3K27me3 at the Rag1 and Rag2
loci and surrounding genomic region encompassing
known regulatory elements Erag and the ASE as well
as the newly identified STAT5/H3K27me3 enriched
binding site (PRCC). Genomic location of these elements is indicated.
involved in the transcriptional regulation of the Rag genes (2, 19).
To test whether Erag is responsible for Rag locus positioning
relative to PCH within the nucleus, we analyzed Rag locus recruitment to PCH in Erag null mice relative to their wild-type
counterparts. Deletion of Erag has been previously been shown
to result in reduced B lineage-specific transcription of Rag (19).
Intriguingly, despite reduced transcription within Erag null proB cells (Fig. 4A), we observed no difference in Rag locus association with PCH (Fig. 4B, Supplemental Table III). These data
allow us to draw two important conclusions. First, association of
the Rag locus with PCH is not always linked to transcriptional
repression because deletion of Erag does not affect nuclear localization. Second, an alternate STAT5-dependent regulatory element is involved in Rag locus repositioning.
To identify putative repressive STAT5 binding sites within the
110-kb region of the genome containing the Rag genes and their
regulators, we analyzed ChIP-seq data sets of H3K27me3 and
STAT5 that we obtained from a recent analysis of these factors
(25). These ChIP-seq experiments were performed by the Clark
laboratory using Rag22/2 pro-B cells cultured in high IL-7 (10 ng/
ml). Using these data sets, we identified several small peaks of
STAT5 within the 110-kb Rag region that encompasses all the
known regulatory elements (Fig. 4C). One of these peaks is located
on Erag. However, only one site, located ∼6 kb downstream from
the Rag1 locus on chromosome 2 at 101495000, was enriched for
both STAT5 and the repressive histone modification H3K27me3
(Fig. 4C). (It should be noted that we cannot rule out that other
H3K27me3-enriched STAT5 binding sites can be found in these
cells within the Rag2 locus because at least part of this locus is
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
In the overall population, phospho-STAT5 levels are slightly increased in transgenic cells (Fig. 3B), but importantly, in contrast to
their wild-type counterparts, phopho-STAT5 levels were not reduced in G0-G1 caStat5 pro-B cells (Fig. 3C). However, increased
levels of phospho-STAT5 did not correlate with an increased
percentage of caStat5 S-G2-M cells (Supplemental Fig. Bii). To
ensure equivalent developmental status, we sorted CD19+CD252
pro-B cell from the two cultures before analyzing Rag expression.
Consistent with our previous observations, we found that in
caStat5 pro-B cells Rag transcripts were reproducibly decreased
compared with wild-type controls (Fig. 3B).
To determine whether STAT5 was responsible for Rag locus
repositioning to PCH, three-dimensional DNA FISH was performed on wild-type and caStat5 sorted pro-B cells using probes
that hybridize to Rag and gamma satellite. As shown in Fig. 3D,
we found an increased frequency of association of Rag with PCH
in caStat5 pro-B cells compared with wild-type counterparts (data
from two independent experiments is shown in Supplemental
Table II). In sum, these data demonstrate that phospho-STAT5
plays a role in repositioning the Rag locus to a repressive compartment of the nucleus and downregulating its expression in
a manner that relates to cell cycle status.
IL-7R/STAT5 SUBDIVIDES THE PRO-B CELL COMPARTMENT
The Journal of Immunology
deleted in these cells.) The site that we have identified downstream
of the Rag1 locus is a previously unidentified site, which we have
named putative new regulatory element (putative regulator of RAG
expression through cell cycle [PRCC]). To validate the role of this
element in downregulating expression of Rag1 in G2-M in proB cells, targeted deletion of this region would be required.
IL-7 concentration influences phospho-STAT5 levels across
cell cycle
5 ng/ml IL-7. During a 6- to 10-d culture, half of the media was
replaced a total of two times. Under these conditions, it is unclear
what the actual IL-7 concentration is at any particular time point
of the culture. We therefore revised these conditions to more accurately control IL-7 levels. Pro-B cell cultures were established
during 6–7 d as described above. We then removed the proB cells, washed them, and replated them in the absence of stroma
in 10, 5, 2.5, 1 ng/ml, or no IL-7 for ∼36 h and directly analyzed
phospho-STAT5 levels within the G0-G1 or S-G2-M compartments. Strikingly, the proportion of cells that contain phosphoSTAT5 within the G0-G1 population remains consistent within
cells cultured in the highest concentrations of IL-7. However, this
proportion is visibly altered between the 2.5 and 1 ng/ml culture
conditions (Fig. 5A). We note that the percentage of cells within
S-G2-M did not change significantly until IL-7 was completely
withdrawn from the cultures. These data again separate phosphoSTAT5 levels and cell cycle status. We conclude that the regulation of phospho-STAT5 levels in G0-G1 is influenced by the
amount of available IL-7. However, additional mechanisms must
also exist to downregulate phospho-STAT5 when IL-7 is present in
excess. Although Rag transcription is reduced at low concentrations of IL-7 within G0-G1 cells, this may be an indirect effect
as IL-7 withdrawal induces progression to the pre-B cell stage.
Discussion
Genetic studies have shown that it is critical to restrict recombination to the G0-G1 phase of the cell cycle to prohibit trans-
FIGURE 5. IL-7 concentration influences phospho-STAT5 levels across cell cycle. (A) Intracellular FACS analysis of phospho-STAT5 levels across the
cell cycle within pro-B cells cultured in decreasing amounts of IL-7. Phospho-STAT5 is shown in blue, and an isotype (IG) control is shown in red. Cell
cycle profiles within each culture condition assessed by DAPI staining are shown within histograms on the far right. (B) Model shows how IL-7 segregates
proliferation and recombination in pro-B cells via cell cycle-mediated control of Rag transcription.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Our data suggest a role for the dynamic regulation of phosphoSTAT5 levels in controlling the expression of Rag1 in a manner
that relates to cell cycle status. However, it remains unclear how
IL-7R/phospho-STAT5 levels are controlled within the pro-B cell
stage. IL-7R and phospho-STAT5 are only ablated in a proportion
of wild-type pro-B cells in G0-G1 (Fig. 1A, 1B), indicating that
levels do not change during each replication cycle. In addition,
constitutive activation of phospho-STAT5 (either via Abl transformation or the presence of a constitutively active transgene)
negates alterations in phospho-STAT5 levels relative to cell cycle
status. We next asked whether increasing IL-7 concentrations
could “mimic” constitutive activation of the pathway, effectively
reducing the proportion of cells that downregulate phosphoSTAT5 levels in G0-G1. For this analysis, we cultured proB cells in increasing concentrations of IL-7 and analyzed
phospho-STAT5 levels by intracellular staining (Fig. 5A). In
previous experiments, pro-B cell cultures were established on
bone marrow stroma (which secrete IL-7) using media containing
7
8
The exact relationship between the IL-7R/phospho-STAT5 low/
negative population and IL-7R/phospho-STAT5 high population
remains to be determined. Although it is possible that IL-7R/
phospho-STAT5 is regulated dynamically during the cell cycle
in individual cells, the bimodal expression of IL-7R/phosphoSTAT5 within G0-G1 is consistent with the existence of a population of IL-7R/phospho-STAT5 low/negative daughter cells that
truly exits the cell cycle (at least temporarily) to allow recombination to occur.
In the B cell lineage, IL-7R/STAT5 is known to have a role
in promoting the sequential ordering of recombination of the Ig
loci (16, 40, 44). Now, we find that IL-7R/STAT5 is also involved
in segregating recombination and cell division by differentially
regulating the transcription of key gene targets involved in the
recombination process in a manner that is linked to cell cycle
progression. Unfortunately, because current Abs do not allow
detection of endogenous RAG1 within ex vivo-derived wild-type
pro-B cells (45) or in ex vivo-cultured cells, we were not able to
confirm these findings at the protein level. Instead, we focused on
transcriptional regulation via STAT5 and regulatory mechanisms
associated with transcriptional control within the nucleus.
STAT5 can act as both an activator and a repressor of transcription (25). Recent studies indicate that H3K27me3 is enriched
at sites where STAT5 acts as a repressor (25). Interestingly,
through analysis of the Clark laboratory ChIP-seq data sets for
STAT5 and H3K27me3, we uncovered a putative new regulatory
element that binds both factors at a site downstream of the Rag1
locus in pro-B cells. Although this PRCC element lies outside
what is considered to be the main promoter region, it is certainly
possible that it functions to control promoter activity, which in
turn could affect transcription levels. We note that the Rag1 promoter is positively regulated by E2A, and the IL-7R/STAT5
pathway has previously been shown to prohibit E2A function
and binding at the Igk locus (16, 44). It will be interesting to
determine whether an important element of STAT5-repressive
function is the antagonism of the E2A protein. Additional genetic targeting studies will be required to determine whether this
element can regulate cell cycle expression of Rag1 through the
repositioning of the latter to PCH.
It is clear that not all of the targets of STAT5 are regulated
similarly across the cell cycle, as indicated by differences in the
behavior of the Igh and Rag loci. We have previously shown that
STAT5 can maintain the accessibility of Igh by keeping it euchromatic (18), whereas STAT5 regulates Rag in an opposite
manner. The cell cycle regulation of Rag by STAT5 occurs at the
transcriptional level and correlates with the repositioning of the
Rag locus to PCH. As with all studies that link gene silencing with
PCH repositioning, there is no clear indication as to whether
a change in nuclear localization is a cause or a consequence of
repression. How STAT5 functions in this context remains to be
determined.
IL-7R/STAT5 has been shown to regulate the expression of
genes involved in survival, proliferation, and lineage specification
(36, 40, 46). Importantly, STAT5 is rendered constitutively active
and independent of extrinsic signals in numerous cancers (47). It
is possible that some STAT5 targets outside of the recombination
pathway are also differentially regulated within pro-B cells in
a manner that relates to cell cycle status. In this context, changes
in gene dosage across a cell cycle could ultimately promote oncogenesis in many different settings as indicated by recent findings that show that alterations in the levels of transcription factors
cooperate with STAT5 to initiate acute lymphoblastic leukemia
(48).
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
locations. Periodic destruction of RAG2 protein during S-phase
entry is one mechanism by which this is accomplished. As with
most important biological processes, multiple layers of protection are required. In this study, we elucidate an additional
regulatory constraint that segregates proliferation from recombination during early B cell development. We find that IL-7R
is expressed within a larger proportion of S-G2-M than G0-G1
pro-B cells and that S-G2-M cells have reduced Rag expression
as a consequence of the action of the downstream IL-7 signaling
effector molecule, STAT5. These data are consistent with a
known role for IL-7 in promoting proliferation as well as inhibiting Rag transcription.
The early development of B and T cells share many parallels;
each proceeds through windows of locus-specific recombination
flanked by bursts of proliferation, thus developmental stages between the two lineages are often compared. Double-negative T cells
are considered to be the counterpart for pro-B cells. Yet, doublenegative T cells are subdivided into four distinct compartments
with different cell cycle profiles. Thus, it is not unexpected that proB cells can also be subdivided into different compartments with
different cell cycle profiles. A role for IL-7 in this subdivision is
supported by studies showing that constitutive expression of IL-7R
within hematopoietic progenitor cells blocks B cell development
at the pre–pro-B cell stage (36). A similar developmental block is
seen within mice that express c-myc and bcl-2 constitutively (37),
a state in which cell cycle exit or pausing in G0-G1 would be
unlikely. In the context of the data presented in this paper, these
phenotypes could in part be explained by an inability of those cells
to exit cell cycle and upregulate Rag expression for recombination. Because D-J rearrangement is initiated within the Igh locus
at the pre–pro-B cell stage, it is tempting to speculate that IL-7
signals are transiently downregulated at each stage in which recombination occurs. In fact, an IL-7R–negative or low population
has been found within pre–pro-B cells (38). However, it is unlikely that the block seen in cells expressing a constitutive IL-7R
would be solely due to differential Rag expression because Ragdeficient mice are able to proceed to the pro-B cell stage. Instead,
it is more likely that dynamic regulation of the IL-7 signaling
pathway is required for regulating additional genes outside of the
recombination pathway.
Interestingly, we found that IL-7 levels regulate the proportion
of cells with phospho-STAT5 in G0-G1. An intriguing possibility
is that dividing cells in the bone marrow move back and forth
between IL-7 expressing and non–IL-7 expressing stroma, resulting in a transient attenuation of IL-7R signaling. Indeed,
IL-7R/phospho-STAT5 levels do not change each time a cell
passes through G0-G1, as evidenced by our data showing a bimodal expression within G0-G1 cells. This suggests that only a
subset of daughter cells downregulate IL-7R/phospho-STAT5,
a result that could be attributed to either limiting concentrations
of IL-7 or asymmetric cell division. One idea is that these two
possibilities are linked. In this model, the process of cell division
causes one daughter cell to be positioned further away from IL-7
stroma cells in a manner that downregulates the IL-7R signaling
pathway. These possibilities need to be further investigated.
Taking into account that IL-7 signals are generally thought to
promote Igh locus accessibility for recombination (39–43), we
propose a model for an ordered subdivision of the pro-B cell
compartment, based on IL-7R expression (Fig. 5B). In this
model, the (first) actively cycling, IL-7R–positive pro-B subpopulation prepares the Igh locus for recombination by enhancing
accessibility. In the subsequent G0-G1 IL-7R–negative population,
Rag is upregulated to recombine the accessible Igh locus. Cells
could transit between these states until recombination is complete.
IL-7R/STAT5 SUBDIVIDES THE PRO-B CELL COMPARTMENT
The Journal of Immunology
Acknowledgments
We thank members of the Skok laboratory for thoughtful discussions and
critical comments on the manuscript. We also thank Mark Schlissel for providing the Erag null animals.
Disclosures
The authors have no financial conflicts of interest.
References
23. Skok, J. A., K. E. Brown, V. Azuara, M. L. Caparros, J. Baxter, K. Takacs,
N. Dillon, D. Gray, R. P. Perry, M. Merkenschlager, and A. G. Fisher. 2001.
Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes.
Nat. Immunol. 2: 848–854.
24. Will, W. M., J. D. Aaker, M. A. Burchill, I. R. Harmon, J. J. O’Neil, C. A. Goetz,
K. L. Hippen, and M. A. Farrar. 2006. Attenuation of IL-7 receptor signaling is
not required for allelic exclusion. J. Immunol. 176: 3350–3355.
25. Mandal, M., S. E. Powers, M. Maienschein-Cline, E. T. Bartom, K. M. Hamel,
B. L. Kee, A. R. Dinner, and M. R. Clark. 2011. Epigenetic repression of the Igk
locus by STAT5-mediated recruitment of the histone methyltransferase Ezh2.
Nat. Immunol. 12: 1212–1220.
26. Langmead, B., C. Trapnell, M. Pop, and S. L. Salzberg. 2009. Ultrafast and
memory-efficient alignment of short DNA sequences to the human genome.
Genome Biol. 10: R25.
27. Micsinai, M., F. Parisi, F. Strino, P. Asp, B. D. Dynlacht, and Y. Kluger. 2012.
Picking ChIP-seq peak detectors for analyzing chromatin modification experiments. Nucleic Acids Res. DOI: 10.1093/nar/gks048.
28. Danial, N. N., A. Pernis, and P. B. Rothman. 1995. Jak-STAT signaling induced
by the v-abl oncogene. Science 269: 1875–1877.
29. Schneider, R., and R. Grosschedl. 2007. Dynamics and interplay of nuclear architecture, genome organization, and gene expression. Genes Dev. 21: 3027–
3043.
30. Brown, K. E., J. Baxter, D. Graf, M. Merkenschlager, and A. G. Fisher. 1999.
Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell
division. Mol. Cell 3: 207–217.
31. Hendzel, M. J., Y. Wei, M. A. Mancini, A. Van Hooser, T. Ranalli,
B. R. Brinkley, D. P. Bazett-Jones, and C. D. Allis. 1997. Mitosis-specific
phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106: 348–360.
32. Hesslein, D. G., S. Y. Yang, and D. G. Schatz. 2006. Origins of peripheral B cells
in IL-7 receptor-deficient mice. Mol. Immunol. 43: 326–334.
33. Erlandsson, L., S. Licence, F. Gaspal, S. Bell, P. Lane, A. E. Corcoran, and
I. L. Mårtensson. 2004. Impaired B-1 and B-2 B cell development and atypical
splenic B cell structures in IL-7 receptor-deficient mice. Eur. J. Immunol. 34:
3595–3603.
34. Yannoutsos, N., V. Barreto, Z. Misulovin, A. Gazumyan, W. Yu, N. Rajewsky,
B. R. Peixoto, T. Eisenreich, and M. C. Nussenzweig. 2004. A cis element in the
recombination activating gene locus regulates gene expression by counteracting
a distant silencer. Nat. Immunol. 5: 443–450.
35. Yu, W., Z. Misulovin, H. Suh, R. R. Hardy, M. Jankovic, N. Yannoutsos, and
M. C. Nussenzweig. 1999. Coordinate regulation of RAG1 and RAG2 by cell
type-specific DNA elements 59 of RAG2. Science 285: 1080–1084.
36. Purohit, S. J., R. P. Stephan, H. G. Kim, B. R. Herrin, L. Gartland, and
C. A. Klug. 2003. Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor. EMBO J. 22: 5511–5521.
37. Strasser, A., A. G. Elefanty, A. W. Harris, and S. Cory. 1996. Progenitor tumours
from Emu-bcl-2-myc transgenic mice have lymphomyeloid differentiation potential and reveal developmental differences in cell survival. EMBO J. 15: 3823–
3834.
38. Allman, D., J. Li, and R. R. Hardy. 1999. Commitment to the B lymphoid lineage occurs before DH-JH recombination. J. Exp. Med. 189: 735–740.
39. Corcoran, A. E., A. Riddell, D. Krooshoop, and A. R. Venkitaraman. 1998.
Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor.
Nature 391: 904–907.
40. Malin, S., S. McManus, C. Cobaleda, M. Novatchkova, A. Delogu, P. Bouillet,
A. Strasser, and M. Busslinger. 2010. Role of STAT5 in controlling cell survival
and immunoglobulin gene recombination during pro-B cell development. Nat.
Immunol. 11: 171–179.
41. Chowdhury, D., and R. Sen. 2001. Stepwise activation of the immunoglobulin m
heavy chain gene locus. EMBO J. 20: 6394–6403.
42. Chowdhury, D., and R. Sen. 2003. Transient IL-7/IL-7R signaling provides
a mechanism for feedback inhibition of immunoglobulin heavy chain gene
rearrangements. Immunity 18: 229–241.
43. Roldán, E., M. Fuxa, W. Chong, D. Martinez, M. Novatchkova, M. Busslinger,
and J. A. Skok. 2005. Locus “decontraction” and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat.
Immunol. 6: 31–41.
44. Mandal, M., S. E. Powers, K. Ochiai, K. Georgopoulos, B. L. Kee, H. Singh, and
M. R. Clark. 2009. Ras orchestrates exit from the cell cycle and light-chain
recombination during early B cell development. Nat. Immunol. 10: 1110–1117.
45. Ji, Y., W. Resch, E. Corbett, A. Yamane, R. Casellas, and D. G. Schatz. 2010.
The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell
141: 419–431.
46. Goetz, C. A., I. R. Harmon, J. J. O’Neil, M. A. Burchill, T. M. Johanns, and
M. A. Farrar. 2005. Restricted STAT5 activation dictates appropriate thymic B
versus T cell lineage commitment. J. Immunol. 174: 7753–7763.
47. Malin, S., S. McManus, and M. Busslinger. 2010. STAT5 in B cell development
and leukemia. Curr. Opin. Immunol. 22: 168–176.
48. Heltemes-Harris, L. M., M. J. Willette, L. B. Ramsey, Y. H. Qiu, E. S. Neeley,
N. Zhang, D. A. Thomas, T. Koeuth, E. C. Baechler, S. M. Kornblau, and
M. A. Farrar. 2011. Ebf1 or Pax5 haploinsufficiency synergizes with STAT5
activation to initiate acute lymphoblastic leukemia. J. Exp. Med. 208: 1135–
1149.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
1. Bassing, C. H., W. Swat, and F. W. Alt. 2002. The mechanism and regulation of
chromosomal V(D)J recombination. Cell 109(Suppl.): S45–S55.
2. Kuo, T. C., and M. S. Schlissel. 2009. Mechanisms controlling expression of the
RAG locus during lymphocyte development. Curr. Opin. Immunol. 21: 173–178.
3. Lee, G. S., M. B. Neiditch, S. S. Salus, and D. B. Roth. 2004. RAG proteins
shepherd double-strand breaks to a specific pathway, suppressing error-prone
repair, but RAG nicking initiates homologous recombination. Cell 117: 171–184.
4. Takata, M., M. S. Sasaki, E. Sonoda, C. Morrison, M. Hashimoto, H. Utsumi,
Y. Yamaguchi-Iwai, A. Shinohara, and S. Takeda. 1998. Homologous recombination and non-homologous end-joining pathways of DNA double-strand
break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17: 5497–5508.
5. Zhu, C., K. D. Mills, D. O. Ferguson, C. Lee, J. Manis, J. Fleming, Y. Gao,
C. C. Morton, and F. W. Alt. 2002. Unrepaired DNA breaks in p53-deficient cells
lead to oncogenic gene amplification subsequent to translocations. Cell 109:
811–821.
6. Difilippantonio, M. J., J. Zhu, H. T. Chen, E. Meffre, M. C. Nussenzweig,
E. E. Max, T. Ried, and A. Nussenzweig. 2000. DNA repair protein Ku80
suppresses chromosomal aberrations and malignant transformation. Nature 404:
510–514.
7. Gao, Y., D. O. Ferguson, W. Xie, J. P. Manis, J. Sekiguchi, K. M. Frank,
J. Chaudhuri, J. Horner, R. A. DePinho, and F. W. Alt. 2000. Interplay of p53 and
DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development. Nature 404: 897–900.
8. Schlissel, M., A. Constantinescu, T. Morrow, M. Baxter, and A. Peng. 1993.
Double-strand signal sequence breaks in V(D)J recombination are blunt, 59phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 7(12B):
2520–2532.
9. Li, Z., D. I. Dordai, J. Lee, and S. Desiderio. 1996. A conserved degradation
signal regulates RAG-2 accumulation during cell division and links V(D)J recombination to the cell cycle. Immunity 5: 575–589.
10. Lin, W. C., and S. Desiderio. 1994. Cell cycle regulation of V(D)J
recombination-activating protein RAG-2. Proc. Natl. Acad. Sci. USA 91: 2733–
2737.
11. Lin, W. C., and S. Desiderio. 1993. Regulation of V(D)J recombination activator
protein RAG-2 by phosphorylation. Science 260: 953–959.
12. Zhang, L., T. L. Reynolds, X. Shan, and S. Desiderio. 2011. Coupling of V(D)J
recombination to the cell cycle suppresses genomic instability and lymphoid
tumorigenesis. Immunity 34: 163–174.
13. Deriano, L., J. Chaumeil, M. Coussens, A. Multani, Y. Chou, A. V. Alekseyenko,
S. Chang, J. A. Skok, and D. B. Roth. 2011. The RAG2 C terminus suppresses
genomic instability and lymphomagenesis. Nature 471: 119–123.
14. Ye, M., and T. Graf. 2007. Early decisions in lymphoid development. Curr. Opin.
Immunol. 19: 123–128.
15. Amin, R. H., and M. S. Schlissel. 2008. Foxo1 directly regulates the transcription
of recombination-activating genes during B cell development. Nat. Immunol. 9:
613–622.
16. Johnson, K., T. Hashimshony, C. M. Sawai, J. M. Pongubala, J. A. Skok,
I. Aifantis, and H. Singh. 2008. Regulation of immunoglobulin light-chain recombination by the transcription factor IRF-4 and the attenuation of interleukin7 signaling. Immunity 28: 335–345.
17. Bertolino, E., K. Reddy, K. L. Medina, E. Parganas, J. Ihle, and H. Singh. 2005.
Regulation of interleukin 7-dependent immunoglobulin heavy-chain variable
gene rearrangements by transcription factor STAT5. Nat. Immunol. 6: 836–843.
18. Hewitt, S. L., B. Yin, Y. Ji, J. Chaumeil, K. Marszalek, J. Tenthorey,
G. Salvagiotto, N. Steinel, L. B. Ramsey, J. Ghysdael, et al. 2009. RAG-1 and
ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci. Nat. Immunol. 10: 655–664.
19. Hsu, L. Y., J. Lauring, H. E. Liang, S. Greenbaum, D. Cado, Y. Zhuang, and
M. S. Schlissel. 2003. A conserved transcriptional enhancer regulates RAG gene
expression in developing B cells. Immunity 19: 105–117.
20. Goetz, C. A., I. R. Harmon, J. J. O’Neil, M. A. Burchill, and M. A. Farrar. 2004.
STAT5 activation underlies IL7 receptor-dependent B cell development. J.
Immunol. 172: 4770–4778.
21. Fuxa, M., J. Skok, A. Souabni, G. Salvagiotto, E. Roldan, and M. Busslinger.
2004. Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev. 18: 411–422.
22. Hewitt, S. L., D. Farmer, K. Marszalek, E. Cadera, H. E. Liang, Y. Xu,
M. S. Schlissel, and J. A. Skok. 2008. Association between the Igk and Igh
immunoglobulin loci mediated by the 39 Igk enhancer induces “decontraction”
of the Igh locus in pre-B cells. Nat. Immunol. 9: 396–404.
9