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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). 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