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Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461,1 Torrey Pines Institute for Molecular Studies, San Diego, California 921212
Received 24 January 2005/ Returned for modification 7 March 2005/ Accepted 30 March 2005
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ABSTRACT |
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1-Mb region in the 5' half of the variable-gene region heavy-chain (Vh) locus regularly colocalizes with the nuclear lamina. The 3' half of the Vh gene region is less frequently colocalized with the periphery, while sequences flanking the Vh gene region are infrequently so. Importantly, in plasmacytomas, VDJ rearrangements that delete most of the Vh locus, including part of the 5' half of the Vh gene region, result in loss of peripheral compartmentalization, while deletion of only the proximal half of the Vh gene region does not. In addition, when Igh-Myc translocations move the Vh genes to a new chromosome, the distal Vh gene region is still associated with the nuclear periphery. Thus, the Igh region that interacts with the nuclear periphery is localized but is likely comprised of multiple sites that are distributed over 1 Mb in the 5' half of the Vh gene region. This 5' Vh gene region that produces peripheral compartmentalization is the same region that is distinguished by requirements for interleukin-7, Pax5, and Ezh2 for rearrangement of the Vh genes. |
INTRODUCTION |
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We and others have found that the mouse immunoglobulin heavy-chain (Igh) locus is positioned at different sites in the nucleus depending on its transcription and rearrangement states. The immunoglobulin (Ig) heavy- and light-chain genes are expressed in B lymphocytes to produce antibodies, the secreted effector molecules of adaptive immunity. To encode a very large diversity of binding sites, Ig genes are assembled from sets of gene segments in the germ line: Ig heavy-chain genes from V, D, and J segments (shown at the top of Fig. 1) and Ig light-chain genes from V and J segments. This assembly takes place during the differentiation of B cells from progenitors in the bone marrow and marks specific developmental stages. In the first stage, pro-B, D-J joining occurs at the Igh locus; in the next stage, pre-B, V-to-DJ joining takes place to complete the assembly of the heavy-chain gene. V-J joining of the light chain marks progression to a B cell capable of expressing immunoglobulin as both its cell surface receptor and secreted serum antibody. Subsequent repeated stimulation by antigen, along with other regulatory signals, can drive further differentiation to the plasmablast, or plasma cell, which is an antibody factory that secretes at a high rate. Remarkably, the physical location of the Igh locus within the nucleus changes several times during this developmental progression.
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While the mechanisms of perinuclear positioning of genes and the organization of such nuclear components are not fully understood, outlines are emerging. Electron microscopy shows only patches of chromatin positioned against the nuclear envelope in a peripheral chromosome (26). By direct analysis of chromatin motion in vivo, the nuclear-envelope-adherent chromatin has been shown to be directly linked to the nuclear periphery (9, 17). FISH studies of Drosophila, protozoan, mouse, and human cells demonstrate that not every part of chromatin adheres to the nuclear envelope but that only selective chromatin regions confer nuclear-envelope attachment (for a review, see reference 22).
The outer boundary of the nucleus is a protein shell, the lamina, underlying the nuclear membrane. The lamina is composed primarily of a number of lamin proteins that can directly interact with chromatin or interact indirectly through the lamin-associated proteins, such as HS95, LAP2ß, and BAF (11, 20, 21, 23, 36). Other components of the nuclear envelope are the nuclear pore complexes that are also associated with chromatin and genes (13, 34). Recent evidence suggests that nuclear pore complexes preferentially interact with transcriptionally active genes rather than inactive genes in Saccharomyces cerevisiae (6).
In multipotential progenitors and T-lineage lymphocytes, the distal (5') Vh genes and flanking sequences are closer to the nuclear periphery than the proximal (3') Vh genes and downstream constant-region genes (12, 19), suggesting that the Igh locus anchors at the nuclear periphery through a site or sites in the distal Vh genes. In this study, we asked if Igh is anchored by the distal Vh region in all the primary cell types and transformed cell lines in which it resides at the periphery. Further, to more specifically identify the anchoring region, we have systematically determined the nuclear locations of individual segments across the Igh locus in EL4 cells by performing three-dimensional fluorescent in situ hybridization (3D FISH) with Igh bacterial artificial chromosomes (BACs). We demonstrate that in either B-lineage cells or non-B cells, different regions of the Igh locus vary in how frequently they are located at the nuclear periphery. We show here that the Igh Vh region, which is closer to the nuclear periphery than the downstream constant region, encompasses at least two domains with different propensities for association with the nuclear envelope. A region of approximately 1 Mb in the 5' half of Igh is most frequently in contact with the nuclear lamin B, while the 3' half is distinctly less often peripheral. We also approached this question with a deletion strategy, examining the Igh nuclear location in a series of plasmacytomas that rearranged Vh genes at sites across the locus, thereby retaining decreasing portions of the Vh gene array. Further, we examined the behavior of Igh-Myc translocations in plasmacytomas. These studies supported the conclusions of the scan across Igh with BAC probes and showed that the distal Vh region can locate peripherally in the absence of the 3' Vh region, and even when placed on a new chromosome.
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MATERIALS AND METHODS |
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All cell lines were maintained as exponential cultures. Most lines were cultured at 37°C with 5% CO2 in RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (GIBCO BRL), penicillin-streptomycin (GIBCO BRL), 2 mM L-glutamate (GIBCO BRL), and 50 µM 2-mercaptoethanol (Sigma). S107, MPC11, and MEL cells were grown in Dulbecco's modified Eagle's medium supplemented only with 10% fetal bovine serum and penicillin-streptomycin. ES cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) (ES cell qualified; Invitrogen) and 103 units/ml of mouse leukemia inhibitory factor.
Primary cell populations. Normal mature B cells and plasmablasts were isolated from the spleens of 6- to 8-week-old C57BL/6J mice as described previously (30). Following red blood cell depletion, the spleen cells were incubated with anti-CD43 (Ly-48)-coupled microbeads (Miltenyi Biotech) according to the manufacturer's instructions. Unbound resting B cells were isolated using magnetic cell separation (MiniMACs depletion column). To activate B cells, enriched resting B cells were cultured in RPMI containing 10% FCS, 2-mercaptoethanol, antibiotics, and lipopolysaccharide (LPS) (50 µg/ml) for 96 h. To isolate S-phase mature B cells from these cultures, we added Hoechst 33342 (Sigma) to 5 µg/ml and cultured them for 30 min. Hoechst-labeled cells were incubated with R-phycoerythrin-conjugated rat anti-mouse B220 monoclonal antibody (1 µg per million cells; BD Biosciences) for 30 min at 4°C. B220-positive and S-phase cells (cells having 120 to 180% diploid DNA content) were isolated by fluorescence-activated cell sorting (FACS). To isolate plasmablast cells, syndecan-positive and S-phase cells were sorted from the activated splenic B-cell culture using R-phycoerythrin-conjugated rat anti-mouse CD138 (syndecan-1) monoclonal antibody (1 µg/106 cells; BD Biosciences).
Pro-B cells came from a short-term culture of bone marrow from 6- to 8-week-old Rag1–/– C57BL/6 mice as described previously (18). The cells were plated at 1.0 x 106/ml in RPMI supplemented with 5% heat-inactivated FCS, penicillin-streptomycin, 50 µM 2-mercaptoethanol, and 12.5 ng/ml interleukin-7 (IL-7) (R&D). Half of the medium was changed every 3 days. Unattached cells were labeled with Hoechst as described above and harvested on day 10, and S-phase pro-B cells were sorted; 94% of the isolated population was B220+.
3D FISH and confocal analysis. Immunostaining dual-probe 3D FISH was carried out as described previously in detail (5). Cells in exponential cultures were placed on polylysine-coated coverslips for 5 min to let the cells attach to the coverslips. The coverslips were placed in fixation buffer (20 mM KH2PO4, 130 mM NaCl, 20 mM KCl, 10 mM EGTA, 2 mM MgCl2, 0.1% [vol/vol] Triton X-100, 0.5% glutaraldehyde) for 30 min and then were immersed in 1 mg/ml NaBH4 (twice, each for 15 min). Nonspecific binding of proteins to the cells was blocked with 10% rabbit serum in phosphate-buffered saline. The cells were incubated with goat anti-lamin B antibodies (C-20 and M-20; Santa Cruz) and Cy5-conjugated rabbit anti-goat IgG (sequential incubations, each for 30 min). The samples were then treated with a 50 mM solution of ethylene glycol bis succinimidyl succinate to preserve the proteins. Chromosomal DNA was subsequently denatured in NaOH (pH 12.8) for 2 min, and the coverslips were hybridized overnight with BAC probes. BACs were labeled with biotin-16-dUTP or digoxigenin-11-dUTP, using a nick translation kit (Roche) as described previously (39). Biotinylated probes were detected with fluorescein isothiocyanate-avidin (Oncor); digoxigenin-labeled probes were detected with rhodamine-conjugated antidigoxigenin antibody (Oncor). The cells were examined using a Provis AX70 microscope equipped with a Cool-SNAP digital camera. Optical sections were typically collected at 0.18-µm steps through each nucleus. There are usually 30 to 60 optical sections for each image stack, depending on the sizes of the nuclei, and the data were analyzed in Slidebook 3.0 (Intelligent Imaging Innovations).
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RESULTS |
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The distal Vh region is most frequently at the nuclear periphery. Igh loci localized at the nuclear periphery in the cells examined, except in pro- and pre-B cells (5, 12, 19, 39). In a 3D FISH experiment, we compared the nuclear positions of the distal (5') and proximal (3') ends of the locus in a variety of cell types. We assigned each peripherally positioned Igh locus to one of three classes: the 5' end of Igh (5' VhJ558 genes; 218B2) is closer to the nuclear lamina (lamin B stain) than the 3' end (3' regulatory region [RR]; downstream enhancer; CT7-199M11), the 3' end is more peripheral than the distal, or the positions of the two regions are equivalent or overlapping. In all cell types, the distal Vh region is most frequently associated with the lamin rim (Fig. 3).
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1 Mb; region 1 in Fig. 4A, including BACs 218B2, 354D10, 257D2, and 197F8), the test BAC was as likely as 218B2 to be most peripheral, but most signal pairs were positioned similarly. When the pair was similarly positioned, both signals always colocalized with the lamin. Adjacent to this region on the 3' side is a second 1-Mb region containing most of the proximal Vh gene families (region 2 in Fig. 4A). In this region, most BAC pairs were again equivalently positioned, but when they were not, 218B2 was more peripheral in most cases (compared with 70F21, 206K17, 147E23, or 373N4). The next region 3' contains the Vh7183 and VhQ52 gene families, the D-J-C gene complex, and the 3' flanking region (region 3-A). In this region, the 218B2 signal was more peripheral most of the time in each BAC pair (145K3, 270B12, 109B20, and 422M13). Similar results were obtained in the region at the 5' end (32J1) and flanking Igh (38P6; 429J21) (region 3-B). These data indicate that a 1-Mb region near the 5' end of the Igh locus is preferentially oriented toward, and often colocalizes with, the nuclear lamina, and the DH, JH, and constant regions are usually oriented away from the lamina.
The absence of the proximal Vh genes does not reduce the probability of the Igh locus locating at the nuclear periphery in plasma cell lines. Although the distal Vh sequences are more consistently colocalized with the lamin rim, the proximal Vh gene regions are found there as well in fully half of the nuclei (Fig. 4, see the comparisons of 218B2 with region 2 BACs). This led us to wonder if the proximal Vh gene region is important for peripheral localization. To address this question, six plasma cell lines that deleted different portions of the Vh locus in VDJ rearrangement were examined for the nuclear positioning of Igh (Fig. 5). 3D FISH analyses were performed with the 5' Vh region BAC 218B2 and the 3' regulatory region BAC CT7-199M11. In these lines, only one allele, the productive VDJ rearrangement, retains both BAC signals; the other (one or more) is involved in Myc translocations or other aberrant rearrangements. In four of six lines (MOPC21, S107, X24, and J558), 74% to 82% of productively rearranged Igh alleles are positioned at the nuclear periphery, while only 18% to 34% of productive Igh alleles are peripheral in MPC11 and NZPC3609 (Fig. 5A, B, and C). In these six cell lines, the productive Igh rearrangements possess upstream Vh gene regions of various lengths, depending on the location of the rearranged Vh gene (Fig. 5D). The MOPC21, S107, and X24 VDJ rearrangements retain most of the Vh gene region as the 5' flank; the rearrangement in J558 cells deleted approximately the 3' half of the Vh gene locus, but most of the VhJ558 gene family was retained. However, in the MPC11 (expressing a more 5' VhJ558 family gene) and NZPC3609 plasma cell lines, sequencing and our in situ hybridization with Vh region BACs indicated that only the most distal part of the VhJ558/Vh3609P family region remains after VDJ rearrangement (14). These data indicate that the absence of the proximal Vh genes does not affect the preferential localization of the Igh locus to the nuclear periphery in plasma cell lines (for example, in the J558 plasmacytoma line). In contrast, when the rearranged Vh gene is near the 5' end of Igh so that almost the entire Vh gene region is deleted, Igh no longer associates with the periphery, thus confirming the importance of the distal 1-Mb region for directing peripheral compartmentalization.
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2a gene; in S107 and J558, c-myc is translocated to the C
gene; and in MOPC21, it is translocated to a constant gene that is located 5' of the C gene. By performing 3D FISH with Igh Vh probe 218B2 (red) and downstream Jag2 region probe 422M13 (green) in these plasmacytomas, we distinguished the productive allele (two probes juxtaposed) and nonproductive allele (two probes separated due to translocation). We found that 72% to 88% of the translocation products that include the Vh region but not the downstream region were still at the nuclear periphery (Fig. 5B and C and 6B). In contrast, by performing another 3D FISH experiment with the c-myc BAC probe 51A4 (green) and the Jag2 region probe 422M13 (red), we found that only 26% to 34% of the translocation products that juxtapose Myc and the region downstream of Vh were associated with the nuclear periphery (Fig. 6B and C). This indicates that the Vh region can be located at the nuclear periphery in the absence of an associated 3' RR and is consistent with the preceding finding that a region in distal Vh is the site that directs peripheral compartmentalization. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 Mb; region 1) that contains much of the VhJ558 and Vh3609 gene families more frequently adheres to the nuclear periphery than other portions of Vh; the proximal half of the Vh region (1 Mb; region 2) also associates with the periphery, but the likelihood is lower than for the distal Vh region; the remaining sequences, including the DH, JH, and constant regions at the 3' end and at least 100 kb of Vh genes at the 5' end of Igh (BAC probe 32J1), are infrequently associated with the lamina. Thus, the strongest determinants of lamin association are located in a 1-Mb region of distal Vh genes near the 5' end of Igh. Independently confirming this, we found that the Igh locus resides at the nuclear periphery in plasma cell lines with VDJ rearrangements that retain much of the J558 gene region but is no longer peripheral when most of the VhJ558 region is deleted. This result indicates that the 3' half of the Vh gene region has no measurable contribution to determining the nuclear position. In further experiments, we examined Myc-Igh translocation products to identify any role of the region downstream of Vh, including the 3' RR and the 3' part of the constant region, in determining the peripheral location in plasmacytoma cells. In four plasma cell lines, when the end of chromosome 12 carrying the Vh genes is translocated to chromosome 15, the distal Vh region still associates with the nuclear periphery, while the Myc-Igh-3'RR complex resides in the interior. This suggests that the 3' RR has no role in determining location at the nuclear envelope.
The distal half of the Vh gene region, which we observed to be preferentially associated with the nuclear lamina, also behaves differently than the 3' half in VDJ recombination. Hesslein et al. (16) reported that in Pax5-deficient pro-B cells, the distal V gene segments rarely recombine, while proximal Vh genes recombine efficiently. Similarly, the distal VDJ rearrangements are also differentially inhibited by loss of the histone methyltransferase Ezh2, a member of the polycomb group protein (PcG) family (33). In a third example, a deficiency in IL-7 signaling has little effect on VDJ recombination in the 3' half of the Vh gene region but strongly depresses rearrangement of the 5' VhJ558 gene family (10, 28). Absence of IL-7 signaling depresses both germ line transcription and histone acetylation in the distal Vh region (8, 10), and in IL-7 receptor-deficient early pro-B cells, Igh loci remain at the nuclear periphery instead of shifting to the interior of the nucleus (19). This suggests that IL-7 signaling could play an important role in Igh localization. In contrast, Igh is shifted to the nuclear interior in PAX5-deficient pro-B cells, as it is in normal pro-B cells (12), so PAX5 apparently has no role in regulating peripheral location.
The Igh locus and the nuclear envelope. Recent work suggests that the nuclear envelope plays a complex role in gene regulation (for a review, see reference 25). For example, the lamina functions as a scaffold that can recruit lamin-associated proteins to attached chromatin (3, 24). Our data clearly show that peripheral Igh loci localize with lamin B through interactions of the Vh region, particularly the 5' Vh region. Since the nuclear envelope has commonly been considered a zone of transcriptional repression, it seems likely that prior to B-cell differentiation, the Igh locus is silenced by its interaction with the nuclear lamina, and that for VDJ rearrangement, the locus must leave this silencing environment. Consistent with this is the observation that in IL-7R-deficient pro-B cells the 5' Vh gene region fails to disengage from the periphery and remains silenced (19).
After VDJ rearrangement, in cycling mature B cells and plasmablasts, the Igh locus again returns to the periphery, but at this time one Igh allele is actively transcribed rather than silenced. It will be interesting to learn whether the active allele has moved to a different, transcriptionally active peripheral compartment, possibly associated with a nuclear pore, or perhaps has returned to the repressive region but has assumed an extended conformation, allowing transcription of the rearranged VDJ while the upstream Vh region is still silenced.
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ACKNOWLEDGMENTS |
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We thank J. Zhou for contributions at the early stages of this work and M. Scharff and B. Birshtein for critical reading of the manuscript. We thank S. Thomas, Z. Guan, and N. Brown for valuable discussions and help with writing of the manuscript. We thank I. Su for advice on 3D FISH and D. Gilbert, P. Brodeur, and H. Worman for very helpful discussions. We also acknowledge the assistance of the Analytical Imaging and FACS Core Facilities of the Albert Einstein Cancer Center, a clinical cancer center designated by the National Cancer Institute, supported in part by P30 CA13330.
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FOOTNOTES |
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