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Dynamic Changes in Accessibility, Nuclear Positioning, Recombination, and Transcription at the Ig Locus^1,2

Sean P. Fitzsimmons^*, Ralph M. Bernstein, Edward E. Max, Jane A. Skok^3, and Marjorie A. Shapiro^4,*

^* Division of Monoclonal Antibodies and Division of Therapeutic Proteins, Food and Drug Administration, Center for Drug Evaluation and Research, Rockville, MD 20852; and Department of Immunology and Molecular Pathology, Division of Infection and Immunity, University College London, London, United Kingdom

	Abstract

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The 3-megabase Ig locus undergoes differentially controlled nuclear positioning events and chromatin structural changes during the course of B cell development. The temporal association of chromatin structural changes, transcription, and recombination at the Ig locus was determined in a murine pre-B cell line that can be induced to recombine at the Ig locus and in ex vivo-cultured murine pre-B cells. Additionally, the timing of nuclear positioning relative to the temporal order of chromatin structural changes and recombination and transcription was determined. We demonstrate that before induction, the Ig locus was poised for recombination; both alleles were in a contracted state, and the enrichment of histone modifications and germline transcripts of specific V genes were observed. Histone modifications of the V genes did not vary upon induction but the levels of modifications correlated with the levels of germline V gene transcripts and recombination. Upon induction, but before VJ recombination, centromeric recruitment of single Ig alleles occurred. DNase I sensitivity of the entire locus increased gradually over the course of differentiation while the enrichment of histone modifications downstream of the V genes was increased in the silencer regions upstream of J1, within the Ig sterile transcript, the constant region, the Ei and E3' enhancers, and the recombining sequence. The ex vivo pre-B cells showed similar patterns of histone modifications across the locus except at the V genes. In this study, H3 acetylation correlated with levels of germline transcripts while H3 methylation correlated with levels of recombination.

	Introduction

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B cell development follows a complex progression of events characterized by the regulated expression of lineage and stage-specific proteins, cell surface markers, and the ordered recombination of variable, diversity, and joining V(D)J genes (1, 2). V(D)J recombination requires expression of proteins encoded by the two recombination activating genes rag1 and rag2. However, expression of these proteins is not sufficient to induce V(D)J recombination at a given locus, since even in the presence of both RAG proteins, recombination occurs at different gene loci depending on the lineage and maturation state of the lymphocyte. Ig gene recombination occurs first in pro-B cells at the H chain (HC)⁵ locus between D and J elements on both alleles. Successful V(D)J recombination on one allele, production of a functional H chain protein, and expression of the pre-BCR result in the down-regulation of RAG proteins and suppression of further HC recombination. At the early pre-B cell stage, RAG proteins are re-expressed and L chain (LC) rearrangement is initiated at the Ig locus. Productive LC recombination ultimately results in the expression of Ab molecules on the surface of immature B cells. The ordered rearrangement of HC genes before LC genes depends on differentially regulated accessibility of the two gene loci to the RAG proteins. Thus, recombination at the Ig locus is blocked in pro-B cells during HC recombination and is later activated by increased accessibility of this locus in pre-B cells.

Changes in nuclear organization accompany Ig recombination and play a role in the control of recombination by positioning alleles in either permissive or repressive chromatin environments (3, 4, 5). In early lymphoid progenitor cells, both Ig alleles are in an extended configuration and are associated with the nuclear periphery (4), an environment which is presumed to be repressive (6). Progression to the pro-B cell stage and expression of Pax5 results in a relocation of both Ig alleles away from the periphery into a central nuclear location (3, 7). In pre-B cells, Ig alleles undergo contraction, mediated by looping, in preparation for recombination (5), and the majority of cells have one allele associated with repressive centromeric heterochromatic regions (3, 5). The timing of Ig recombination in the context of Ig locus contraction and allelic repositioning to centromeric heterochromatin is unknown.

The importance of chromatin context for regulating V(D)J recombination has suggested that the preferential susceptibility of specific V genes to recombination might be explained by differential chromatin modifications at individual germline V genes. Indeed, several studies have demonstrated a correlation between hyperacetylation of histones H3 and H4 at individual IgH and TCR V genes and their recombination potential (8, 9, 10). In temperature-sensitive (ts), virally transformed pre-B cells, chemical blockade of histone deacetylases resulted in increases in total VJ recombination, suggesting that acetylation of histones is an important mechanism in the global control of recombination at the Ig locus (11). However, at the level of individual V genes, it is not known whether enrichments in histone modifications influence the frequency with which they recombine.

Several investigators (3, 12) have examined the chromatin histone modifications of several locations in the Ig locus between J4 and the E3'. A chromatin immunoprecipitation (ChIP) analysis in a ts Abelson-transformed pre-B cell line demonstrated that activation of the Ig locus was associated with increased H3K4 methylation over the region of the Ig germline transcript on the recombining allele (12). Other experiments (3) showed increased enrichments of AcH3 and H3K4 methylation on the allele that was not associated with heterochromatin in developing B cells. These studies examined changes in histone modifications within a limited region of the 3.2-megabase (Mb) Ig locus in the context of B cell development, but they did not assess the temporal relation of these changes to germline transcription or the appearance of recombination products.

To further define changes in nuclear positioning and locus accessibility with respect to developmental events that occur at the Ig locus, we used an inducible murine ts Abelson-transformed pre-B cell line and ex vivo-cultured murine pre-B cells in experiments designed to determine the time course of nuclear positioning, changes in locus-wide DNase I sensitivity, histone modifications, recombination, and germline transcription at the Ig locus. Our analysis spans the entire 3.2-Mb locus and includes 13 sites that encompass 4 V genes, a putative pre/pro-B silencer element named silencer-intervening sequence (Sis) (13), a germline transcript initiation site, the intronic and 3' enhancers, C, and the recombining sequence (RS).

	Materials and Methods

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Cells and cell lines

103/BclX7 (hereafter referred to as 103X7), was a gift from Drs. J. Curry and M. Schlissel (14). It is a ts Abl-MuLV-transformed pre-B cell line that has been stably transfected with the Bcl-x expression construct. Germline transcription and rearrangement were induced in these cells by a shift in growth temperature from 34 to 39°C. Cells were expanded at 34°C, shifted to 39°C, and analyzed at 0, 2, 4, 6, 12, 24, 36, and 48 h. M1 myeloblast cells (ATCC TIB 192) and WEHI 7.1 T lymphoma cells (ATCC TIB 53) were grown at 37°C. ⁺ B cells were isolated from BALB/c spleen by flow cytometry. Ex vivo pre-B cells were obtained by culturing magnetically selected BALB/c CD19⁺ bone marrow cells on an OP-9 stromal cell layer in the presence of 1 ng/ml IL-7 (PeproTech) for 9 days. Bone marrow cells were obtained under a protocol approved by the Institutional Animal Care and Use Committee. The OP-9 cells were a gift from Dr. J. C. Zuniga-Pflucker from the University of Toronto (Toronto, Canada). After expansion, ex vivo pre-B cells were examined by FACS analysis for the surface expression of CD19 (FITC clone 1D3; BD Pharmingen), B220 (PE clone A3/6B2; BD Pharmingen), BP-1 (PE clone BP-1; BD Pharmingen), CD25 (PE or allophycocyanin clone PC61; BD Pharmingen), and IgM (PE clone DS-1, allophycocyanin clone 11/41; BD Pharmingen).

RT-PCR for Rag1, Rag2, 5, Vpre-B, -actin, and ° (germline C) transcripts

RNA was isolated from 103X7 cells using the TRIzol procedure (Invitrogen Life Technologies) and digested with DNase I (Stratagene) before cDNA synthesis to remove potentially contaminating genomic DNA. cDNA was synthesized by the Omniscript procedure (Qiagen) with 2 µg of total RNA and oligo(dT) primer. One-tenth (2 µl) of each reverse transcriptase (RT) reaction was utilized in PCR to detect the presence of rag1, rag2, 5, Vpre-B, -actin, and ° (germline) transcripts. PCR were performed in 100-µl reaction volumes containing 1.5 mM MgCl₂, 0.05 mM dNTPs, 50 pmol of each primer, and 2.5 units of AmpliTaq (Applied Biosystems). For rag1, Vpre-B, and 5, PCR conditions were 95°C for 4 min (95°C for 30 s/55°C for 30 s/72°C for 30 s x 30 cycles), 72°C for 10 min, and 4°C hold. For rag2, -actin, and ° transcripts, the cycling parameters were identical to those above except that the annealing temperature was 62°C. The RT-PCR primers used in this study have been previously described (15, 16) The primer sets for rag1, rag2, 5, and Vpre-B were designed to amplify across introns (15). In the case of ° transcripts, only spliced products (17) were detected. Thus, no-RT controls were not included. All products were of the expected size and there was no evidence of amplification products from genomic DNA. The primer sequences for all PCR, including those described in the following sections, and oligonucleotide probes are listed in the Table I.

VJ and RS recombination PCR

Genomic DNA was obtained from 1 to 2 x 10⁷ 103X7 cells, BALB/c spleen, WEHI 7.1 T lymphoma cells, and ex vivo pre-B cells with a genomic DNA isolation kit (Amersham Biosciences).

To assess LC rearrangements on a broad scale, PCR were prepared with primers (VJU and VJD) designed to amplify many VJ LC rearrangements (18, 19). Triplicate reactions were prepared with 100 ng of genomic DNA per reaction. Cycling conditions were 95°C for 4 min (95°C for 30 s/63°C for 30 s/72°C for 90 s x 30 cycles), 72°C for 10 min, and 4°C hold. Triplicate reactions were pooled, precipitated, and resuspended in 30 µl of distilled H₂O. After electrophoresis and transfer to nylon membranes, VJ recombination PCR products (VJU/VJD) in 103X7, BALB/c spleen, and WEHI 7.1 were detected with a ³²P-labeled HindIII-XbaI fragment from plasmid pcJ, which contains germline J genes. Membranes were washed two times for 10 min at 65°C in 2x SSC/0.1% SDS followed by three times for 15 min at 65°C in 0.2x SSC/0.1% SDS.

The individual V genes chosen for analysis roughly reside at the 5' end (hf24), central (gn33 and 12-38), and 3' end (21-3) of the locus. According to the nomenclature described in Ref. 20 , these genes are also named 24-140, 33–85, 12-38, and 21-3 and are the 140th, 85th, 38th, and 3rd V genes respectively, 5' of J1. To determine the frequency of hf24, gn33, 12-38, and 21-3 V gene recombination in 103X7 cells after 24 h in culture at 39°C and in ex vivo pre-B cells, we used PCR with100 ng of genomic DNA templates and the VJU2/J2SP primer set. The VJU2 primer was adapted from the VJU primer (18) to match the sequence of hf24, gn33, 12-38, and 21-3 in framework 3 and eliminate the preferential amplification of any individual gene. The J2SP primer binds 3' of J2. PCRs were performed as described above for assessment of global VJ recombination, with the exception that the annealing temperature of the reaction was decreased to 60°C. PCR products were cloned and bacterial colonies were lifted to nylon membranes. Membranes were blotted with the pcJ probe to obtain the total number of J-positive colonies and then blotted sequentially with ³²P-labeled V gene-specific oligonucleotide probes using wash temperatures for each probe that ensured unique binding to the sequences of hf24, gn33, 12-38, and 21-3. Washes (2x SSC/0.1% SDS) consisted of two room temperature washes followed by three 15-min washes at 42°C (hf24), 60°C (gn33), 45°C (12-38), and 72°C (21-3). Wash temperatures were determined by blotting positive control clones for each V gene. Membranes were stripped between probes by incubation at 45°C in 0.4 N NaOH for 30 min followed by two 10-min washes at room temperature in 200 mM Tris (pH 8.0)/0.1% SDS/0.1x SSC. Positive colonies from each oligonucleotide blotting were confirmed by sequencing.

VJ-intron RS recombination products (21) were detected using nested PCR. The primary reactions contained 200 ng of genomic DNA and utilized the external 5' rsC primer (21) and the 3' rsD primer. One microliter (1%) of the primary reaction product was used as template in the secondary PCR with the internal 5' rsC-nested and 3' rsD-nested primer set. Cycling conditions for both PCRs were as follows: 95°C for 4 min, (95°C for 30 s/55°C for 30 s/72°C for 45 s x 30 cycles), 72°C for 10 min, and 4°C hold.

V sterile transcripts

V sterile transcripts from 103X7 cells and ex vivo pre-B cells for the hf24, gn33, 12-38, and 21-3 V germline genes were detected using SYBR green real-time PCR. The 5' and 3' primers were specific for each gene. PCR products were sequenced to confirm the specificity of the reactions. The 3' primers bound downstream of the recombination signaling sequence (RSS). One-tenth of each RT reaction, the no-RT control, and 1, 5, 10, 20, and 50 ng of 103X7 genomic DNA (from cells grown at 34°C) or distilled H₂O were used as templates in 50-µl reactions containing 1x AmpliTaq Gold buffer, 12.5 pmol each of primer, 0.05 mM dNTPs, 1.5 mM MgCl₂, SYBR green (Molecular Probes), and 1.25 units of AmpliTaq Gold. Reactions were run on an Applied Biosystems Prism 7900 HT machine using 50°C for 2 min, 95°C for 10 min (95°C for 15 s/67°C for 30 s/72°C for 30 s x 45 cycles). A standard curve for each V gene-specific primer pair was constructed using the threshold cycle (Ct) values of the genomic DNA standard reactions. Values for each cDNA sample relative to genomic DNA were calculated by interpolation of the Ct values into the genomic DNA standard curve.

Three-dimensional (3D) DNA fluorescence in situ hybridization (FISH)

Probes were prepared from bacterial artificial chromosomes (BAC) purchased from BACPAC (http://bacpac.chori.org). RP23-101G13, RP23-49M20, and RP23-139M1 correspond to the 5', middle, and 3' areas of the V region of the locus. The probe used for gamma satellite DNA and all probes were labeled as previously described (5). 103X7 cells were grown for 0, 6, 12, 24, or 30 h at the nonpermissive temperature and fixed onto poly-L-lysine slides for three-color, 3D DNA FISH as described elsewhere (22, 23, 24).

DNase I accessibility

103X7 cells, cultured at 39°C for 0, 2, 12, and 24 h, were lysed and nuclei were isolated (25) before digestion with 5 units and 1 unit of DNase I (Roche). Accessibility of the Ig locus to DNase I digestion was measured by analytical real-time PCR amplification of various amplicons in the locus, in a technique known as chromatin accessibility real-time PCR (26). In this assay, the degree of accessibility, as measured by the amount of digestion, was inversely proportional to the amount of PCR product generated. Standard curves were generated for each primer set using Ct values from non-DNase I-digested 103X7 cell genomic DNA (400, 200, 100, 50, 25, and 10 ng/reaction) from cells maintained at 34°C. The DNA concentration for each DNase I-digested sample was calculated by interpolation into the standard curve. Relative percent accessibility was calculated by comparing the DNA concentration of the DNase I-treated samples generated from DNA isolated at 2, 12, and 24 h with the value of the DNase I-digested sample at 34°C (0 h). Three to five replicate PCRs were performed for each amplicon at each time point and DNase I (5 units or 1 unit) concentration. The primer sets and cycling conditions for these experiments were the same as those used in ChIP real-time PCR (see below), except that SYBR green was used to monitor amplification instead of amplicon-specific probes. A primer set derived from the caveolin gene, which resides outside of the locus and is not expressed in B lymphocytes (27), was used as a control.

ChIP

Mono- and dinucleosomes were prepared from 2 x 10⁸ cells and ChIP were performed as described previously (28). M1 cells were used as controls. Abs used were anti-AcH3 (no. 06-599), anti-H3K4me2 (no. 07-030), anti-H3K4me3 (no. 07-473), and anti-H3K79me2 (no. 07-366) (Upstate Biotechnology). DNA content in each fraction was calculated with picogreen (Molecular Probes).

Real-time PCR for histone modification enrichments

PCR volumes were 50 µl and consisted of 1x AmpliTaq Gold buffer, 1.5 mM MgCl₂, 0.05 mM dNTPs, 12.5 pmol of each primer, 7.7 pmol of FAM-TAMRA-labeled probe, 1.25 units of AmpliTaq Gold, and 2 ng of template DNA. The probe and primer sets were specific for each of the V genes. Reactions were performed in an Applied Biosystems 7900HT using 96-well optical plates with optical plate sealers. Cycling conditions for all primer/probe sets were 50°C for 2 min, 95°C for 10 min, and 95°C for 15 s/55°C for 30 s/72°C for 30 s x 45 cycles. PCRs were performed in duplicate or triplicate (depending on yield) from nucleosomes derived from two independent time courses (103X7 cells) and from nucleosomes derived from ex vivo pre-B cells (pool of four mice). The relative enrichment of target DNA in immunoprecipitated fractions vs input DNA was calculated by using the formula 2^{(Ct input – Ct ChIP DNA)} (29).

RS information content score

To estimate the impact of sequence differences in the RSS on recombination, we used a mathematical model (30) that estimates recombination potential of a RSS, which is reported as RS information content scores (RIC). The four V genes (hf24, gn33, 12-38, and 21-3) have consensus heptamer sequences and hf24 and 21-3 have consensus nonamer sequences. gn33 and 12-38 have identical nonamers that differ from consensus sequences at the fourth position and the spacer for each V gene each was unique. Higher RIC are associated with higher recombination potential. The RIC for the V genes we studied were similar (hf24 = –9.53, gn33 = –12.04, V12–38 = –11.12, V21-3 = –14.29).

	Results

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Differentiation events in 103X7 and pre-B cells

To study the steps associated with initiation of Ig recombination in pre-B cells, we used a ts Abl-MuLV transformed pre-B cell line, 103/BclX7 (14). In such cell lines, which have been used by several groups as models for pre-B cell differentiation (12, 31, 32, 33), v-Abl expression arrests development at a stage similar to an early pre-B cell in which V(D)J recombination has assembled a µ HC but LC rearrangement has not begun. Inhibition of v-Abl activity by culture at an elevated temperature (39°C) induces Ig germline transcription and recombination, as well as other events that are characteristic of pre-B cell development (31, 33, 34). To correlate the changes in Ig locus accessibility with differentiation events in 103X7 cells, we verified the timing of transcription and recombination events after temperature shift. RT-PCR revealed that rag1 and rag2 transcripts increased within 2 h of temperature shift, while transcripts for the components of the surrogate LC, 5, and Vpre-B were present before the temperature shift (Fig. 1A).

View larger version (66K): [in this window] [in a new window]   FIGURE 1. Transcription and recombination in 103X7 cells and ex vivo pre-B cells. Ethidium bromide gel images in A, B, and D were inverted and levels adjusted in Adobe Photoshop CS for clarity. A, rag1, rag2, 5, Vpre-B, and -actin RT-PCR from 103X7 cells and WEHI 7.1 (T) lymphoma T cells. B, RT-PCR of germline transcripts from 103X7 cells and control T cells. C, VJ rearrangements were detected by PCR from 103X7, BALB/c spleen, and T cell genomic DNA followed by Southern blotting with a 32P-labeled pcJ HindIII-XbaI fragment containing germline J genes. D, Inverted gel image of RS recombination products detected by nested PCR. + = DNA from +-sorted B cells obtained from BALB/c spleen. E, V germline transcription as detected by real-time PCR. Values are expressed in nanograms relative to a genomic DNA standard curve. F, Individual V gene recombination frequency. Frequency of each V gene is expressed as percentage of total J-positive colonies.

VJ rearrangements were assessed by PCR using a degenerate 5' primer that binds to the framework 3 segment of many different V genes and a 3' primer in J4. In 103X7 cells, very low levels of VJ recombination were observed between 0 and 12 h, but recombination events increased dramatically between 12 and 24 h (Fig. 1C). At 36 h, the level of recombination was indistinguishable from the level observed in BALB/c spleen DNA. As expected, ex vivo-cultured pre-B cells also exhibited VJ rearrangements (data not shown). Using nested PCR, we also observed RS recombination events in both 103X7 cells 24 h after temperature shift and in ⁺ cells isolated from BALB/c spleen (Fig. 1D).

FACS analysis of 103X7 cells revealed that CD25 was expressed on 12.4% of cells at 0 h, increasing to 70% of cells at 24 h (data not shown). Together, the differentiation events that occurred in 103X7 cells were consistent with events that occur in developing pre-B cells and provided a kinetic profile of events we could compare with nuclear positioning and locus accessibility changes. FACS analysis of the ex vivo pre-B cells revealed that after 9 days in culture, 97% of the cells were CD19⁺B220⁺BP1⁺IgM^– and, of these, 41% expressed CD25 (data not shown). CD25⁺BP1⁺ pre-B cells represent both early (fraction C') and late (fraction D) pre-B cells (2). LC recombination occurs in late pre-B cells.

V gene germline transcription and recombination frequency

Using real-time PCR of cDNA, we measured germline V transcripts from four V genes during the course of differentiation in 103X7 cells and in ex vivo pre-B cells (Fig. 1E). These genes were chosen to sample V genes across the locus; however, they may not be representative of all genes in their vicinity. In both 103X7 cells and ex vivo pre-B cells, substantial levels of V germline transcripts were found for hf24 (5' end of the locus) and 21-3 (3' end of the locus), even at time 0 in 103X7 cells, while transcripts from the more central V genes (gn33 and 12-38) were barely detectable at any time (Fig. 1E).

Because differences in chromatin structure are likely to predict the frequency of recombination of individual V genes, we assessed the recombination frequency of the hf24, gn33, 12-38, and 21-3 V genes in 103X7 cells after 24 h of growth at the elevated temperature and in ex vivo pre-B cells (Fig. 1F). We estimated the recombination frequency of the four V genes by cloning VJ PCR amplification products and hybridizing the clones with gene-specific oligonucleotide probes. The results are presented as a percentage of all J-positive colonies. In 103X7 cells, the highest recombination frequencies were observed for hf24 and 21-3, which were 5- to 6-fold greater than those for gn33 and 10- to12-fold greater than 12-38. In ex vivo pre-B cells, hf24 was the most frequently recombining of these V genes, followed by gn33, 21-3, and 12-38. It was not possible to design a probe capable of differentiating between gn33 and gm33; therefore, both members of this family are represented.

The differences in V recombination frequency among hf24, gn33, 12-38, and 21-3 are not likely to be due to sequence differences at the RSS. The four V genes (hf24, gn33, 12-38, and 21-3) have consensus heptamer sequences and hf24 and 21-3 have consensus nonamer sequences. gn33 and 12-38 have identical nonamers that differ from consensus sequences at the fourth position and the spacer for each V gene was unique. Analysis of the RSS for these four V genes with a mathematical model that estimates recombination potential (30) revealed no significant differences.

Nuclear organization of Ig alleles

The Ig locus undergoes nuclear repositioning and contraction during the course of B cell development, and there is evidence that movement between repressive and accessible compartments within the nucleus is involved in regulating monoallelic recombination (3, 4, 5). Contraction of the Ig locus is thought to be important for enabling synapse formation of gene segments separated by large distances (3, 4, 5).

In non-B cells and multipotential hematopoetic progenitor cells, Ig alleles are located at the nuclear periphery and are associated with the nuclear lamina (4). In pro-B cells, both Ig alleles are in an extended conformation and are located predominantly in a central nuclear position away from heterochromatin (3, 4). At the pre-B cell stage, both Ig alleles undergo contraction and one allele is repositioned to centromeric heterochromatin located near the nuclear periphery (3, 4, 5).

To determine the order of events at the Ig locus in 103X7 cells, we assessed nuclear positioning and locus contraction over the course of 36 h of culture at the elevated temperature. Nuclear positioning of the Ig locus was examined using 3D DNA FISH with BAC probes for the 5', middle, and 3' V gene regions of the locus (Fig. 2). Recruitment of one Ig allele to heterochromatin was initially low (35%) between 0 and 6 h, but more than doubled to 75% at 12 h and remained high (80%) through 30 h (Fig. 2C). These results provide new information about the order in which these events occur and show that recruitment to heterochromatin takes place before the appearance of detectable VJ recombination events at 24 h (Fig. 1C).

View larger version (61K): [in this window] [in a new window]   FIGURE 2. Centromeric recruitment of single Ig alleles in 103X7 cells. A, Cells were grown for the indicated times at 39°C and subjected to 3D DNA FISH analysis using BAC probes specific for the 5' end (red) and 3' end (blue) of the V gene segment of the locus and gamma satellite DNA (green) for heterochromatin. The cells were analyzed using a Leica SP2 AOBS (acoustical optical beam splitter) confocal microscope. Single optical sections show the position of each allele within the nucleus. B, Ig locus (not drawn to scale) indicating positions of BAC probes. C, Percentage of cells with one Ig allele positioned at centromeric heterochromatin. n = 31 for 0 h and n = 50 for all additional time points. The star above the bar at 24 h indicates the time point at which VJ recombination products were detectable.

View larger version (47K): [in this window] [in a new window]   FIGURE 3. Ig locus contraction during differentiation. A, Ig locus (not drawn to scale) indicating positions and linear order of BAC probes. B, 103X7 cells were grown for the indicated times at 39°C and subjected to 3D DNA FISH with BAC probes specific for the 5' (red), middle (green), and 3' (blue) segments of the V gene region of the locus. A single confocal section showing a looped/contracted locus is shown after 24 h at 39°C with a scheme depicting the looped order of gene segments. C, The physical distance separating the probes was measured in 100 cells for each time point. The percentage of cells with probes in close contact (0.3 µm), separate (0.5–1 µm), or far apart (1–1.5 µm) are shown by gray, white, and black bars, respectively.

Increases in DNase I sensitivity and alterations in chromatin structure at the Ig locus have been reported to be associated with germline transcription and recombination (35, 36, 37, 38). We measured accessibility of the Ig locus to DNase I during induction of 103X7 cells by real-time PCR using primers for 13 amplicons spanning the Ig locus (Fig. 4). Caveolin, which is not expressed in B lymphocytes (27), was examined and did not show an increase in DNase I sensitivity (data not shown). The V amplicons included the hf24, gn33, 12-38, and 21-3 genes. HS3, 4, 5, and 6 coincide with Sis, a region 6 kb downstream of V genes that negatively regulates Ig transcription and recombination events (13, 39). 02 is 5' of J1 near the 4.7- kb germline transcript promoter site and Ei, E3', C, and RS correspond to the intronic enhancer, 3' enhancer, constant region gene, and RS, respectively.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. DNase I accessibility of the locus. A, Accessibility to DNase I at different developmental time points was measured by chromatin accessibility real-time PCR. PCR amplicons are listed on the x-axis. Percent accessibility for each time point was calculated relative to 0 h DNase I-digested DNA. B, Ig locus (not drawn to scale). Arrows, Promoter sites for the 8.4- and 4.7-kb germline Ig transcripts. Circles, 2 h; squares, 12 h; triangles, 24 h.

Histone modifications at the Ig locus during pre-B cell differentiation

We next analyzed changes in chromatin structure at the level of histone modifications using ChIP and real-time PCR (Figs. 5 and 6). The histone modifications H3K4me2, AcH3, H3K4me3, and H3K79me2 were assessed in 103X7 cells and M1 myeloblast cells at the same amplicons examined in the DNase I experiments. AcH3 and H3K4me2 modifications were examined in ex vivo pre-B cells. M1 data are not shown.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Histone modifications at V genes in 103X7 cells (A) and ex vivo pre-B cells (B). 103X7 cells were grown for the indicated times at 39°C and assayed by ChIP real-time PCR using FAM-TAMRA-labeled probes. Ex vivo pre-B cells were harvested after 9 days in culture with OP-9 stromal cells and IL-7. Note scale differences in y-axes. C, Scheme of the Ig locus (not drawn to scale). Arrows, Promoter sites for the 8.4- and 4.7-kb germline Ig transcripts.

View larger version (41K): [in this window] [in a new window]   FIGURE 6. A, Histone modifications in the intervening sequence between V and J in 103X7 cells and ex vivo pre-B cells. B, Histone modifications from the Ei through RS in 103X7 cells and ex vivo pre-B cells. C, Scheme of the Ig locus (not drawn to scale). Arrows, Promoter sites for the 8.4- and 4.7-kb germline Ig transcripts.

Although we observed minimal changes in histone modifications of the V genes in 103X7 cells, increases in histone modifications were seen in three of the four Sis amplicons, HS4–6 (Fig. 6A). The largest response to temperature shift was in HS6, which showed a 4-fold increase in both AcH3 and H3K4me2; HS4 and HS5 showed smaller responses in AcH3 and almost no response in H3K4me2. The AcH3 profile at all four Sis amplicons in ex vivo pre-B cells was virtually indistinguishable from that of 103X7 cells cultured for 24 h at elevated temperatures (Fig. 6A).

In 103X7 cells, the 02 and C amplicons both showed a substantial response to temperature shift, especially with increases in H3K4me2 and AcH3 modifications at 24 h (Fig. 6B). Additionally, 02 showed an increase in H3K4me3 at 24 h. Before induction, two enhancers, Ei and E3', showed significant enrichment of H3K4me2 and comparatively lower levels of AcH3 (Fig. 6B). Enrichments in both modifications increased between 6 and 24 h at Ei and between 12 and 24 h at E3'. The RS amplicon, which is located 25 kb 3' of C, showed a 3-fold increase in AcH3 at 24 h, which coincided with the appearance of RS recombination products. In ex vivo pre-B cells, the overall enrichments levels of AcH3 and H3K4me2 between 02 and RS were elevated compared with 103X7 cells at 24 h postinduction, but the ratios of AcH3:H3K4me2 were very similar (Fig. 6B). In M1 cells, there were only barely detectable levels of histone modifications at all of the amplicons in the Ig locus (data not shown).

The 103X7 data demonstrate two patterns of specific histone modifications evident at the Ig locus. The first pattern consisted of enrichment of histone modifications at V genes and the enhancers that occurred prior to nuclear repositioning events and the onset of recombination. The second pattern consisted of increased histone modification enrichment that occurred during the course of differentiation. The similarity in the overall patterns of histone modification enrichments observed between ex vivo-cultured pre-B cells and 103X7 cells at 24 h supports the use of 103X7 cells as a model for the examination of epigenetic changes at the Ig locus during pre-B cell differentiation.

	Discussion

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We examined the temporal association of nuclear positioning events and chromatin structural changes at the Ig locus with transcription and recombination in a temperature-inducible pre-B cell line. For comparison, histone modifications, V gene recombination, and V gene germline transcription were analyzed in ex vivo-cultured pre-B cells, establishing 103X7 cells as a relevant model for normal events during pre-B cell differentiation.

As defined by histone modifications, we demonstrated that the V region and two enhancers (Ei and E3') within the Ig locus are in an accessible conformation in 103X7 cells before nuclear repositioning of one Ig allele to the repressive centromeric heterochromatin environment and the onset of recombination. Additionally, we have shown for the first time that nuclear repositioning of single Ig alleles to centromeric heterochromatin occurs before VJ recombination. Furthermore, our analysis suggests a correlation between H3 acetylation and germline V transcription and between H3 methylation and VJ recombination. The enrichment of histone modifications at the V region before the onset of recombination is in contrast with the enrichment of histone modifications coincident with recombination at J (12) and RS. Overall, the distinct patterns of histone modifications during differentiation and the gradual, locus-wide shift in DNase I sensitivity indicate that there is a differential control of accessibility across the 3-Mb locus.

The Ig alleles in 103X7 cells are in a contracted form at 0 h, before recombination and recruitment of single alleles to centromeric heterochromatin. This is consistent with previous findings demonstrating that Ig alleles can be found in a contracted state early in development (5). Locus contraction of the IgH locus has been shown to be Pax5 dependent (5, 22) and, additionally, Pax5 has been shown to induce relocation of the Ig locus from the periphery to the center of the nucleus (7), but the factor(s) required for initiation of contraction/looping at the Ig locus are unknown. Sequences in or near distinct V genes may act as preferred interaction sites for the enhancers and/or transcription factors and enrichment of H3K4me2 and AcH3 at the enhancers, and these V regions early in development could facilitate long distance looping interactions. If histone modifications are involved in locus contraction, then it would be expected that the enrichments in histone acetylation and methylation we observed before recombination and recruitment occurred on both alleles before targeting of one allele to heterochromatin. This is in contrast to the subsequent enrichment of histone modifications we observed across the locus in response to temperature shift, which likely result from the nonheterochromatin-associated or recombining allele, since active histone modifications along the transcription unit have been shown to be enriched at these alleles (3, 12).

Acetylation of histones has been correlated with either an increase or a bias in V gene rearrangements at other immune receptor loci (8, 9, 10, 11, 40, 41), but aside from histone acetylation, little information is available regarding the relationship among alternative histone modifications, germline transcription, and recombination of V genes.

Enrichments of histone modifications at the V genes we studied were present before temperature shift and locus wide changes in DNase I accessibility. The only previous study to examine alternative histone modifications of V genes used a similar ts v-Abl-transformed pre-B cell line and examined H3K4me2 and H3K4me3 enrichment of a V4 gene near the middle of the locus and 21-1, the most 3' V gene (12). H3K4me2 and H3K4me3 enrichment at V4 and 21-1 were shown to be low both before and after induction of recombination, but transcription and recombination of these genes was not assessed. These findings could be interpreted to suggest that H3K4me2 and H3K4me3 modifications are not important for RAG accessibility. In contrast, our positive correlation between H3K4me2 enrichment and recombination frequency in both the inducible cell line and ex vivo pre-B cells suggests that this modification may explain some of the variance between V genes in a native chromatin environment and their accessibility to RAG proteins. In addition, we observed increases in both AcH3 and H3K4me2 enrichments at the RS element 24 h after induction, which correlated with the appearance of RS recombination products. Our results suggest that in addition to histone acetylation, H3K4me2 enrichments may facilitate access for the recombination machinery or interaction of the enhancers with the promoters of V genes, resulting in elevated levels of both transcription and recombination.

In contrast to the V genes, increases in histone modification, principally H3 acetylation, were induced by temperature shift in three of four amplicons in Sis. Our observation of histone modifications that are tied to the timing of germline transcription and VJ recombination suggests some role for the silencer related to Ig locus activation. Sis was originally shown to be involved in pre- and pro-B cell-specific transcriptional silencing in transient transfection assays (13). Additionally, deletion of Sis from Ig minilocus transgenes was shown to increase transgene VJ joining, reduce levels of transgene germline transcripts, and target transgenes to centromeric heterochromatin (39). Additional experiments will be necessary to determine which of the Sis-mediated functions are associated with alterations in histone modifications.

Chromatin structural changes have been shown to both precede transcription and occur concurrently with its onset (12, 42) and our data are in agreement with these findings. However, transcription as a mechanism for the active recruitment of histone modification enzymes cannot explain the 12-h delay in histone modification at 02 relative to two downstream elements in the same transcription unit. The KI-KII site, which lies just upstream of J1 and can bind Pax5, has been shown to be important in the regulation of recombination efficiency but not germline transcription (43, 44). The 02 amplicon contains the KI sequence of the KI-KII complex and the timing of modifications to histones in this region that coincide precisely with significant increases in VJ recombination products may reflect its importance for this function.

This work has provided new insight into the temporal order of chromatin structural changes and nuclear positioning events that occur at the locus during pre-B cell development and has shown that accessibility of the locus is regulated in a segmented fashion. The factor(s) responsible for mediating locus contraction and the mechanism of allelic selection for centromeric recruitment remain unknown, but our work raises the possibility that individual V genes enriched in histone modifications may be sites that are involved in locus contraction. The correlation between specific histone modifications and germline transcription and/or recombination of individual V genes is intriguing and raises the question as to whether specific histone modifications influence a differential accessibility of RAG proteins to RSS.

	Acknowledgments

 
We thank Drs. John Curry and Mark Schlissel from the University of California, Berkeley, for providing the 103/BclX7 cell line used in this study. We also thank Dr. Francis Flomerfelt of the Experimental Immunology Branch, National Institutes of Health (Bethesda, MD) and Dr. Juan Carlos Zuniga-Pflucker of the University of Toronto for supplying the OP-9 stromal cell line. We are grateful to Drs. Mark Schlissel, Mate Tolnay, and Frederick Mills for critical review of this manuscript and Kathleen Clark for help with bone marrow preparation and FACS analysis.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Intramural Research Program of the Center for Drug Research and Review, Food and Drug Administration. J.S. was supported by a Wellcome Trust Project Grant.

² Opinions expressed in this publication reflect the professional views of the authors and should not be viewed as official policy of the U.S. Food and Drug Administration or the government of the United States.

³ Current address: Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016.

⁴ Address correspondence and reprint requests to Dr. Marjorie Shapiro, Division of Monoclonal Antibodies, U.S. Food and Drug Administration, Center for Drug Evaluation and Research, 5600 Fishers Lane, Rockville, MD 20857. E-mail address: marjorie.shapiro{at}fda.hhs.gov

⁵ Abbreviations used in this paper: HC, H chain; LC, L chain; ts, temperature sensitive; ChIP, chromatin immunoprecipitation; Mb, megabase; Sis, silencer intervening sequence; RS, recombining sequence; FISH, fluorescence in situ hybridization; RSS, recombination signal sequence; RIC, RS information content score; Ct, threshold cycle; BAC, bacterial artificial chromosome.

Received for publication April 26, 2007. Accepted for publication August 8, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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