Molecular Visualization of Immunoglobulin Switch Region RNA/DNA Complex by Atomic Force Microscope*

Ryushin MizutaDagger §, Kousuke Iwai, Masatsugu Shigeno||, Midori MizutaDagger , Takeshi UemuraDagger , Tatsuo Ushiki, and Daisuke KitamuraDagger **

From the Dagger  Research Institute for Biological Sciences and the ** Genome and Drug Research Center, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan, the  Department of Anatomy and Histology, Faculty of Medicine, Niigata University, Asahimachi-dori, Niigata 951-8510, Japan, and || Seiko Instruments, Matsudo, Chiba 270-2222, Japan

Received for publication, September 10, 2002, and in revised form, December 2, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two subsequent DNA recombination steps are involved in immunoglobulin (Ig) production (1-3). First, V(D)J recombination assembles the V, D, and J segments into a variable region exon and contributes to the diversification of antigen-binding sites. Second, class switch recombination (CSR)1 converts the constant (CH) region of the immunoglobulin heavy chain (IgH) from Cµ to other classes and alters physiological functions that differ in their properties of complement fixation, binding to class-specific Fc receptors, and polymerization. Thus, class switching is a mechanism for diversifying Ig effector functions with the same antigen specificity.

The mouse IgH locus of the IgM-expressing B lymphocyte contains a rearranged V(D)J exon and 8 CH exons of the following organization: 5'-V(D)J-Cµ-Cdelta -Cgamma 3-Cgamma 1-Cgamma 2b-Cgamma 2a-Cepsilon -Calpha -3'. CSR is initiated by DNA breakage in the S-region featuring tandem repetitive nucleotide sequences that are located upstream of each CH exon except for the Cdelta exon. In the case of CSR from IgM to IgE, DNA breakage initially occurs in both Sµ and Sepsilon and results in deletion of the intervening region containing the Cµ to Cgamma 2a exons, and religation of Sµ and Sepsilon causes juxtaposition of the V(D)J exon and the Cepsilon exon. Despite the recent discovery of the activation-induced deaminase (AID) as an indispensable factor for CSR (2, 4), the molecular mechanism of CSR still remains elusive. In particular, the initiation step of DNA cleavage is completely unknown.

Previous studies have revealed that the generation of germline transcripts from the sites upstream of each S-region is a prerequisite for CSR, and the higher order DNA structure involving S-region RNAs that are spliced out from the primary transcripts has been proposed as a critical factor for CSR (1, 5-7). Moreover, biochemical studies, including an agarose gel analysis, suggested that a persistent RNA/DNA hybrid is formed between S-region transcripts and its template DNA (8-11). Thus, the S-region RNA may be catalytically involved in DNA breakage and CSR, and the characterization of the RNA/DNA hybrid may be critical for the understanding of CSR. However, the precise structure and the physical characteristic of both the RNA/DNA hybrid and the S-region RNA are not yet fully established.

To directly determine the potential role and the physical characteristic of both the S-region transcript and the RNA/DNA hybrid at the single molecular level, we examined the S-region RNA transcribed in vitro using an atomic force microscope (AFM) (12). This approach revealed the specific characteristic of the nascent S-region transcript and the higher order structure of the RNA/DNA hybrid at the single molecular level.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid-- The pGD231 plasmid, a generous gift from M. Lieber (9), contains an immunoglobulin Sgamma 3-region DNA (2.2 kb) in a pBSKS vector (Stratagene).

In Vitro Transcription-- Standard reactions were carried out using a RiboProbe in vitro transcription system (Promega). Briefly, the pGD231 plasmid DNA (1 µg) was transcribed with 10 units of T3 or T7 RNA polymerase in 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl2, 10 mM dithiothreitol, 2 mM spermidine, 0.05% Tween 20, 20 units of recombinant ribonuclease inhibitor, and 0.5 mM each of rATP, rGTP, rCTP, and rUTP in a volume of 20 µl for 1 h at 37 °C. Reactions were stopped by heat inactivation for 20 min at 65 °C. Some samples were incubated with 1 µg of RNase A (Wako, Osaka, Japan) for an additional 30 min at 37 °C. Where indicated, 2 units of RNase H (Toyobo, Osaka, Japan) were added. Samples for AFM analysis were purified with MicroSpin S-400 HR columns (Amersham Biosciences). Unless otherwise specified, all samples were subjected to phenol/chloroform extraction and ethanol precipitation and suspended in TE buffer (10 mM Tris-HCl, pH 7.9, and 1 mM EDTA).

AFM Imaging-- Samples were diluted in 10 mM Tris-HCl (pH 7.9) and 1 mM NaCl to 1 ng/µl DNA concentration. 5 µl of each solution was deposited onto the center of a freshly cleaved mica disk. Just before deposition, the sample solution was supplied with MgCl2 to a final concentration of 2.5 mM. After 5 min, the mica was blown dry with compressed air. To remove salts on the mica surface, 10 µl of distilled water was dropped on the mica followed immediately by a quick blow for drying. This rinsing step was repeated twice. AFM studies were performed using the SPM-400 scanning probe microscope controlled by an SPI 3700 probe station (Seiko Instruments). This microscope was equipped with a piezo translator with maximum x-y scan range of 20 µm width and a z range of 1.2 µm. The cantilevers used were rectangular, with a force constant of 35-40 N m-1 and a resonance frequency of 320-400 kHz (SI-DF40, Seiko Instruments). Unless otherwise specified, all images were obtained as height mode images in a dynamic force mode in the air at room temperature. The resonance frequency shift was measured using the slope detection method at a slightly higher frequency of the cantilever than its resonance frequency.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Altered DNA Mobility upon in Vitro Transcription of Repetitive Sequence-- To confirm previous results (8-11), we examined the conformational change of the plasmid (pGD231, Fig. 1A) containing an immunoglobulin Sgamma 3-region DNA after inducing Sgamma 3-region transcription on either strand of the DNA. The guanine-rich (G-rich) sense RNA and the cytosine-rich (C-rich) antisense RNA were transcribed by T7 and T3 RNA polymerases, respectively. Thereafter, free RNA was digested with RNase A. Consistent with the previous report (9), almost all of the plasmids transcribed with T7 RNA polymerase migrated slowly in an electrophoretic gel as smears (Fig. 1B, lane 3), but those transcribed with T3 RNA polymerase migrated normally as supercoiled plasmids (Fig. 1B, lane 1). Treatment with RNase H, which specifically degrades the RNA in the RNA/DNA hybrid, restored those plasmids transcribed with T7 RNA polymerase to their original mobility (Fig. 1B, lane 4). These results suggest that the sense RNA, but not the antisense RNA transcribed from the Sgamma 3 region, forms the RNA/DNA hybrid and is responsible for the mobility shift. However, from results of this analysis, it was not sufficiently clear whether the mobility shift is caused by the conformational change of the plasmids or merely by the added molecular weight of the associated transcripts.


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Fig. 1.   Conformational change of plasmid DNA upon in vitro transcription of the mouse IgH repetitive switch sequence. A, diagram of the plasmid DNA (pGD231) containing Sgamma 3 sequence (2.2 kb). The long arrow and the bent arrows indicate the direction of physiological transcription in the IgH gene and the direction of transcription by T3 or T7 RNA polymerase, respectively. The Sgamma 3 sequence is indicated by a box. B, altered mobility of pGD231 on an agarose gel after in vitro transcription. Supercoiled pGD231 plasmids were transcribed by T3 (lanes 1 and 2) or T7 RNA polymerase (lanes 3 and 4), treated with RNase A (+, lanes 1-4) and/or RNase H (+, lanes 2 and 4), and run on an agarose gel. Untranscribed pGD231 plasmids and a 1-kb ladder (M) were run on lanes 5 and 6, respectively. Positions of supercoiled (SC) and open circle (OC) forms of the plasmids are indicated. C, AFM views of the conformational change of pGD231 after transcription by the T3 or T7 RNA polymerase. The samples examined above (B, lanes 1 and 3) were purified and visualized under AFM. Scale bars, 200 nm.

Conformational Change of Plasmid DNA upon in Vitro Transcription-- To visualize a possible conformational change of the pGD231 plasmid at single molecular resolution, the same samples used in the agarose gel analysis were subjected to AFM analysis after purification by phenol/chloroform extraction and ethanol precipitation. As clearly seen in Fig. 1C, plasmids treated with T3 RNA polymerase formed the supercoiled structure (left panel), but those treated with T7 RNA polymerase formed the relaxed open circle structure to various extents (right panel). These data clearly indicate that the sense RNA transcribed from the S-region forms a persistent hybrid with its substrate DNA and relaxes its conformation, which may represent a phenomenon known as chromatin opening. Chromatin opening in the S-region, despite the lack of direct evidence, is proposed as the initial step for the access of some endonucleases to this region in CSR (1, 3, 5).

Spermidine Stabilizes the RNA/DNA Hybrid-- In the course of the purification of the RNA/DNA hybrid, we found that a cation and some salt is indispensable for establishing and maintaining this hybrid. In our in vitro transcription, the standard buffer contains spermidine, a trivalent cation at normal cellular pH. Spermidine is one of the polyamines (putrescine, spermidine, and spermine) and is known to be critical for cell cycle progression and induction of nucleic acid and protein syntheses (13). Polyamines are also known to preferentially bind to the GC-rich region of RNA and to change its structure (13). We found that spermidine at a physiological concentration in a reaction significantly increases the extent of RNA/DNA hybrid formation (Fig. 2, top panel) without affecting total RNA production (bottom panel). Furthermore, the purified RNA/DNA complex was found to be unstable in water, and the addition of some salt increased its stability (data not shown). These results suggest that the formation and maintenance of the RNA/DNA hybrid is regulated by spermidine and some salt.


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Fig. 2.   Spermidine is required for RNA/DNA hybrid formation. Spermidine (SP) concentration was varied (0-2 mM) in the in vitro transcription reaction (lanes 1-4). Aliquots of samples before (bottom panel) and after RNase A treatment (top panel) were run on agarose gels and stained with ethidium bromide. A 1-kb ladder was used as a molecular weight marker (M).

RNA/DNA Hybrid Formation Proven by RNase H Binding-- Biochemical studies have suggested that the S-region RNA/DNA hybrid forms a structure called R-loop, a protrusion of a non-template DNA strand as a result of RNA/DNA hybridization (10, 11). The single-stranded region of the R-loop is supposed to serve as a substrate for some endonucleases that may cause DNA cleavage and subsequent recombination. To inspect the RNA/DNA hybrid without the helical stress from the double-stranded circular DNA, we digested the plasmid with SacI or KpnI, and then subjected the linearized plasmid to in vitro transcription and AFM analysis (Fig. 3A). However, we did not detect any visible alteration in structure, including an R-loop, by the transcription on either strand (Fig. 3B, top panels). Possibly, the R-loop collapsed and clamped onto the nascent RNA, forming an intramolecular triplex as suggested by Reaban et al. (10), and/or the R-loop is too small to be visualized using AFM. To detect RNA/DNA hybrid formation in a linear plasmid, we incubated the purified sample with RNase H. As mentioned above, RNase H specifically degrades the RNA in the RNA/DNA hybrid, and this catalytic activity requires Mg2+. Therefore, RNase H is supposed to maintain its association with the nascent RNA of the RNA/DNA hybrid in the absence of Mg2+. After incubation with RNase H without MgCl2, we detected some aggregation on each T7 (but not T3) RNA polymerase-treated DNA (Fig. 3B, bottom panels). Also, the apparent length of the linear DNA in the T7 RNA polymerase-treated sample became shorter, and the aggregation was often associated with DNA loop or branch formation. These data suggest that the sense S-region RNA actually forms a persistent hybrid with the template DNA and that the binding of RNase H to each hybrid facilitates the secondary structure formation.


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Fig. 3.   Evidence of RNA/DNA hybrid formation after in vitro transcription. A, diagram of the linear pGD231 plasmid after digestion by SacI or KpnI restriction enzymes. The long arrow and bent arrows indicate the direction of physiological transcription and the direction of transcription by T3 or T7 RNA polymerase, respectively. B, RNA/DNA hybrid formation probed by RNase H binding. Transcribed and RNase A-treated linear plasmids were incubated with (+, bottom panels) or without (-, top panels) RNase H and then purified with spin columns and examined under AFM. Scale bars, 200 nm.

Direct Visualization of S-region RNAs-- In the above experiments, we used RNase A-treated samples to eliminate the nonhybridized free RNA. We next sought to determine the physical properties of the free S-region RNA transcribed in the sense or antisense direction. Thus, the in vitro transcribed samples were subjected to AFM analysis without RNase A treatment (Fig. 4). We visualized RNA molecules and identified a clear difference between the sense and antisense RNAs. An elongated form of the antisense RNA, possibly forming an intramolecular secondary structure, was released from the substrate DNA (Fig. 4, left T3 panels). In contrast, the sense RNA formed a large aggregate (Fig. 4, right T7 panels). The RNA/DNA hybrid examined in Fig. 3 was supposed to serve as a core of this aggregate. The height profiles of the RNA, DNA, and RNA/DNA molecules are shown in the bottom panel of Fig. 4. The largest height of the right side T7 panel (~3.3 nm) was significantly greater than that of the left side T3 panel (~1.3 nm) or the height of the double-stranded DNA (~0.4 nm). The large aggregate of the RNA/DNA complex consisted of small globular molecules, which was evident from the phase mode image (Fig. 5, right panel). Interestingly, several DNA fragments were linked via this aggregate (Fig. 5), raising a possibility that the RNA aggregate functions as an anchor for synapsing two S-region DNAs. From these results, we propose that the S-region RNA, transcribed in the sense direction, forms a persistent hybrid with the template DNA, as well as a globular form, and forms a large complex with other RNA molecules to juxtapose the other S-region DNAs.


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Fig. 4.   Elongated form of the antisense RNA and the aggregated form of the sense RNA. Linear pGD231 plasmids were transcribed in vitro, purified without RNase A treatment, and visualized under AFM. Plasmids transcribed by T3 RNA polymerase (antisense orientation) or T7 RNA polymerase (sense orientation) are shown in the left and right panels, respectively, and enlarged images are shown in the middle panels. The height profiles, indicated as lines and arrows in the middle panels, are shown in the bottom panel. The dotted line at 0.4 nm indicates the height of a double-stranded DNA. Scale bars, 200 nm.


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Fig. 5.   Synapsed DNA fragments via a large aggregate composed of small globular sense RNA molecules. Left panel, height mode image. Right panel, phase mode image. Scale bars, 200 nm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an AFM. The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate.

The globular form and the large RNA/DNA complex formed by the sense S-region RNA are intriguing, because nucleic acids are negatively charged, and electric repulsion should have an adverse effect on the aggregation. Several possibilities might explain the aggregation. One possibility is that some cations can neutralize the negative charge. As mentioned earlier, spermidine, a trivalent cation in solution, is indispensable for the S-region RNA/DNA hybrid formation (Fig. 2), and the addition of salt also increases the stability of the purified RNA/DNA hybrid (data not shown). Thus, neutralization of a charge by a cation may be indispensable for the generation of the globular RNA or RNA/DNA complex. Another possibility is that the unique structure of the S-region might facilitate the aggregate formation. Two possible structures might be critical for this phenomenon. One is a four-stranded structure (G4-DNA) in which G-rich DNA strands are linked by a Hoogsteen-bonded guanine quartet (14, 15). G-rich RNA strands may be linked in the same manner and form G4-RNA and/or G4-RNA/DNA. The second is the stem-loop structure generated from palindromic sequences of the S-region, which has recently been proposed as a possibility because of its role in CSR (2, 16). Further analysis of the sequence requirement for the complex formation will verify these possibilities.

What is the physiological role of this RNA/DNA complex in CSR? We propose the following possibilities. 1) The RNA/DNA complex maintains the S-region of the IgH gene open, as shown in Fig. 1C, which facilitates the access of some endonucleases and CSR machinery. 2) This complex serves as an anchor for synapsing S-region RNA/DNA complexes, as shown in Fig. 4, which is a critical step in CSR. 3) This complex stalls the movement of the replication fork, which may also create a target for some endonucleases. It was reported previously that some repetitive sequences become highly recombinogenic by stalling the replication fork with their transcription and that a stable higher order structure of DNA and RNA, despite the lack of direct evidence, is the cause of the stalling (17). Our data directly showed that the RNA/DNA complex at repetitive sequences causes such a higher order structure, which may well explain the replication-coupled recombination of repetitive sequences. This is particularly relevant in CSR, because several reports have suggested the correlation between CSR and replication (5, 18) and the increased level of spermidine, a critical factor for the RNA/DNA hybrid, in cells entering replication (19).

Here, at the single-molecular level, we showed the unique physical characteristics of S-region transcripts that may be crucial for CSR. A similar intron-delivered repetitive RNA may be important in various aspects of cellular functions.

    ACKNOWLEDGEMENTS

We thank M. R. Lieber for the pGD231 plasmid and T. Kawai and H. Tanaka (the Institute of Scientific and Industrial Research (ISIR)-Sanken, Osaka University) and M. J. Shulman for discussions.

    FOOTNOTES

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

§ To whom correspondence should be addressed. Tel.: 81-4-7123-9883; Fax: 81-4-7124-1561; E-mail: mizuta@rs.noda.tus.ac.jp.

Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M209262200

    ABBREVIATIONS

The abbreviations used are: CSR, class switch recombination; CH, constant region of IgH; S-region, switch region; AFM, atomic force microscope.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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