Switch Junction Research Articles

Class Switch Recombination Efficiency and Junction Microhomology Patterns in Msh2-, Mlh1-, and Exo1-Deficient Mice Depend on the Presence of μ Switch Region Tandem Repeats

The Msh2 mismatch repair (MMR) protein is critical for class switch recombination (CSR) events that occur in mice that lack the Smu tandem repeat (SmuTR) region (SmuTR(-/-) mice). The pattern of microhomology among switch junction sites in Msh2-deficient mice is also dependent on the presence or absence of SmuTR sequences. It is not known whether these CSR effects reflect an individual function of Msh2 or the function of Msh2 within the MMR machinery. In the absence of the SmuTR sequences, Msh2 deficiency nearly ablates CSR. We now show that Mlh1 or Exo1 deficiencies also eliminate CSR in the absence of the SmuTR. Furthermore, in SmuTR(-/-) mice, deficiencies of Mlh1 or Exo1 result in increased switch junction microhomology as has also been seen with Msh2 deficiency. These results are consistent with a CSR model in which the MMR machinery is important in processing DNA nicks to produce double-stranded breaks, particularly in sequences where nicks are infrequent. We propose that double-stranded break paucity in MMR-deficient mice leads to increased use of an alternative joining pathway where microhomologies are important for CSR break ligation. Interestingly, when the SmuTR region is present, deficiency of Msh2 does not lead to the increased microhomology seen with Mlh1 or Exo1 deficiencies, suggesting that Msh2 might have an additional function in CSR. It is also possible that the inability to initiate MMR in the absence of Msh2 results in CSR junctions with less microhomology than joinings that occur when MMR is initiated but then proceeds abnormally due to Mlh1 or Exo1 deficiencies.

Genomic Instability Resulting from Blm Deficiency Compromises Development, Maintenance, and Function of the B Cell Lineage

The RecQ family helicase BLM is critically involved in the maintenance of genomic stability, and BLM mutation causes the heritable disorder Bloom's syndrome. Affected individuals suffer from a predisposition to a multitude of cancer types and an ill-defined immunodeficiency involving low serum Ab titers. To investigate its role in B cell biology, we inactivated murine Blm specifically in B lymphocytes in vivo. Numbers of developing B lymphoid cells in the bone marrow and mature B cells in the periphery were drastically reduced upon Blm inactivation. Of the major peripheral B cell subsets, B1a cells were most prominently affected. In the sera of Blm-deficient naive mice, concentrations of all Ig isotypes were low, particularly IgG3. Specific IgG Ab responses upon immunization were poor and mutant B cells exhibited a generally reduced Ab class switch capacity in vitro. We did not find evidence for a crucial role of Blm in the mechanism of class switch recombination. However, a modest shift toward microhomology-mediated switch junction formation was observed in Blm-deficient B cells. Finally, a cohort of p53-deficient, conditional Blm knockout mice revealed an increased propensity for B cell lymphoma development. Impaired cell cycle progression and survival as well as high rates of chromosomal structural abnormalities in mutant B cell blasts were identified as the basis for the observed effects. Collectively, our data highlight the importance of BLM-dependent genome surveillance for B cell immunity by ensuring proper development and function of the various B cell subsets while counteracting lymphomagenesis.

Analysis of clonotypic switch junctions reveals multiple myeloma originates from a single class switch event with ongoing mutation in the isotype-switched progeny

AbstractMultiple myeloma (MM) is a cancer of plasma cells (PCs) expressing immunoglobulin heavy chain (IgH) postswitch isotypes. The discovery of earlier stage cells related to postswitch PCs, called preswitch clonotypic IgM (cIgM) cells led to the hypothesis that cIgM cells may be MM progenitors, replenishing the tumor throughout malignancy. cIgM cells may do this by undergoing class switch recombination (CSR), a process detectable in postswitch PCs as multiple IgH switch junctions associated with a single clonotypic IgH V/D/J. We addressed this with a specific clonotypic-switch polymerase chain reaction (PCR), informative for 32 of 41 cases. Here we made 2 significant discoveries: (1) in all cases, we detected only a single clonotypic switch fragment that persists over time (1-7.6 years), and (2) we detected ongoing mutation upstream of the switch junction in 5 of 6 patients, often targeting the intronic enhancer, a key control region in IgH expression. The presence of a single, unchanging clonotypic switch junction suggests that cIgM cells are not MM-PC progenitors; rather, postswitch PCs arise from a single cIgM cell, and MM-PC progenitors reside in the postswitch population. Furthermore, mutations revealed here provide a new marker to identify MM-PC progenitors and aggressive clones that evolve throughout malignancy.

Structure and Consequences of IGH Switch Breakpoints in Burkitt Lymphoma

The t(8;14) MYC/IGH breakpoint is the hallmark translocation of human Burkitt lymphoma (BL). The translocation breakpoint most often involves the immunoglobulin heavy-chain switch regions and is thought to be brought about by an aberrant class switch recombination (CSR) event. During CSR in normal germinal center B cells, DNA double-stranded breaks are introduced in Smu and one of the downstream switch regions (Sgamma, Salpha, or Sepsilon) that are juxtaposed and ligated to form the switch junction, with deletion of the intervening DNA. In contrast, aberrant switch recombination in BL exclusively involves only one switch region, resulting in a perfect reciprocal translocation. A functional consequence of this type of translocation is that IgM expression from the chromosome affected by the translocation is not necessarily disrupted.

Zero-Voltage-Switching DC–DC Converters With Synchronous Rectifiers

Active resonant tank (ART) cells are proposed in this paper to achieve zero-voltage-switching (ZVS) and eliminate body-diode conduction in DC-DC converters with synchronous rectifiers (SRs). In low-output-voltage DC-DC converters, SRs are widely utilized to reduce rectifier conduction loss and improve converter efficiency. However, during switches' transition, SRs' parasitic body diodes unavoidably carry load current, which decreases conversion efficiency because voltage drop across body diodes is much higher than that across SRs. Moreover, body diodes' reverse recovery leads to increased switching losses and electromagnetic interference. With the proposed cells of an ART, the body diode conduction of the SR is eliminated during the switching transition from a SR to an active switch, and thus body diode reverse-recovery-related switching and ringing losses are saved. An ART cell consists of a LC resonant tank and an auxiliary switch. A resonant tank cell is charged in a resonant manner and energy is stored in the capacitor of the tank. Prior to a switching transition from a SR to an active switch, the energy stored in the tank capacitor is released and converted to inductor current, which forces the SR current changes direction to avoid conduction of the body diode and related reverse recovery when the SR turns off. Moreover, at the help of energy released from the ART, the active switch's junction capacitance is discharged, which allows the active switch turns on at ZVS. Since energy commutation occurs only during switching transition, conduction loss in the ART cell is limited. Moreover, the auxiliary switch turns off at ZVS and the SR operates at ZVS. The concept of ART cells is generally introduced and detailed analysis is presented based on a synchronous buck converter. Experimental results show the proposed ART cell improves conversion efficiency due to the reduced switching loss, body diodes' conduction, and reverse-recovery losses.

Perfectly matched layer implementation using bilinear transform for microwave device applications

This paper presents an extensive study on the perfectly matched layer (PML) implementation using bilinear transform in the finite-difference time-domain (FDTD) simulation. The bilinear transform is used to implement both the stretched coordinate PML (SC-PML) and the uniaxial PML (UPML) with the complex frequency-shifted (CFS) equations. It is shown that with the CFS factor, the implemented SC-PML and UPML attain significantly lower relative reflection error over wide frequency range with both superior in performance to the split-field PML. The FDTD algorithm incorporating these PMLs is applied to analyze wide-band responses of some complex microwave devices, including RF microelectromechanical systems switch and coplanar waveguide crossover junction.

Impact of DNA ligase IV on nonhomologous end joining pathways during class switch recombination in human cells

Class switch recombination (CSR) is a region-specific, transcriptionally regulated, nonhomologous recombinational process that is initiated by activation-induced cytidine deaminase (AID). The initial lesions in the switch (S) regions are subsequently processed and resolved, leading to recombination of the two targeted S regions. The mechanisms by which repair and ligation of the broken DNA ends occurs is still elusive. Recently, a small number of patients lacking DNA ligase IV, a critical component of the nonhomologous end joining (NHEJ) machinery, have been identified. We show that these patients display a considerably increased donor/acceptor homology at Sμ–Sα junctions compared with healthy controls. In contrast, Sμ–Sγ junctions show an increased frequency of insertions but no increase in junctional homology. These altered patterns of junctional resolution may be related to differences in the homology between the Sμ and the downstream isotype S regions, and could reflect different modes of switch junction resolution when NHEJ is impaired. These findings link DNA ligase IV, and thus NHEJ, to CSR.

VDJ-Switch Region Analysis in Multiple Myeloma Patients Reveals Homogeneity and Long-Term Stability of Switch Junctions, and Ongoing Mutation Upstream of Switch Mu.

During class switch recombination (CSR) B cells change their immunoglobulin heavy chain (IgH) constant region genes by a deletional recombination mechanism involving well characterized switch (S) regions several Kb downstream of IgH variable region (VDJ) sequences. Unlike VDJ recombination, the junction formed by CSR between pre-switch S-mu (IgM) and post-switch S-gamma (IgG) is imprecise, and the key factors mediating this process are not fully understood. In multiple myeloma, a bone marrow cancer characterized by post-switch plasma cells bearing a unique clonotypic VDJ, switch regions have been shown to mediate translocations in a subset of MM patients, suggesting a role in transformation. In this study we cloned and sequenced the VDJ-hybrid immunoglobulin switch (VDJ-S) regions of post-switch multiple myeloma (MM) cells at diagnosis and a second time point to determine if the switch junction was normal, homogeneous within a time point, and stable over the course of malignancy.The VDJ-S of five stage III IgG MM patients were examined in this study. Each patient has a specific clonotypic VDJ region and downstream hybrid switch junction (S-mu/S-gamma) arising after class switch recombination (CSR) from IgM to IgG. For each patient two samples were tested: a diagnosis sample and a second sample taken between 1.6 and 4.2 years later. The 5–7 Kb VDJ-S was amplified by long distance PCR using bulk DNA samples from bone marrow and blood as template, CDR2 specific sense primers and S-gamma specific antisense primers. In each case a single DNA product was amplified, cloned, and sequenced with primers covering the VDJ-S region. Patient specific CDR2 primers were used to confirm that the fragments were clonotypic. Unique switch junctions for patients 1–5 were detected at positions 125, 251, 435, 590, and 750 of S-mu, respectively. This agrees with previous studies mapping MM switch junctions to the 5′ portion of S-mu. The sequence and mutation profile of the switch junctions, with more frequent mutations in the S-mu region upstream of the hybrid junction, suggests that these junctions arise from normal CSR. A single hybrid junction was detected in all patients, corresponding to the single VDJ-Switch fragment amplified by long-distance PCR. The switch junction sequences also remained constant between time point samples, suggesting switch junction stability. Interestingly, new mutations were observed in the 3.5 Kb region upstream of the switch region, in the second time point samples of 2/5 patients. Most notably, 19 new mutations were detected in one patient, including 3 bp and 64 bp deletions 135 and 223 bp downstream of the intronic enhancer, respectively. The significance of these mutations is unclear, considering previous studies showing mutations, insertions, and deletions in this region in normal CSR. Nevertheless, it seems this process continues in clonotypic cells of some patients throughout malignancy. This ongoing mutation may lead to ‘remodeling' of the VDJ-S region, which is significant in the context of immunoglobulin translocations where proximity with immunoglobulin enhancers may confer a selective advantage.

Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination

Isotype switching involves a region-specific, nonhomologous recombinational deletion that has been suggested to occur by nonhomologous joining of broken DNA ends. Here, we find increased donor/acceptor homology at switch junctions from PMS2-deficient mice and propose that class switching can occur by microhomology-mediated end-joining. Interestingly, although isotype switching and somatic hypermutation show many parallels, we confirm that PMS2 deficiency has no major effect on the pattern of nucleotide substitutions generated during somatic hypermutation. This finding is in contrast to MSH2 deficiency. With MSH2, the altered pattern of switch recombination and hypermutation suggests parallels in the mechanics of the two processes, whereas the fact that PMS2 deficiency affects only switch recombination may reflect differences in the pathways of break resolution.

Characterizing IGBT Losses for Switched Mode Operation

Accurate loss analysis of switch mode circuits that use IGBTs as a switching device is difficult. Switching losses are a function of switch current, collector voltage and junction temperature. These three-dimensional parameters increase equation complexity and are often time consuming in their determination. However, computer programs such as MathcadTM MatlabTM ExcelTM and Table CurveTM provide practical means of curve fitting empirical data. This paper outlines a methodology for recording and converting empirical IGBT performance data into accurate and useful equations. These equations may then be utilized as simple function calls for loss analysis calculations. The examples covered in this paper are the IGBT clamped inductive turn-off energy and VCE(SAT) on-state drop. The losses predicted by the empirical equations are compared with in-circuit measured losses. IGBTs representing two different technologies are modeled.

Targeting of human switch recombination breakpoints: implications for the mechanism of mu-gamma isotype switching.

We recently characterized the allelic variants of the human Sgamma4 region which makes it possible to accurately identity and map Smu-Sgamma4 fragments from in vivo switched B cells. Twenty-six fragments were identified and a comparison was made with all previously published Smu-Sgamma sequences ( n = 82). Switch recombination outside the region flanking the Sgamma repeat sequence is a rare event in vivo and differences previously observed in patterns between in vitro and in vivo switched B cells appear to be artefactual and due to constraints of the methods used. Furthermore, internal deletions in the switch regions are common, but do not appear to be involved in isotype stabilization. A slight preference for switching to the B (SNIP) site was observed, suggesting a limited importance of both the B and A (SNAP) in the switching process. Mutations can be identified on either one or both sides of the switch junction, showing involvement of an error-prone process, and the pattern of mutations/substitutions at and around the junctions shows non-random nucleotide replacements by the enzyme(s) involved which may help in its future identification.

Refined structure of an intact IgG2a monoclonal antibody.

The structure of an intact, anti-canine lymphoma monoclonal antibody (Mab231) was determined by molecular replacement and refined in a triclinic cell to an R-value of 20.9%, using synchrotron diffraction data from 2.8 to 20 A resolution. All segments of the antibody, including the hinge region and carbohydrate component, are visible in electron density maps. There is no overall symmetry to the antibody, as the Fc is disposed in an entirely oblique manner with respect to the Fabs. The CH2 and CH3 domains do, however, possess a nearly exact, local 2-fold relationship. The Fab segments are related by a second, independent, local dyad axis, exact only with respect to constant domains. Variable domains exhibit no symmetry relationship as a consequence of the 16 degrees difference in Fab elbow angles. Variable domain pair associations VL:VH for the Fabs are virtually the same, and corresponding CDRs of the two Fabs also are nearly identical in structure. CDR-H3 displays the greatest difference. Hypervariable loops of both Fabs are involved in contacts with symmetry-related Fc segments at the CH2-CH3 switch junction, suggesting a "complex" structure. The hinge segment connecting Fabs with the Fc is quite extended and exhibits thermal factors indicative of a high degree of mobility. It consists of a well-defined upper hinge that partially maintains dyad symmetry and a fairly rigid core bounded above and below by fluid polypeptides that provide segmental flexibility. This structure represents the first visualization by X-ray analysis of a murine Fc segment, and its CH2 domains exhibit substantial rigid body conformational changes with respect to the human Fc used as an initial molecular replacement model. The oligosaccharides were found by difference Fourier syntheses to be very similar to those of the free human Fc fragment, although differences are present in the terminal residues. The detailed structure of the IgG presented here, and the distribution of effector binding sites, appears consistent with effector activation mechanisms involving translocation and/or aggregation of the Fc following antigen binding by the Fabs.

Asymmetric mutation around the recombination break point of immunoglobulin class switch sequences on extrachromosomal substrates

Junctions at class switch recombination sites in the genome are characterized by a unique sequence feature. Nucleotide substitutions and small deletions are common on either of the two sides of the switch junction, but not on both together. We have previously reported an extrachromosomal substrate assay system for analyzing the recombination of class switch sequences. Here we have sequenced nine junctions on each side of the break point and compared these to 17 recombination junctions of control substrates from the same cells. Five of the nine switch recombination junctions have nucleotide substitutions and deletions, with multiple nucleotide changes being more common. Furthermore, mutations were found only on a single side of the junction, just as for the recombination of switch sequences in the genome. In contrast, only one of 17 control substrate junctions had a mutation, and this was a single nucleotide insertion. This difference is highly significant (P < 0.00007) and indicates that the fundamental recombination mechanism is likely to be similar for switch sequences in the chromosome and on minichromosome substrates.

Sequential switching from mu to epsilon via gamma 4 in human B cells stimulated with IL-4 and hydrocortisone.

The molecular events leading to IgE synthesis in human B cells stimulated with IL-4 and hydrocortisone were analyzed. IL-4, but not hydrocortisone, induced C epsilon germ line transcription. However, hydrocortisone increased the levels of IL-4-induced germ line C epsilon transcripts by twofold and delivered the signal required for transcription of mature C epsilon mRNA. Nested primer polymerase chain reaction of high m.w. DNA revealed deletional switch recombination occurring in B cells sorted for lack of expression of surface IgE and stimulated with both IL-4 and hydrocortisone, but not in B cells stimulated with IL-4 alone or hydrocortisone alone. DNA sequence analysis of 10 switch fragments revealed direct joining of S mu to S epsilon in eight fragments, one of which exhibited an 876-bp deletion in S mu. The ninth fragment contained a 50-bp insertion at the S mu/S epsilon junction, which was likely to be derived from S gamma 4. The sequence of the 10th fragment was consistent with either a 17-bp insertion at the S mu/S epsilon junction derived from S gamma 4 or with a complex 38-bp deletion within S epsilon. Mapping of the switch junction sites showed "hot spots" for recombination within S mu but not within S epsilon. These findings indicate that hydrocortisone induces S mu-S epsilon deletional switch recombination in IL-4-treated B cells, and support a model of sequential isotype switching from IgM to IgE via IgG4.

Ig mu-epsilon isotype switch in IL-4-treated human B lymphoblastoid cells. Evidence for a sequential switch.

IgE is produced by B lymphocytes that have undergone a deletional rearrangement of their Ig H chain gene locus, a rearrangement that joins the switch region of the mu gene, S mu, with the corresponding region of the epsilon gene, S epsilon. To examine the resulting composite S mu-S epsilon junctions of human lymphoid cells, we have used a polymerase chain reaction strategy to clone the switch regions of the human myeloma U266 and of two IgE-producing human cell lines generated by treatment of lymphocytes with EBV plus IL-4. The switch junction of one of the EBV lines is a complex rearrangement in which a fragment of S gamma is interposed between S mu and S epsilon. This finding suggested that the switch to epsilon in this human lymphoid cell was preceded by a S mu-S gamma recombination. To determine whether this sequential switch rearrangement represented a unique event or occurred with some regularity in human B cells switching to IgE production, DNA samples from bulk cultures of lymphocytes treated with IL-4 were subjected to polymerase chain reaction amplification of their S mu-S epsilon junctions. When the resulting fragments were examined by Southern blotting, a substantial fraction hybridized to an S gamma probe. This finding suggests that sequential recombination involving S gamma is not rare in the switch to epsilon production in humans. Our polymerase chain reaction strategy should be useful in studying isotype switching at the DNA level.

Molecular analysis of the induction of immunoglobulin E synthesis in human B cells by interleukin 4 and engagement of CD40 antigen.

The molecular events leading to immunoglobulin E (IgE) synthesis in human sIgE- B cells stimulated with interleukin 4 (IL-4) and anti-CD40 monoclonal antibody (mAb) 626.1 were analyzed. Anti-CD40 mAb increased the levels of IL-4-induced germline C epsilon transcripts and induced the production of mature C epsilon mRNA. These effects were dependent on the presence of IL-4. Nested primer PCR revealed deletional switch recombination occurring only in B cell stimulated with both IL-4 and anti-CD40 mAb. DNA sequence analysis of switch fragments showed direct S mu/S epsilon joining, without the deletions or duplications within S mu often found in B cells stimulated with IL-4 and Epstein-Barr virus. Analysis of the switch junction map sites showed "hot spots" for recombination within S mu, but not within S epsilon. These findings indicate that IL-4 provides a signal to B cells to induce germline C epsilon transcription and concurrent CD40 engagement induces S mu/S epsilon deletional switch recombination, production of mature C epsilon mRNA, and IgE synthesis.

Deletional switch recombination occurs in interleukin-4-induced isotype switching to IgE expression by human B cells.

There is controversy as to whether deletional rearrangement occurs between the IgM and IgE switch regions (S mu and S epsilon, respectively) during switching to the IgE isotype. We have addressed the issue by stimulating normal human B cells, sorted for lack of expression of surface IgE, to produce IgE by infection with Epstein-Barr virus (EBV) in the presence of interleukin 4 (IL-4). Genomic DNA was amplified for S mu/S epsilon switch junction fragments by utilizing the nested-primer polymerase chain reaction. Switch junction fragments were amplified from B cells infected with EBV in the presence of IL-4 but not from B cells infected with EBV alone. The DNA sequence of these "switch fragments" revealed direct joining of S mu to S epsilon in each case. The recombination sites within S mu were clustered within 900 base pairs at the 5' end of the switch region, suggesting that there are "hot spots" for recombination within S mu. The S epsilon recombination sites were scattered throughout the S epsilon region. These findings indicate that IL-4-induced isotype switching to IgE production in human B cells is accompanied by DNA rearrangements with joining of S mu to S epsilon.