Class Switch Regions Research Articles

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) invitro and onto rDNA invivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

Complexities due to single-stranded RNA during antibody detection of genomic rna:dna hybrids.

BackgroundLong genomic R-loops in eukaryotes were first described at the immunoglobulin heavy chain locus switch regions using bisulfite sequencing and functional studies. A mouse monoclonal antibody called S9.6 has been used for immunoprecipitation (IP) to identify R-loops, based on the assumption that it is specific for RNA:DNA over other nucleic acid duplexes. However, recent work has demonstrated that a variable domain of S9.6 binds AU-rich RNA:RNA duplexes with a KD that is only 5.6-fold weaker than for RNA:DNA duplexes. Most IP protocols do not pre-clear the genomic nucleic acid with RNase A to remove free RNA. Fold back of ssRNA can readily generate RNA:RNA duplexes that may bind the S9.6 antibody, and adventitious binding of RNA may also create short RNA:DNA regions. Here we investigate whether RNase A is needed to obtain reliable IP with S9.6.FindingsAs our test locus, we chose the most well-documented site for kilobase-long mammalian genomic R-loops, the immunoglobulin heavy chain locus (IgH) class switch regions. The R-loops at this locus can be induced by using cytokines to stimulate transcription from germline transcript promoters. We tested IP using S9.6 with and without various RNase treatments. The RNase treatments included RNase H to destroy the RNA in an RNA:DNA duplex and RNase A to destroy single-stranded (ss) RNA to prevent it from binding S9.6 directly (as duplex RNA) and to prevent the ssRNA from annealing to the genome, resulting in adventitious RNA:DNA hybrids. We find that optimal detection of RNA:DNA duplexes requires removal of ssRNA using RNase A. Without RNase A treatment, known regions of R-loop formation containing RNA:DNA duplexes can not be reliably detected. With RNase A treatment, a signal can be detected over background, but only within a limited 2 or 3-fold range, even with a stable kilobase-long genomic R-loop.ConclusionAny use of the S9.6 antibody must be preceded by RNase A treatment to remove free ssRNA that may compete for the S9.6 binding by forming RNA:RNA regions or short, transient RNA:DNA duplexes. Caution should be used when interpreting S9.6 data, and confirmation by independent structural and functional methods is essential.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1092-1) contains supplementary material, which is available to authorized users.

ADAR1 Protein Induces Adenosine-targeted DNA Mutations in Senescent Bcl6 Gene-deficient Cells

Somatic mutations accumulate in senescent cells. Bcl6, which functions as a transcriptional repressor, has been identified as a potent inhibitor of cell senescence, but a role of Bcl6 in the accumulation of somatic mutations has remained unclear. Ig class-switch recombination simultaneously induces somatic mutations in an IgM class-switch (Ig-Sμ) region of IgG B cells. Surprisingly, mutations were detected in the Ig-Sμ region of Bcl6-deficient IgM B cells without class-switch recombination, and these mutations were mainly generated by conversion of adenosine to guanosine, suggesting a novel DNA mutator in the B cells. The ADAR1 (adenosine deaminase acting on RNA1) gene was overexpressed in Bcl6-deficient cells, and its promoter analysis revealed that ADAR1 is a molecular target of Bcl6. Exogenous ADAR1 induced adenosine-targeted DNA mutations in IgM B cells from ADAR1-transgenic mice and in wild-type mouse embryonic fibroblasts (MEFs). These mutations accumulated in senescent MEFs accompanied with endogenous ADAR1 expression, and the frequency in senescent Bcl6-deficient MEFs was higher than senescent wild-type MEFs. Thus, Bcl6 protects senescent cells from accumulation of adenosine-targeted DNA mutations induced by ADAR1.

G Clustering Is Important for the Initiation of Transcription-Induced R-Loops In Vitro, whereas High G Density without Clustering Is Sufficient Thereafter

R-loops form cotranscriptionally in vitro and in vivo at transcribed duplex DNA regions when the nascent RNA is G-rich, particularly with G clusters. This is the case for phage polymerases, as used here (T7 RNA polymerase), as well as RNA polymerases in bacteria, Saccharomyces cerevisiae, avians, mice, and humans. The nontemplate strand is left in a single-stranded configuration within the R-loop region. These structures are known to form at mammalian immunoglobulin class switch regions, thus exposing regions of single-stranded DNA for the action of AID, a single-strand-specific cytidine deaminase. R-loops form by thread-back of the RNA onto the template DNA strand, and here we report that G clusters are extremely important for the initiation phase of R-loop formation. Even very short regions with one GGGG sequence can initiate R-loops much more efficiently than random sequences. The high efficiencies observed with G clusters cannot be achieved by having a very high G density alone. Annealing of the transcript, which is otherwise disadvantaged relative to the nontemplate DNA strand because of unfavorable proximity while exiting the RNA polymerase, can offer greater stability if it occurs at the G clusters, thereby initiating an R-loop. R-loop elongation beyond the initiation zone occurs in a manner that is not as reliant on G clusters as it is on a high G density. These results lead to a model in which G clusters are important to nucleate the thread-back of RNA for R-loop initiation and, once initiated, the elongation of R-loops is primarily determined by the density of G on the nontemplate DNA strand. Without both a favorable R-loop initiation zone and elongation zone, R-loop formation is inefficient.

Mechanistic Aspects of Lymphoid Chromosomal Translocations

Chromosomal translocations require double-strand breakage at two sites, followed by joining of the ends. The joining is usually done by nonhomologous DNA end-joining, though homologous recombination and single-strand annealing play roles in cases where there is homology. The mechanism of breakage can be more difficult to understand at sites other than the antigen receptor loci. Some breakage events in pre-B or pre-T cells are due to the RAG proteins recognizing heptamer/nonamer-like sequences, but most breaks are not. Rather, some of these breaks are due to RAG nicking at non-B DNA conformations. Translocation events in mature B cells, when RAGs are not expressed, may be due to the activation-induced deaminase (AID). But AID only acts on single-stranded DNA, and it is not yet clear how this single-stranded DNA arises at some transcribed sites and not others. During the physiologic process of class switch recombination, R-loops form at transcribed class switch regions, thereby accounting for how single strandedness is facilitated at these sites of AID action.

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the Smu locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The Smu R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core Smu repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core Smu repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core Smu repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.

Downstream boundary of chromosomal R-loops at murine switch regions: Implications for the mechanism of class switch recombination

R-loops form at Sgamma3 and Sgamma2b Ig class switch regions in the chromosomes of stimulated murine primary B cells and are suspected to be a general feature of mammalian class switch regions. The in vivo upstream boundary of the R-loops is known to begin within the switch repeats. To determine how precisely the R-loop structure conforms to the repetitive zone of the murine Sgamma3 and Sgamma2b switch regions, a chemical probing method was used to obtain structural information on the downstream boundary. We find that only 61-67% of the R-loops terminate within the Sgamma3 and the Sgamma2b repetitive zones, and the remainder terminate downstream, usually within the first 600 bp immediately downstream of the core switch repeats. Interestingly, the nontemplate strand G density falls to the random level gradually through this same region. Hence, the R-loops terminate as the G-richness of the nascent RNA strand falls. This finding is consistent with thermodynamic predictions for RNA:DNA duplex strength relative to that of DNA:DNA duplexes. This result contrasts with the location of known recombination breakpoints, which correlate not with G-richness and R-loop location but rather with AGCT density. The implications of these findings are discussed in the context of models for the targeting of class switch recombination.

Sa.102. Formation of R-Loops at Immunoglobulin Heavy Chain Class Switch Regions

Sa.102. Formation of R-Loops at Immunoglobulin Heavy Chain Class Switch Regions

Fine-Structure Analysis of Activation-Induced Deaminase Accessibility to Class Switch Region R-Loops

Activation-induced deaminase (AID) is essential for class switch recombination and somatic hypermutation, and it has the ability to deaminate single-stranded DNA at cytidines. Mammalian class switch regions form R-loops upon transcription in the physiological orientation. The displaced DNA strand of an R-loop is forced to wrap around the RNA-DNA hybrid; hence, it may not have complete exposure to proteins. A fundamental question concerns the extent to which AID is accessible to the displaced strand of a transcription-generated R-loop. We used a minimal R-loop to carry out high-resolution analysis of the precise locations of AID action. We found that AID deaminates on the displaced DNA strand across the entire length of the R-loop. Displaced strand locations with a WRC (where W is A or T and R is A or G) sequence are preferred targets, but there are clear exceptions. These WRC deviations may be due to steric constraints on the accessibility of AID to these sites as the displaced strand twists around the RNA-DNA duplex. This phenomenon may explain the lack of WRC site preference at the mutations surrounding class switch recombination junctions.

R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells

The mechanism responsible for immunoglobulin class switch recombination is unknown. Previous work has shown that class switch sequences have the unusual property of forming RNA-DNA hybrids when transcribed in vitro. Here we show that the RNA-DNA hybrid structure that forms in vitro is an R-loop with a displaced guanine (G)-rich strand that is single-stranded. This R-loop structure exists in vivo in B cells that have been stimulated to transcribe the gamma3 or the gamma2b switch region. The length of the R-loops can exceed 1 kilobase. We propose that this distinctive DNA structure is important in the class switch recombination mechanism

Internal IgH class switch region deletions are position-independent and enhanced by AID expression.

Ig heavy chain class switch recombination (CSR) involves a recombination/deletion mechanism that exchanges the expressed C(H) gene with a downstream C(H) gene. CSR is mediated by highly repetitive switch (S) region sequences and requires the activation-induced deaminase (AID). The S region 5' of the C mu gene (S mu) can undergo high-frequency internal deletions in normal B cells and B cell lines activated for CSR, although the relationship of these deletions and CSR has not been elucidated. In this study, we introduced constitutively transcribed S mu or S gamma 2b regions into a pro-B cell line that can be activated for AID expression, CSR, and endogenous S mu deletions. We find that randomly integrated S region transcription units in these cells also undergo increased levels of internal rearrangements after cellular activation, indicating that the deletion process is independent of location within the Ig heavy chain locus and potentially AID-promoted. To test the latter issue, we generated hybridomas from wild-type and AID-deficient activated B cells and assayed them for internal S mu deletions and S region mutations. These studies demonstrated that efficient intra-S region recombination depends on AID expression and that internal S region deletions are accompanied by frequent mutations, indicating that most S region deletions occur by the same mechanism as CSR.

Chicken immunoglobulin μ-chain gene: germline organization and tandem repeats characteristic of class switch recombination

We have isolated the phage clones covering the region spanning from the heavy (H)-chain joining (J) region to the end of the μ-chain gene of the chicken immunoglobulin (Ig). The distance from J H to the first exon of the μ-chain constant (C) region is approximately 13 kb, and introns between the C region exons measure more than 3 kb. These distances are significantly larger than those of known μ-chain genes. We found a region cross-hybridizing to the switch regions of the mouse Cμ and Cα genes just in front of the first exon of Cμ. Partial nucleotide sequencing of this region revealed that this region consists of tandem repeats of pentamers ( C T )( C A ) CAG complementary to the mammalian switch repetitive region. These findings suggest that this region is a good candidate for a class switch region of the μ-chain gene of chicken Ig.

Autoantigenic proteins that bind recombinogenic sequences in Epstein-Barr virus and cellular DNA.

We have identified conserved autoantigenic cellular proteins that bind to G-rich sequence motifs in recombinogenic regions of Epstein-Barr virus (EBV) DNA. This binding activity, called TRBP, recognizes the EBV terminal repeats, a locus responsible for interconversion of linear and circular EBV DNA. We found that TRBP also binds to EBV DNA sequences involved in deletion of EBNA2, a gene product required for immortalization. We show that TRBP binds sequences present in repetitive cellular DNA, such as variable-number tandem repeats (VNTR) and immunoglobulin heavy-chain class switch regions. We propose that EBV utilizes cellular DNA recombination systems to mediate several types of viral genome alterations. These findings may lead to an understanding of the mechanism of rearrangements of EBV DNA that are a central feature of the biology of the virus.

The complete murine immunoglobulin class switch region of the alpha heavy chain gene-hierarchic repetitive structure and recombination breakpoints.

A 7255-base pair (bp) sequence, including the previously sequenced murine segments of I alpha, S alpha, and C alpha, has been completed. Homology matrix comparison revealed a switch repetitive region of 4.2 kilobases (kb) composed of 20-80-bp homology runs, including the previously assigned S alpha region. We distinguished several stretches of duplication, i.e. the central 0.8-kb repetitive region, with some 80-bp staggered consensus repeats containing 20-30-bp subsets, made up of the primordial pentamers CTG(A/G)G. All the break-points of the S alpha switch recombination, including those generated by the translocation of the c-myc protooncogene and those catalyzed by bacterial extracts, are located within the consensus sequence subsets of the 4.2-kb repetitive region.

Sequences of human immunoglobulin switch regions: implications for recombination and transcription

We have sequenced the entire human S mu and S gamma 4 immunoglobulin heavy chain class switch regions, and have also completed the sequence of human S epsilon. S mu is composed predominantly of GAGCT and GGGCT pentameric repeats, with these units also being found in S epsilon at a much lower density. S mu-S gamma 4 matches are infrequent, but S gamma 4 contains a cluster of repeated sequences similar to units in mouse gamma switch sites and unrelated to the S mu repeats, suggesting that S mu-S gamma homology is not important in mu-gamma switching. We examined our epsilon and gamma 4 sequences for features that could regulate production of 'sterile' transcripts preceding switch recombination. There is an Evolutionarily Conserved Sequence (ECS) upstream from the human and mouse S epsilon regions that overlaps and extends 5' to the start sites for human and mouse epsilon sterile transcripts. Similarly, an ECS upstream from S gamma 4 is homologous to a mouse sequence that overlaps and extends 5' to the start sites for mouse gamma 2b sterile transcripts. The epsilon and gamma 4 conserved segments contain potential Interferon Stimulable Response Elements (ISRE's) that are identical between human epsilon and gamma 4.

Sigma region located between C mu and C delta genes of human immunoglobulin heavy chain: possible involvement of tRNA-like structure in RNA splicing.

Noncoding regions within the cluster of immunoglobulin heavy chain constant genes in the human genome contained a number of repeats. In the mu-delta intron, two repeating units were contained. One 442-base-long fragment located JH-mu intron (defined as "sigma mu(sigma mu)") occupied the position in the mu-delta intron. The other 1166-base-long fragment located somewhere in front of S (class switch) region of C gamma gene was also found in the mu-delta intron. We defined the repeats in the mu-delta intron as "SIGMA (sigma)". The polarities of the longer repeats in the genome were opposite between the mu-delta intron and the upstreams of C gamma genes. These inverted copies (defined as sigma gamma 3 and sigma gamma 4), located 6 kb upstream of their respective C gamma's, were apparently transcribed in vitro, via RNA polymerase III and transcripts should have contained tRNA-like structures. Small DNA fragments capable of encoding tRNA-like structures were also found in corresponding regions of mouse Ig C gamma cluster.

Conserved organization of the murine immunoglobulin epsilon gene region: restriction endonuclease maps and switch-region nucleotide sequence.

Various inbred mouse strains showed remarkable conservation of organization in the epsilon germline region as determined by restriction mapping. Restriction fragment length polymorphisms (RFLP) were seen in the epsilon gene region for only two of eight restriction enzymes tested. Furthermore, the RFLP did not correlate with the IgE-response phenotype for the murine strains SJL, SJA, C57BL/6, BALB/c, A/ST, and A/J. The IgE class-switch region (S epsilon) DNA from an SJL genomic clone was sequenced and was compared with S epsilon sequences from BALB/c mice. These S epsilon sequences were at least 95% homologous. Most of the S epsilon sequence differences observed between the two strains were single base pair substitutions, deletions, or insertions. The largest difference between the S epsilon sequences resulted from an insertion of seven contiguous bases seen in the SJL S epsilon region.

IgE heavy chain constant region genes in atopic dermatitis and senile erythroderma patients.

By Southern hybridization using a genomic DNA fragment carrying a human IgE heavy chain constant region gene (C epsilon) as a probe, we analyzed the organization of human C epsilon genes and their flanking regions in 23 atopic dermatitis and 6 senile erythroderma patients with elevated serum IgE levels, and 6 atopic dermatitis patients with normal IgE levels. On Bam HI, Hind III, and Eco RI digestions, we detected three hybridizable fragments containing three human C epsilon genes, C epsilon 1, C epsilon 2, and C epsilon 3, respectively, in all leukocyte DNAs. These fragments were almost identical in size among patients and healthy donors. Pst I digestion generated a genetic polymorphism. We, however, could find no correlation between this polymorphism and the disorders. It was concluded that among the patients and healthy donors, there was no marked difference in the organization of the functional C epsilon gene and its flanking region containing a class switch region. Our conclusion cannot rule out the presence of genetic abnormalities of this region in some atopic dermatitis patients which are not resolvable by our method. In the course of this study, we found a novel C epsilon-like gene in placenta DNA which differs from the three C epsilon genes commonly present in normal human DNA.

Molecular basis of heavy-chain class switching and switch region deletion in an Abelson virus-transformed cell line.

We demonstrated that a subclone of an Abelson murine leukemia virus-transformed B-lymphoid cell line switched from mu to gamma 2b expression in vitro, by the classical recombination-deletion mechanism. In this line, the expressed VHDJH region and the C gamma 2b constant region gene were juxtaposed by a recombination event which linked the highly repetitive portions of the S mu and S gama 2b regions and resulted in the loss of the C mu gene from the intervening region. An additional recombination event in this subclone involved an internal deletion in the S mu region of the expressed (switched) allele. One end of this deletion occurred very close to the switch recombination point. Despite the recombination-deletion mechanism of switching, the gamma 2b-producing line retained two copies of the C mu gene and two copies of the sequence just 5' to the S gamma 2b recombination point. The possible significance of the retention of these sequences to the mechanism of class switching is discussed.

Molecular basis of heavy-chain class switching and switch region deletion in an Abelson virus-transformed cell line.

We demonstrated that a subclone of an Abelson murine leukemia virus-transformed B-lymphoid cell line switched from mu to gamma 2b expression in vitro, by the classical recombination-deletion mechanism. In this line, the expressed VHDJH region and the C gamma 2b constant region gene were juxtaposed by a recombination event which linked the highly repetitive portions of the S mu and S gama 2b regions and resulted in the loss of the C mu gene from the intervening region. An additional recombination event in this subclone involved an internal deletion in the S mu region of the expressed (switched) allele. One end of this deletion occurred very close to the switch recombination point. Despite the recombination-deletion mechanism of switching, the gamma 2b-producing line retained two copies of the C mu gene and two copies of the sequence just 5' to the S gamma 2b recombination point. The possible significance of the retention of these sequences to the mechanism of class switching is discussed.