Detection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutation

Ronai, Diana; Iglesias-Ussel, Maria D.; Fan, Mingming; Li, Ziqiang; Martín, Alberto; Scharff, Matthew D.

doi:10.1083/jcb1763oia7

Cited by 11 publications

(17 citation statements)

References 23 publications

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“…Also, we cannot exclude the possibility that some fraction of the antisense transcripts that we described within the J H region originate from the promoters in S , in addition to the potential start sites that we tentatively identified within the J H region by 5ЈRACE. Antisense transcripts from the J H region and within the rearranged IgH variable region exon have been described in human lymphoma cell lines (30,40). Two recent studies also describe antisense transcripts in the D H J H region of bone marrow pro-B cells.…”

Section: Discussionmentioning

confidence: 99%

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Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions

Perlot

Alt

2008

Proc. Natl. Acad. Sci. U.S.A.

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Activation-induced cytosine deaminase (AID) is essential for both somatic hypermutation (SHM) and class switch recombination (CSR), two processes involved in antibody diversification. Previously, various groups showed both in vitro and in vivo that AID initiates SHM and CSR by deaminating cytosines in DNA in a transcription-dependent manner. Although in vivo both DNA strands are equally targeted by AID, many in vitro and bacterial experiments found that AID almost exclusively targets the nontemplate strand of a transcribed substrate. Here, we report the detection of antisense transcripts in assembled Ig heavy chain (IgH) variable region exons and their immediate downstream region, as well as in switch regions, sequences that, respectively, are targets for SHM and CSR in vivo. In contrast, we did not detect antisense transcripts from the C constant region exons, which lie between the IgH variable region exons and downstream S regions and which are not normally an AID target. Expression of the antisense variable region/flanking region and the S-region transcripts were found in all lymphocytes that transcribe these sequences in the sense direction. Steady-state levels of antisense transcripts appeared very low, and start sites potentially appeared heterogeneous. We discuss the potential implications of antisense IgH locus transcription for AID targeting or other processes.class switch recombination ͉ somatic hypermutation ͉ activation-induced cytosine deaminase ͉ bidirectional transcription

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Section: Discussionmentioning

confidence: 99%

“…Another possibility is that differences in chromatin structure or supercoiled DNA in vivo could allow AID to access both strands (29). Finally, it is conceivable that antisense transcription through variable regions and S regions could target AID to both strands (30).…”

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confidence: 99%

Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions

Perlot

Alt

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition, the authors report much lower steady-state levels of the antisense transcripts than of the spliced sense transcript. This was also observed in Ramos cells (9). However, when they compared the abundance of unspliced sense and antisense pre-mRNA, they were similar.…”

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confidence: 81%

Does antisense make sense of AID targeting?

Roa

Kuang

Scharff

2008

Proc. Natl. Acad. Sci. U.S.A.

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V ertebrates make high-affinity antibodies to protect themselves against pathogenic organisms and their products. The generation of these antibodies requires the highly mutagenic enzyme activation-induced cytidine deaminase (AID) (1). AID deaminates dC to dU in ssDNA, and this mutation recruits additional error-prone repair to carry out somatic hypermutation (SHM) of the antibody variable (V) regions that encode the antigen-binding site, and class switch recombination (CSR), which allows those antigen binding sites to be expressed with different constant regions that mediate effector functions. However, it is still unclear how the genomic instability that is initiated by AID is restricted to only discrete parts of the Ig genes but spares the rest of the genome (2-4). In this issue of PNAS, Perlot et al. (5) provide a partial answer to this puzzle by identifying both sense and antisense transcripts of the V and switch (S) regions, both of which are targeted for mutation by AID; but they find only sense transcripts of the constant (C ) region, which is not targeted by AID (Fig. 1).The finding of antisense transcripts of the V and S regions in primary murine B cells is important because it addresses the unsolved problem of why both the lower (transcribed) and the upper (nontranscribed) strands of the V and S region DNA are highly mutated in mice and humans (6). This has been confusing because biochemical studies with semipurified AID (3, 4) and experiments in which AID was expressed in bacterial cells (7) have found AIDinduced mutations mostly on the upper strand of the targeted DNA. This suggested that something like the large transcription apparatus was protecting the lower strand, whereas the upper strand was accessible to AID ( Fig. 1). A number of explanations have been proposed for why both strands of the V and S regions are mutated in vivo, but not in vitro, such as differences between the mammalian and the bacterial RNA polymerases used in vitro (4), the accessibility of both strands in the supercoiled DNA at the edges of the transcription bubble (8), or ssDNA-binding replication protein A (RPA) participating with AID in mutation (4). Antisense transcription of the V and S regions provides an attractive, although not exclusive, alternative.Antisense transcripts had previously been observed in the V region of a human BurkittЈs lymphoma cell line (9), in S regions within the context of chromosomal translocations (10-12), and in pro-B cells during V(D)J recombination (13), but Perlot et al. (5) are the first to identify such transcripts from a rearranged V region in primary B cells. In the S regions, transcription of the C rich lower strand results in R-loops, where the very G rich nascent RNA hybridizes the template strand and prevents AID action while leaving the upper strand single stranded and accessible to AID (14). However, the G:C content of the V region does not predispose it to R-loop formation, and R-loops have not been found there.There is a considerable body of data that suggests that high rates o...

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“…Alternatively, it has been proposed that AID binds directly to short stretches of ssDNA exposed by RNA polymerase. Yet, although the consensus in the field is that the availability of ssDNA substrate is likely AID-independent and precedes enzyme-based diversification (Ramiro et al, 2003;Ronai et al, 2007), the transcriptionally driven structural elements recognized by AID have not been characterized.…”

Section: Introductionmentioning

confidence: 99%

“…A number of studies indicate that transcription plays a key role in SHM by exposing ssDNA and thus allowing access to enzyme-based mutation Chaudhuri et al, 2003;Dickerson et al, 2003;Lebecque and Gearhart, 1990;Pham et al, 2003;Ramiro et al, 2003;Ronai et al, 2007;Shen and Storb, 2004;Yang et al, 2006;Yoshikawa et al, 2002). Transcription is required for SHM, and the initiation site defines the 5′ boundary of the hypermutable region (Betz et al, 1994;Peters and Storb, 1996;Rada et al, 1998;TumasBrundage and Manser, 1997).…”

Section: Introductionmentioning

confidence: 99%

I. VH gene transcription creates stabilized secondary structures for coordinated mutagenesis during somatic hypermutation

Wright

Schmidt

Minnick

et al. 2008

Molecular Immunology

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During the adaptive immune response, antigen challenge triggers a million-fold increase in mutation rates in the variable region antibody genes. The frequency of mutation is causally and directly linked to transcription, which provides ssDNA and drives supercoiling that stabilizes secondary structures containing unpaired, intrinsically mutable bases. Simulation analysis of transcription in VH5 reveals a dominant 65 nt secondary structure in the non-transcribed strand containing six sites of mutable ssDNA that have also been identified independently in human B cell lines and in primary mouse B cells. This dominant structure inter-converts briefly with less stable structures and is formed repeatedly during transcription, due to periodic pauses and backtracking. In effect, this creates a stable yet dynamic "mutability platform" consisting of ever-changing patterns of unpaired bases that are simultaneously exposed and therefore able to coordinate mutagenesis. Such a complex of secondary structures may be the source of ssDNA for enzyme-based diversification, which ultimately results in high affinity antibodies.

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Detection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutation

Cited by 11 publications

References 23 publications

Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions

Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions

Does antisense make sense of AID targeting?

I. VH gene transcription creates stabilized secondary structures for coordinated mutagenesis during somatic hypermutation

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