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

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

Section: Correlation Of Sense and Antisense Transcript Expression Patmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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

Perlot

Alt

2008

“…First, mammalian S regions are GC-rich and G-rich on their nontemplate strand, a property that allows them to generate singlestranded DNA in the form of R loops when transcribed (18,19). Such R loops are effective AID substrates in vitro (20,21). Second, in activated B cells, AID is phosphorylated, allowing it to interact with replication protein A (RPA) (22).…”

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confidence: 99%

Influence of switch region length on immunoglobulin class switch recombination

Zarrin

Tian

Wang

et al. 2005

The class and effector functions of antibodies are modulated through the process of Ig heavy chain class switch recombination (CSR). CSR occurs between switch (S) regions that lie upstream of the various Ig heavy chain constant region exons. Molecular analyses of S-region functions have been hampered by their large size and repetitive nature. To test potential relationships between S-region size and efficiency of CSR, we generated normal B lymphocytes in which the 12-kb S region flanking the C␥1 exons (S␥1) was replaced with synthetic or endogenous S regions of various lengths. Replacement of S␥1 with 1-and 2-kb synthetic sequences representing the S␥1 core repeats or a 4-kb portion of the core endogenous S␥1 region supported CSR frequencies that directly correlated with S-region length. These findings indicate that Sregion size is an important factor in determining endogenous CSR efficiency. Moreover, these results also will allow the development of a systematic system to test the function of various S-region motifs by replacing endogenous S regions with synthetic S regions controlled for size effects.IgH switch region ͉ activation-induced deaminase ͉ synthetic switch region ͉ IgG1

“…Because of its high G content, the NT DNA strand at S regions could be stabilized by the formation of parallel four-stranded G quartets (17, 18). Indeed, AID appears to bind specifically to G-loops within transcribed S regions (19) and can deaminate the displaced strand of a transcription-induced R-loop in vitro (20).Interestingly, cotranscriptional messenger ribonucleoprotein (mRNP) biogenesis is an essential step in gene expression that may influence genetic integrity (21,22). In the yeast Saccharomyces cerevisiae, hyperrecombination can be triggered by transcription in mutants depleted of THO/TREX, a conserved protein complex that functions at the interface between transcription and mRNA export (23,24).…”

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confidence: 99%

Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex

Gómez-González

Aguilera

2007

102

111

Activation-induced cytidine deaminase (AID) is a B cell enzyme essential for Ig somatic hypermutation and class switch recombination. AID acts on ssDNA, and switch regions of Ig genes, a target of AID, form R-loops that contain ssDNA. Nevertheless, how AID action is specifically targeted to particular DNA sequences is not clear. Because mutations altering cotranscriptional messenger ribonucleoprotein (mRNP) formation such as those in THO/TREX in yeast promote R-loops, we investigated whether the cotranscriptional assembly of mRNPs could affect AID targeting. Here we show that AID action is transcription-dependent in yeast and that strong and transcription-dependent hypermutation and hyperrecombination are induced by AID if cells are deprived of THO. In these strains AID-induced mutations occurred preferentially at WRC motifs in the nontranscribed DNA strand. We propose that a suboptimal cotranscriptional mRNP assembly at particular DNA regions could play an important role in Ig diversification and genome dynamics.hyperrecombination ͉ hypermutation ͉ messenger ribonucleoprotein biogenesis ͉ R-loops A ctivation-induced cytidine deaminase (AID) is a specific B cell enzyme believed to be responsible for the initiation of somatic hypermutation (SHM) and class switch recombination (CSR) during B cell differentiation (1-3). Many studies have shown that AID acts directly on DNA (4), the natural target for AID action in the variable and switch (S) regions for SHM and CSR, respectively. The specific mechanisms of AID function are still unclear, but evidence suggests that the preferential target of AID may be ssDNA (5, 6). Thus, in vitro experiments have shown that AID deaminates ssDNA and dsDNA that is transcribed (5-7), and transcription has been shown to be required for both SHM and CSR in vivo (8,9).Studies have shown a strand preference for the action of AID during in vitro transcription with AID-induced mutations detected preferentially in the nontranscribed (NT) strand (6,(10)(11)(12). Analysis of the products of SHM in B cells, however, reveals that both DNA strands are mutated (13). Other in vitro studies have shown that AID can deaminate both strands within regions of supercoiled DNA (14) and that the ssDNA-binding replication protein A is required for deamination of SHM targets (15). All of these findings are consistent with the idea that transient formation of ssDNA during transcription facilitates AID action.Along the same line, formation of R-loops during transcription of the S regions has been proposed (16). Such R-loops would possibly provide AID with ssDNA substrates at its target region because there the transcribed (T) strand hybridizes with the nascent RNA and the NT strand is present as ssDNA. Because of its high G content, the NT DNA strand at S regions could be stabilized by the formation of parallel four-stranded G quartets (17, 18). Indeed, AID appears to bind specifically to G-loops within transcribed S regions (19) and can deaminate the displaced strand of a transcription-induced R-loop in vitro ...