Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line

Kanayama, Naoki; Todo, Kagefumi; Takahashi, Satoko; Ohmori, Hitoshi

doi:10.1093/nar/gnj013

Cited by 21 publications

(18 citation statements)

References 36 publications

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“…The frequency of mutation events was calculated by dividing all mutation events by the total analyzed nucleotide numbers. An artificial GCV substrate, G/B construct was also used to analyze hypermutation frequency in DT40-SW cells as described previously (39). AID expression in DT40-SW cells were switched on, and single cells expressing AID (AID-ON cells) were sorted as described previously (40).…”

Section: Methodsmentioning

confidence: 99%

“…4C). In addition, reporterbased assays for GCV using the G/B construct showed that overexpression of SRSF1-3 in wild-type DT40 cells did not induce GCV in the reporter construct placed in the ovalbumin locus, in which the AID-dependent hypermutation machinery has been shown to be inactive (39) (Fig. 3E).…”

Section: Srsf1-3 Has a Critical Role In Targeted Induction Of Hypermumentioning

confidence: 99%

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Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1

Kanehiro

Todo

Negishi

et al. 2012

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

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Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.gene conversion | genomic instability S omatic hypermutation (SHM) and class switch recombination (CSR) of Ig genes are key processes needed to generate functional antibodies (Abs) with high affinity in humoral immune responses. Activation-induced cytidine deaminase (AID) is crucial for both SHM and CSR (1, 2), which are initiated by AIDcatalyzed deamination of dC to dU in the variable (V) region and the switch (S) region DNA strands, respectively (3, 4). However, AID has been recently shown to bind numerous chromosomal locations genome-wide and to mutate non-Ig genes, although the mutation frequency of non-Ig genes is much lower than that of the Ig genes (5, 6). It has been proposed that specific cofactors interact with AID and regulate its activity in a target-specific manner (7). AID has been reported to associate with several proteins including RPA (8, 9), protein kinase A (10), RNA polymerase II (Pol II) (11), DNA-PKcs (12), MDM2 (13), Spt6 (14), 14-3-3 (15), Spt5 (16), CTNNBL1 (17), PTBP2 (18), RNA exosome subunits (19), KAP1 (20), and HP1 (20). However, these investigations were mostly focused on CSR, and, thus, the identified factors themselves are not sufficient to explain how AID activity is specifically targeted to the IgV genes during SHM.SHM occurs in IgV region exons starting 100-200 bp downstream of the promoter and extending over 1-2 kb, preferentially targeting WRC/GYW hotspot motifs (3, 4). Importantly, the activity of the SHM machinery strictly depends on transcription of the IgV genes (21, 22). In vitro experiments have shown that replication protein A (RPA) binds to and stabilize...

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Section: Methodsmentioning

confidence: 99%

Section: Srsf1-3 Has a Critical Role In Targeted Induction Of Hypermumentioning

confidence: 99%

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1

Kanehiro

Todo

Negishi

et al. 2012

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

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“…The on-going Ig gene conversion and hypermutation in RAD51 paralogue mutants have likewise been employed to optimize the antigen specificity of antibodies (Cumbers et al 2002). Furthermore, a blue fluorescent protein gene together with a GFP pseudogene has been inserted into the IgL locus demonstrating that non-Ig transgenes can be diversified by gene conversion when inserted into the IgL locus together with a conversion donor sequence (Kanayama et al 2006).…”

Section: Application To Biotechnologymentioning

confidence: 99%

Activation-induced cytidine deaminase-mediated hypermutation in the DT40 cell line

Arima

Buerstedde

2008

Phil. Trans. R. Soc. B

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Depending on the species and the developmental stage of B cells, activation-induced cytidine deaminase (AID) triggers immunoglobulin (Ig) gene diversification by gene conversion, hypermutation or switch recombination. The bursal B cell line DT40 usually diversifies its rearranged Ig light chain (IgL) gene by gene conversion, but disruption of the RAD51 gene paralogues or deletion of the jV conversion donors induces hypermutation. Although not all aspects of somatic hypermutation can be studied in DT40, the compact size of the chicken IgL locus and the ability to modify the genome by targeted integration are powerful experimental advantages. We review here how the studies in DT40 contributed to understanding how AID initiates Ig gene diversification and how AID-induced uracils are subsequently processed by uracil DNA glycosylase, proliferating cell nuclear antigens and error-prone polymerases. We also discuss the on-going research on the Ig locus specificity of hypermutation and the possibility of using hypermutation for the artificial evolution of proteins and regulatory sequences in DT40.

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“…A recent report used a stop codon reversion assay to examine GCV of an artificial substrate containing a transcription cassette and an upstream homologous sequence donor in DT40 cells (27); results showed that the unit underwent GCV in the IgL locus but not in a non-Ig locus, suggesting that GCV is limited to Ig regions. There could be two explanations for the differences in the results of that study compared with those reported here.…”

Section: Generation and Integration Of Heterologous Transcriptionmentioning

confidence: 99%

Activation-induced Cytidine Deaminase-mediated Sequence Diversification Is Transiently Targeted to Newly Integrated DNA Substrates

Yang¹,

Fugmann

Gramlich³

et al. 2007

Journal of Biological Chemistry

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The molecular features that allow activation-induced cytidine deaminase (AID) to target Ig and certain non-Ig genes are not understood, although transcription has been implicated as one important parameter. We explored this issue by testing the mutability of a non-Ig transcription cassette in Ig and non-Ig loci of the chicken B cell line DT40. The cassette did not act as a stable long term mutation target but was able to be mutated in an AID-dependent manner for a limited time post-integration. This indicates that newly integrated DNA has molecular characteristics that render it susceptible to modification by AID, with implications for how targeting and mis-targeting of AID occurs. Gene conversion (GCV)4 and somatic hypermutation (SHM) are two mechanisms crucial for diversifying the variable region sequence of rearranged Ig loci of B cells. In many organisms, V(D)J recombination is able to generate a large primary repertoire of highly diversified antigen receptors, and SHM is used to expand and fine-tune antibody specificities after antigen encounter in a process called affinity maturation. In the chicken Ig light chain (IgL) and heavy chain loci, combinatorial diversity is severely limited, and GCV and SHM are employed to diversify the primary antibody receptor pool (reviewed in Ref. 1). In the chicken IgL locus, there are 25 pseudo V elements with sequence similarities to the sole functional V region, and they serve as sequence donors in the GCV pathway to create sequence variability. Nontemplated SHM events, which occur at a lower frequency, also contribute to sequence diversity of the primary Ig receptor repertoire (2). In the chicken B cell line DT40, Ig GCV and SHM have been shown to arise from a common intermediate (3).Activation-induced cytidine deaminase (AID) is required for GCV/SHM and is thought to initiate these reactions by deamination of cytosine to generate uracil in DNA (4 -8). In DT40 cells, the uracils are processed predominantly by uracil DNAglycosylase (UNG), and the resulting abasic site is channeled into either homology-based repair using pseudo V elements to produce GCV events or error-prone repair to produce SHM events.Little is known about the molecular characteristics that render a gene accessible to AID-mediated diversification processes. Mouse and cell line studies of SHM have demonstrated that transcription is essential, whereas the endogenous Ig promoters and the Ig variable region sequences themselves are not required (9 -13). Non-Ig genes have also been reported to undergo SHM, albeit at frequencies much lower than Ig genes, and such mis-targeting of SHM is thought to contribute to B cell malignancies (14 -18).We tested the ability of a non-Ig expression cassette to undergo sequence diversification in Ig and non-Ig loci of DT40 cells and found that it underwent AID-dependent mutation in a locus-independent fashion to generate predominantly transition mutations at G/C bp. In addition, despite stable levels of transcription, the mutability of the cassettes was transient and waned i...

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Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line

Cited by 21 publications

References 36 publications

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1

Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1

Activation-induced cytidine deaminase-mediated hypermutation in the DT40 cell line

Activation-induced Cytidine Deaminase-mediated Sequence Diversification Is Transiently Targeted to Newly Integrated DNA Substrates

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