Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire

Kanehiro, Yuichi; Todo, Kagefumi; Ikeda, Mika; Kanayama, Naoki; Ohmori, Hitoshi

doi:10.1016/j.bbrc.2010.04.096

Cited by 8 publications

(12 citation statements)

References 24 publications

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“…The IgVL and IgVH genes were amplified from genomic DNA by PCR and analyzed to find mutations. Mutations were categorized as GCV or point mutation by comparing mutated sequences with published V pseudogene sequences as reported previously (49). The frequency of mutation events was calculated by dividing all mutation events by the total analyzed nucleotide numbers.…”

Section: Methodsmentioning

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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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%

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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“…Chicken IgY can be made by several companies using immunization, and phage display of chicken antibody fragments has been accomplished against multiple antigens [31][32][33]. From a protein engineering standpoint, the DT40 cell line has been manipulated to serve as a host for SH of antigen receptor genes [34], and human pseudogenes have been engineered into DT40 and used to create human repertoires using gene conversion, a process where a donor DNA sequence replaces a homologous sequence in the genome [35]. In this regard, chickens with the antibody loci knocked out have been produced [36 ], which may enable further transgenic engineering of human antibody V-regions or pseudogenes to produce transgenic chickens with human antibodies that utilize the novel gene conversion diversity generating systems of birds.…”

Section: Current Opinion In Structural Biologymentioning

confidence: 99%

Structural and genetic diversity in antibody repertoires from diverse species

Rios¹,

Criscitiello

Smider

2015

Current Opinion in Structural Biology

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The antibody repertoire is the fundamental unit that enables development of antigen specific adaptive immune responses against pathogens. Different species have developed diverse genetic and structural strategies to create their respective antibody repertoires. Here we review the shark, chicken, camel, and cow repertoires as unique examples of structural and genetic diversity. Given the enormous importance of antibodies in medicine and biological research, the novel properties of these antibody repertoires may enable discovery or engineering of antibodies from these non-human species against difficult or important epitopes.

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“…Wild-type DT40 cells and 293T cells were obtained from the RIKEN Cell Bank. DT40-ASF cells [7], DT40-SW cells [11], and DT40-SWDC cells [12] were described previously. DT40 cells were cultured as described previously [11].…”

Section: Cell Linesmentioning

confidence: 99%

“…The frequency of cells developing a strong green fluorescence signal was analyzed using FACS Array (BD Biosciences). The nucleotide sequence analysis of the IgVH gene was carried out after 3-week cultures of DT40-SW cells and DT40-SWDC cells, with or without overexpression of SRSF1-3, as described previously [6,12].…”

Section: Analysis Of Igv Diversificationmentioning

confidence: 99%

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SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus

Kawaguchi

Nariki

Kawamoto

et al. 2017

Biochemical and Biophysical Research Communications

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Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSF1-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSF1-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, overexpression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSF1-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C-terminus-dependent manner.

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Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire

Cited by 8 publications

References 24 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

Structural and genetic diversity in antibody repertoires from diverse species

SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus

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