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Attracting AID to targets of somatic hypermutation.

Tanaka A, Shen HM, Ratnam S, Kodgire P, Storb U - J. Exp. Med. (2010)

Bottom Line: The CA versus AA effect requires AID.CAGGTG does not enhance transcription, chromatin acetylation, or overall target gene activity.Collectively with other recent findings, we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context.

View Article: PubMed Central - HTML - PubMed

Affiliation: Committee on Immunology, University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes requires activation-induced cytidine deaminase (AID). Although mistargeting of AID is detrimental to genome integrity, the mechanism and the cis-elements responsible for targeting of AID are largely unknown. We show that three CAGGTG cis-elements in the context of Ig enhancers are sufficient to target SHM to a nearby transcribed gene. The CAGGTG motif binds E47 in nuclear extracts of the mutating cells. Replacing CAGGTG with AAGGTG in the construct without any other E47 binding site eliminates SHM. The CA versus AA effect requires AID. CAGGTG does not enhance transcription, chromatin acetylation, or overall target gene activity. The other cis-elements of Ig enhancers alone cannot attract the SHM machinery. Collectively with other recent findings, we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context. Ig genes are the most highly mutated genes, presumably because of multiple CAGGTG motifs within the Ig genes, high transcription activity, and the presence of other cooperating elements in Ig enhancers.

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Mutation analysis of sorted GFP+ C-GFP clones. Sorted GFP+ cells were cultured for 1 wk, and 736 bp of the eGFP gene were cloned for sequencing analyses. From a total of 141 sequences, 121 contained a mutation at the G nucleotide (bp 1,099) of the premature stop codon. The x and y axes indicate the basepair of the transgene and the number of mutations, respectively. These were mostly G-to-C transversions. GFP+ cells were selected after either 2 or 4 wk of culture.
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fig5: Mutation analysis of sorted GFP+ C-GFP clones. Sorted GFP+ cells were cultured for 1 wk, and 736 bp of the eGFP gene were cloned for sequencing analyses. From a total of 141 sequences, 121 contained a mutation at the G nucleotide (bp 1,099) of the premature stop codon. The x and y axes indicate the basepair of the transgene and the number of mutations, respectively. These were mostly G-to-C transversions. GFP+ cells were selected after either 2 or 4 wk of culture.

Mentions: To analyze GFP sequences for mutations, sorted GFP+ cells from C-GFP and A-GFP subclones were cultured for 1 wk before DNA cloning and sequencing (Fig. 4 A). Although there were a few A-GFP clones with GFP+ cells, the A2-4 subclone (Fig. 3) was the only A-GFP subclone from which we were able to sort enough viable GFP+ cells to culture and expand for genomic DNA isolation. Sequencing analyses of the GFP region of C-GFP clones showed that 86% of the sequences contained a mutation at the premature stop codon (121 out of 141 sequences; Fig. 5). These were mostly G-to-C transversions at the third nucleotide of the stop codon. The SHM frequency of the GFP-expressing C-GFP clones was 1.38 × 10−3 mutations/bp. This is comparable to the SHM frequency of 3.05 × 10−3 in DT40 cells selected for loss of membrane Ig because of mutations of IgL genes (Arakawa et al., 2004). Because the latter cells overexpressed AID, the mutation frequency would be expected to be somewhat higher than in the cells we used that produce limiting amounts of AID (Arakawa et al., 2004). In accord with the SHM pattern of the IgL gene in DT40 cells, transversions were frequent.


Attracting AID to targets of somatic hypermutation.

Tanaka A, Shen HM, Ratnam S, Kodgire P, Storb U - J. Exp. Med. (2010)

Mutation analysis of sorted GFP+ C-GFP clones. Sorted GFP+ cells were cultured for 1 wk, and 736 bp of the eGFP gene were cloned for sequencing analyses. From a total of 141 sequences, 121 contained a mutation at the G nucleotide (bp 1,099) of the premature stop codon. The x and y axes indicate the basepair of the transgene and the number of mutations, respectively. These were mostly G-to-C transversions. GFP+ cells were selected after either 2 or 4 wk of culture.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2822603&req=5

fig5: Mutation analysis of sorted GFP+ C-GFP clones. Sorted GFP+ cells were cultured for 1 wk, and 736 bp of the eGFP gene were cloned for sequencing analyses. From a total of 141 sequences, 121 contained a mutation at the G nucleotide (bp 1,099) of the premature stop codon. The x and y axes indicate the basepair of the transgene and the number of mutations, respectively. These were mostly G-to-C transversions. GFP+ cells were selected after either 2 or 4 wk of culture.
Mentions: To analyze GFP sequences for mutations, sorted GFP+ cells from C-GFP and A-GFP subclones were cultured for 1 wk before DNA cloning and sequencing (Fig. 4 A). Although there were a few A-GFP clones with GFP+ cells, the A2-4 subclone (Fig. 3) was the only A-GFP subclone from which we were able to sort enough viable GFP+ cells to culture and expand for genomic DNA isolation. Sequencing analyses of the GFP region of C-GFP clones showed that 86% of the sequences contained a mutation at the premature stop codon (121 out of 141 sequences; Fig. 5). These were mostly G-to-C transversions at the third nucleotide of the stop codon. The SHM frequency of the GFP-expressing C-GFP clones was 1.38 × 10−3 mutations/bp. This is comparable to the SHM frequency of 3.05 × 10−3 in DT40 cells selected for loss of membrane Ig because of mutations of IgL genes (Arakawa et al., 2004). Because the latter cells overexpressed AID, the mutation frequency would be expected to be somewhat higher than in the cells we used that produce limiting amounts of AID (Arakawa et al., 2004). In accord with the SHM pattern of the IgL gene in DT40 cells, transversions were frequent.

Bottom Line: The CA versus AA effect requires AID.CAGGTG does not enhance transcription, chromatin acetylation, or overall target gene activity.Collectively with other recent findings, we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context.

View Article: PubMed Central - HTML - PubMed

Affiliation: Committee on Immunology, University of Chicago, Chicago, IL 60637, USA.

ABSTRACT
The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes requires activation-induced cytidine deaminase (AID). Although mistargeting of AID is detrimental to genome integrity, the mechanism and the cis-elements responsible for targeting of AID are largely unknown. We show that three CAGGTG cis-elements in the context of Ig enhancers are sufficient to target SHM to a nearby transcribed gene. The CAGGTG motif binds E47 in nuclear extracts of the mutating cells. Replacing CAGGTG with AAGGTG in the construct without any other E47 binding site eliminates SHM. The CA versus AA effect requires AID. CAGGTG does not enhance transcription, chromatin acetylation, or overall target gene activity. The other cis-elements of Ig enhancers alone cannot attract the SHM machinery. Collectively with other recent findings, we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context. Ig genes are the most highly mutated genes, presumably because of multiple CAGGTG motifs within the Ig genes, high transcription activity, and the presence of other cooperating elements in Ig enhancers.

Show MeSH