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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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C-GFP and A-GFP clones in an AID-deficient background. Six transgenic clones each from Cre DT40 or AID−/− Cre DT40 lines were monitored for GFP+ cells by flow cytometry. The 24 subclones were analyzed by FACS weekly for 4 wk. Green boxes are as in Fig. 3.
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fig6: C-GFP and A-GFP clones in an AID-deficient background. Six transgenic clones each from Cre DT40 or AID−/− Cre DT40 lines were monitored for GFP+ cells by flow cytometry. The 24 subclones were analyzed by FACS weekly for 4 wk. Green boxes are as in Fig. 3.

Mentions: To determine whether C-GFP transgenes were mutated by an AID-dependent mechanism, C-GFP and A-GFP constructs were transfected into AID−/− Cre DT40 or the parental clone, AID+ Cre DT40, and six independent clones were selected for each of the four combinations (Fig. 6). There were no significant differences in GFP mRNA levels between AID−/− and AID+ cells (Fig. S1). The AID+ but not the AID−/− cells expressed AID mRNA (Fig. S1). The flow cytometry assay of GFP showed no GFP+ cells in the AID−/− background in both C-GFP and A-GFP clones. However, in the AID+ cells, mutations were significant for C-GFP clones. Out of the six A-GFP clones, only two showed sporadic, very low numbers of GFP+ cells. We assume that, as in the experiments shown in Fig. 3, in the A-GFP clones of Fig. 6, the transgene was inserted near CAGGTG sites (see CAGGTG motifs near… and Fig. 10). The data indicate that the mutability differences of C-GFP and A-GFP transgenes are dependent on AID-mediated SHM (Fig. 6).


Attracting AID to targets of somatic hypermutation.

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

C-GFP and A-GFP clones in an AID-deficient background. Six transgenic clones each from Cre DT40 or AID−/− Cre DT40 lines were monitored for GFP+ cells by flow cytometry. The 24 subclones were analyzed by FACS weekly for 4 wk. Green boxes are as in Fig. 3.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig6: C-GFP and A-GFP clones in an AID-deficient background. Six transgenic clones each from Cre DT40 or AID−/− Cre DT40 lines were monitored for GFP+ cells by flow cytometry. The 24 subclones were analyzed by FACS weekly for 4 wk. Green boxes are as in Fig. 3.
Mentions: To determine whether C-GFP transgenes were mutated by an AID-dependent mechanism, C-GFP and A-GFP constructs were transfected into AID−/− Cre DT40 or the parental clone, AID+ Cre DT40, and six independent clones were selected for each of the four combinations (Fig. 6). There were no significant differences in GFP mRNA levels between AID−/− and AID+ cells (Fig. S1). The AID+ but not the AID−/− cells expressed AID mRNA (Fig. S1). The flow cytometry assay of GFP showed no GFP+ cells in the AID−/− background in both C-GFP and A-GFP clones. However, in the AID+ cells, mutations were significant for C-GFP clones. Out of the six A-GFP clones, only two showed sporadic, very low numbers of GFP+ cells. We assume that, as in the experiments shown in Fig. 3, in the A-GFP clones of Fig. 6, the transgene was inserted near CAGGTG sites (see CAGGTG motifs near… and Fig. 10). The data indicate that the mutability differences of C-GFP and A-GFP transgenes are dependent on AID-mediated SHM (Fig. 6).

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