Limits...
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.

Show MeSH
Percentage of GFP+ cells from C-GFP and A-GFP clones. 60 subclones each from C-GFP and A-GFP lines were monitored for GFP+ cells by flow cytometry every week for 6 wk. Green boxes are subclones with numbers of GFP+ cells ≥0.01% or 100 GFP+ cells/106 cells. Bolded borders indicate subclones that were sorted for GFP+ cells; red (black) borders indicate sorted subclones that did (did not) expand or survive for subsequent analyses.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Percentage of GFP+ cells from C-GFP and A-GFP clones. 60 subclones each from C-GFP and A-GFP lines were monitored for GFP+ cells by flow cytometry every week for 6 wk. Green boxes are subclones with numbers of GFP+ cells ≥0.01% or 100 GFP+ cells/106 cells. Bolded borders indicate subclones that were sorted for GFP+ cells; red (black) borders indicate sorted subclones that did (did not) expand or survive for subsequent analyses.

Mentions: From each transgenic clone, 12 subclones were derived by single-cell sorting of GFP−IgM+ cells. IgM+ cells were chosen to eliminate potential differences in growth efficiency between IgM+ and IgM− cell cultures. To determine the effect of the CAGGTG motif on SHM targeting, C-GFP and A-GFP subclones were cultured individually and GFP fluorescence was measured by flow cytometry weekly for 6 wk. When assayed in this manner, all C-GFP transgenic clones showed GFP+ subclones and, overall, 54 out of 60 subclones showed GFP+ cells. On the other hand, only two of the AAGGTG clones showed any significant numbers of GFP+ cells and, overall, only 18 out of 60 AAGGTG subclones showed any GFP+ cells during the 6-wk-long experiment (Fig. 3). The differences of observed GFP+ cells between C-GFP subclones and A-GFP subclones were highly significant (P < 0.00001). The mean frequencies of GFP+ cells in 106 cells were also higher in C-GFP clones, ranging between 20 and 60, whereas A-GFP clones were consistently <10 GFP+ cells/106 cells (Fig. 4 B).


Attracting AID to targets of somatic hypermutation.

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

Percentage of GFP+ cells from C-GFP and A-GFP clones. 60 subclones each from C-GFP and A-GFP lines were monitored for GFP+ cells by flow cytometry every week for 6 wk. Green boxes are subclones with numbers of GFP+ cells ≥0.01% or 100 GFP+ cells/106 cells. Bolded borders indicate subclones that were sorted for GFP+ cells; red (black) borders indicate sorted subclones that did (did not) expand or survive for subsequent analyses.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Percentage of GFP+ cells from C-GFP and A-GFP clones. 60 subclones each from C-GFP and A-GFP lines were monitored for GFP+ cells by flow cytometry every week for 6 wk. Green boxes are subclones with numbers of GFP+ cells ≥0.01% or 100 GFP+ cells/106 cells. Bolded borders indicate subclones that were sorted for GFP+ cells; red (black) borders indicate sorted subclones that did (did not) expand or survive for subsequent analyses.
Mentions: From each transgenic clone, 12 subclones were derived by single-cell sorting of GFP−IgM+ cells. IgM+ cells were chosen to eliminate potential differences in growth efficiency between IgM+ and IgM− cell cultures. To determine the effect of the CAGGTG motif on SHM targeting, C-GFP and A-GFP subclones were cultured individually and GFP fluorescence was measured by flow cytometry weekly for 6 wk. When assayed in this manner, all C-GFP transgenic clones showed GFP+ subclones and, overall, 54 out of 60 subclones showed GFP+ cells. On the other hand, only two of the AAGGTG clones showed any significant numbers of GFP+ cells and, overall, only 18 out of 60 AAGGTG subclones showed any GFP+ cells during the 6-wk-long experiment (Fig. 3). The differences of observed GFP+ cells between C-GFP subclones and A-GFP subclones were highly significant (P < 0.00001). The mean frequencies of GFP+ cells in 106 cells were also higher in C-GFP clones, ranging between 20 and 60, whereas A-GFP clones were consistently <10 GFP+ cells/106 cells (Fig. 4 B).

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