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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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Relative mRNA expression of C-GFP and A-GFP transgenes. Relative mRNA levels among five single-copy clones for eGFP and AID from C-GFP and A-GFP lines were measured by real-time PCR. The data show the mean of three independent experiments. β-Actin mRNA was used to normalize eGFP and AID mRNA levels. The y axis shows mRNA levels relative to clone C6.
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fig2: Relative mRNA expression of C-GFP and A-GFP transgenes. Relative mRNA levels among five single-copy clones for eGFP and AID from C-GFP and A-GFP lines were measured by real-time PCR. The data show the mean of three independent experiments. β-Actin mRNA was used to normalize eGFP and AID mRNA levels. The y axis shows mRNA levels relative to clone C6.

Mentions: Because SHM depends on transcription levels as well as transcription initiation (Peters and Storb, 1996), we further selected clones with equivalent levels of transgene transcription. To assess the levels of stable transcripts as a measure of transcription of the transgenes, we measured GFP mRNA levels by quantitative real-time PCR. Five C-GFP clones and five A-GFP clones were selected that expressed very similar levels of GFP mRNA (P > 0.1; Fig. 2).


Attracting AID to targets of somatic hypermutation.

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

Relative mRNA expression of C-GFP and A-GFP transgenes. Relative mRNA levels among five single-copy clones for eGFP and AID from C-GFP and A-GFP lines were measured by real-time PCR. The data show the mean of three independent experiments. β-Actin mRNA was used to normalize eGFP and AID mRNA levels. The y axis shows mRNA levels relative to clone C6.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Relative mRNA expression of C-GFP and A-GFP transgenes. Relative mRNA levels among five single-copy clones for eGFP and AID from C-GFP and A-GFP lines were measured by real-time PCR. The data show the mean of three independent experiments. β-Actin mRNA was used to normalize eGFP and AID mRNA levels. The y axis shows mRNA levels relative to clone C6.
Mentions: Because SHM depends on transcription levels as well as transcription initiation (Peters and Storb, 1996), we further selected clones with equivalent levels of transgene transcription. To assess the levels of stable transcripts as a measure of transcription of the transgenes, we measured GFP mRNA levels by quantitative real-time PCR. Five C-GFP clones and five A-GFP clones were selected that expressed very similar levels of GFP mRNA (P > 0.1; Fig. 2).

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