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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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DNase I hypersensitive sites of GFP transgenes. (A) Southern blot of two C-GFP and two A-GFP subclones. DNA from DNase I–digested cells was digested with Pst I and Bcl I and hybridized with a GFP probe. 0, 10, and 50 U DNase I are indicated by the filled horizontal arrows. (B) Ethidium bromide–stained gel of DNA fragments before hybridization for A. (C) An ovalbumin probe was used to reprobe the blot in A. (D) Vertical arrows indicate DNase I hypersensitive sites observed with GFP for C-GFP and A-GFP clones. The horizontal blue box indicates the probe. The experiment was repeated with DNase I digestion of different C-GFP and A-GFP cell clones showing no difference in DNase hypersensitivity.
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fig7: DNase I hypersensitive sites of GFP transgenes. (A) Southern blot of two C-GFP and two A-GFP subclones. DNA from DNase I–digested cells was digested with Pst I and Bcl I and hybridized with a GFP probe. 0, 10, and 50 U DNase I are indicated by the filled horizontal arrows. (B) Ethidium bromide–stained gel of DNA fragments before hybridization for A. (C) An ovalbumin probe was used to reprobe the blot in A. (D) Vertical arrows indicate DNase I hypersensitive sites observed with GFP for C-GFP and A-GFP clones. The horizontal blue box indicates the probe. The experiment was repeated with DNase I digestion of different C-GFP and A-GFP cell clones showing no difference in DNase hypersensitivity.

Mentions: Between rearranged Ig alleles, which are mutable, and unrearranged Ig alleles, there are dramatic differences in DNase I hypersensitive sites (Storb et al., 1986; Thompson and Neiman, 1987). To investigate factors leading to differences in C-GFP and A-GFP mutation frequencies, the overall accessibility of DNA between C-GFP and A-GFP clones within the transgene-specific region was examined by treating nuclei with DNase I (Fig. 7). The digestibility of the two C-GFP clones appears greater than that of the A-GFP clones (Fig. 7 A). However, the ethidium bromide–stained gel showed that there was less DNA in the C-GFP clones, and overall, the DNA was more digested with lower amounts of DNA (Fig. 7 B). Thus, the digestability is equal. This is also supported by reprobing the Southern blot with an ovalbumin probe: the ovalbumin gene is also more digested in the C-GFP clones (Fig. 7 C). The ovalbumin gene is not expressed in DT40; therefore, one does not see DNase I hypersensitive sites but an overall increasing digestion with increasing DNase I. Among C-GFP clones, there was no variability in hypersensitive sites despite the differences in integration sites (Fig. 7 D). This was true among A-GFP clones as well. However, contrary to our expectation, DNase I hypersensitive sites for C-GFP and A-GFP clones also showed identical patterns (Fig. 7 D). Thus, it appears that the C-GFP and A-GFP transgenes exist in equally accessible chromatin.


Attracting AID to targets of somatic hypermutation.

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

DNase I hypersensitive sites of GFP transgenes. (A) Southern blot of two C-GFP and two A-GFP subclones. DNA from DNase I–digested cells was digested with Pst I and Bcl I and hybridized with a GFP probe. 0, 10, and 50 U DNase I are indicated by the filled horizontal arrows. (B) Ethidium bromide–stained gel of DNA fragments before hybridization for A. (C) An ovalbumin probe was used to reprobe the blot in A. (D) Vertical arrows indicate DNase I hypersensitive sites observed with GFP for C-GFP and A-GFP clones. The horizontal blue box indicates the probe. The experiment was repeated with DNase I digestion of different C-GFP and A-GFP cell clones showing no difference in DNase hypersensitivity.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: DNase I hypersensitive sites of GFP transgenes. (A) Southern blot of two C-GFP and two A-GFP subclones. DNA from DNase I–digested cells was digested with Pst I and Bcl I and hybridized with a GFP probe. 0, 10, and 50 U DNase I are indicated by the filled horizontal arrows. (B) Ethidium bromide–stained gel of DNA fragments before hybridization for A. (C) An ovalbumin probe was used to reprobe the blot in A. (D) Vertical arrows indicate DNase I hypersensitive sites observed with GFP for C-GFP and A-GFP clones. The horizontal blue box indicates the probe. The experiment was repeated with DNase I digestion of different C-GFP and A-GFP cell clones showing no difference in DNase hypersensitivity.
Mentions: Between rearranged Ig alleles, which are mutable, and unrearranged Ig alleles, there are dramatic differences in DNase I hypersensitive sites (Storb et al., 1986; Thompson and Neiman, 1987). To investigate factors leading to differences in C-GFP and A-GFP mutation frequencies, the overall accessibility of DNA between C-GFP and A-GFP clones within the transgene-specific region was examined by treating nuclei with DNase I (Fig. 7). The digestibility of the two C-GFP clones appears greater than that of the A-GFP clones (Fig. 7 A). However, the ethidium bromide–stained gel showed that there was less DNA in the C-GFP clones, and overall, the DNA was more digested with lower amounts of DNA (Fig. 7 B). Thus, the digestability is equal. This is also supported by reprobing the Southern blot with an ovalbumin probe: the ovalbumin gene is also more digested in the C-GFP clones (Fig. 7 C). The ovalbumin gene is not expressed in DT40; therefore, one does not see DNase I hypersensitive sites but an overall increasing digestion with increasing DNase I. Among C-GFP clones, there was no variability in hypersensitive sites despite the differences in integration sites (Fig. 7 D). This was true among A-GFP clones as well. However, contrary to our expectation, DNase I hypersensitive sites for C-GFP and A-GFP clones also showed identical patterns (Fig. 7 D). Thus, it appears that the C-GFP and A-GFP transgenes exist in equally accessible chromatin.

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