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The transcriptional promoter regulates hypermutation of the antibody heavy chain locus.

Tumas-Brundage K, Manser T - J. Exp. Med. (1997)

Bottom Line: However, while the distribution of mutation in such loci appears normal, the frequency of mutation does not.Conversely, moving the VH promoter 750 bp upstream of its normal location results in a commensurate change in the site specificity of hypermutation in H chain loci, and the foreign DNA inserted into the VH leader intron to produce this promoter displacement is hypermutated in a manner indistinguishable from natural Ig DNA.These data establish a direct mechanistic link between the IgH transcription and hypermutation processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

ABSTRACT
A somatic process introduces mutations into antibody variable (V) region genes at a high rate in many vertebrates, and is a major source of antibody diversity. The mechanism of this hypermutation process remains enigmatic, although retrospective studies and transgenic experiments have recently suggested a role for transcriptional regulatory elements. Here, we demonstrate that mouse heavy (H) chain loci in which the natural VH promoter has been replaced by a heterologous promoter undergo hypermutation. However, while the distribution of mutation in such loci appears normal, the frequency of mutation does not. Conversely, moving the VH promoter 750 bp upstream of its normal location results in a commensurate change in the site specificity of hypermutation in H chain loci, and the foreign DNA inserted into the VH leader intron to produce this promoter displacement is hypermutated in a manner indistinguishable from natural Ig DNA. These data establish a direct mechanistic link between the IgH transcription and hypermutation processes.

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Mutation frequency distribution in enlarged  leader intron hybrid loci, wild  type 36-65VH hybrid loci, and  canonical endogenous IgH loci  from normal A/J mice. Hybridomas expressing all enlarged leader  intron and wild-type 36-65VH  hybrid loci, and most endogenous IgH loci were isolated using the hyperimmunized primary  protocol described in Materials  and Methods. Mutation frequency was determined as described in Fig. 4. Seven enlarged  leader intron hybrid loci were  analyzed. The data obtained  from intervals in the D. melanogaster intron from these hybrid  loci (Fig. 4) are not shown. Sequences of the four wild-type  36-65VH hybrid loci used to  generate this graph have been  previously described (18). The  eighteen normal A/J VH locus  sequences used were also previously described (17). For these  endogenous A/J loci, data was  not available for the first 5′ interval in the VDJ, and in the first  interval flanking the 3′ side of  VDJ. *, 3′ flanking interval containing the two mutations from  the hybridoma 4ACPM1-3F1,  which had a 350-bp intron deletion and the most VH mutations  (12) of any hybrid locus expressing hybridoma analyzed.
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Figure 5: Mutation frequency distribution in enlarged leader intron hybrid loci, wild type 36-65VH hybrid loci, and canonical endogenous IgH loci from normal A/J mice. Hybridomas expressing all enlarged leader intron and wild-type 36-65VH hybrid loci, and most endogenous IgH loci were isolated using the hyperimmunized primary protocol described in Materials and Methods. Mutation frequency was determined as described in Fig. 4. Seven enlarged leader intron hybrid loci were analyzed. The data obtained from intervals in the D. melanogaster intron from these hybrid loci (Fig. 4) are not shown. Sequences of the four wild-type 36-65VH hybrid loci used to generate this graph have been previously described (18). The eighteen normal A/J VH locus sequences used were also previously described (17). For these endogenous A/J loci, data was not available for the first 5′ interval in the VDJ, and in the first interval flanking the 3′ side of VDJ. *, 3′ flanking interval containing the two mutations from the hybridoma 4ACPM1-3F1, which had a 350-bp intron deletion and the most VH mutations (12) of any hybrid locus expressing hybridoma analyzed.

Mentions: Sequencing of DNA flanking the 5′ side of the VH gene in seven enlarged leader intron hybrid loci revealed high frequencies of mutation in both the Drosophila intron and natural Ig intron regions (Fig. 4). However, few mutations were observed in the region flanking the 3′ side of VH in these hybrid loci, a region that, as discussed above, displays a high frequency of mutation in wild-type 36-65VH hybrid and endogenous canonical VH loci (Fig. 5). These data indicate that the distribution, but not the rate, of mutation was altered due to the lengthening of the leader intron.


The transcriptional promoter regulates hypermutation of the antibody heavy chain locus.

Tumas-Brundage K, Manser T - J. Exp. Med. (1997)

Mutation frequency distribution in enlarged  leader intron hybrid loci, wild  type 36-65VH hybrid loci, and  canonical endogenous IgH loci  from normal A/J mice. Hybridomas expressing all enlarged leader  intron and wild-type 36-65VH  hybrid loci, and most endogenous IgH loci were isolated using the hyperimmunized primary  protocol described in Materials  and Methods. Mutation frequency was determined as described in Fig. 4. Seven enlarged  leader intron hybrid loci were  analyzed. The data obtained  from intervals in the D. melanogaster intron from these hybrid  loci (Fig. 4) are not shown. Sequences of the four wild-type  36-65VH hybrid loci used to  generate this graph have been  previously described (18). The  eighteen normal A/J VH locus  sequences used were also previously described (17). For these  endogenous A/J loci, data was  not available for the first 5′ interval in the VDJ, and in the first  interval flanking the 3′ side of  VDJ. *, 3′ flanking interval containing the two mutations from  the hybridoma 4ACPM1-3F1,  which had a 350-bp intron deletion and the most VH mutations  (12) of any hybrid locus expressing hybridoma analyzed.
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Related In: Results  -  Collection

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Figure 5: Mutation frequency distribution in enlarged leader intron hybrid loci, wild type 36-65VH hybrid loci, and canonical endogenous IgH loci from normal A/J mice. Hybridomas expressing all enlarged leader intron and wild-type 36-65VH hybrid loci, and most endogenous IgH loci were isolated using the hyperimmunized primary protocol described in Materials and Methods. Mutation frequency was determined as described in Fig. 4. Seven enlarged leader intron hybrid loci were analyzed. The data obtained from intervals in the D. melanogaster intron from these hybrid loci (Fig. 4) are not shown. Sequences of the four wild-type 36-65VH hybrid loci used to generate this graph have been previously described (18). The eighteen normal A/J VH locus sequences used were also previously described (17). For these endogenous A/J loci, data was not available for the first 5′ interval in the VDJ, and in the first interval flanking the 3′ side of VDJ. *, 3′ flanking interval containing the two mutations from the hybridoma 4ACPM1-3F1, which had a 350-bp intron deletion and the most VH mutations (12) of any hybrid locus expressing hybridoma analyzed.
Mentions: Sequencing of DNA flanking the 5′ side of the VH gene in seven enlarged leader intron hybrid loci revealed high frequencies of mutation in both the Drosophila intron and natural Ig intron regions (Fig. 4). However, few mutations were observed in the region flanking the 3′ side of VH in these hybrid loci, a region that, as discussed above, displays a high frequency of mutation in wild-type 36-65VH hybrid and endogenous canonical VH loci (Fig. 5). These data indicate that the distribution, but not the rate, of mutation was altered due to the lengthening of the leader intron.

Bottom Line: However, while the distribution of mutation in such loci appears normal, the frequency of mutation does not.Conversely, moving the VH promoter 750 bp upstream of its normal location results in a commensurate change in the site specificity of hypermutation in H chain loci, and the foreign DNA inserted into the VH leader intron to produce this promoter displacement is hypermutated in a manner indistinguishable from natural Ig DNA.These data establish a direct mechanistic link between the IgH transcription and hypermutation processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

ABSTRACT
A somatic process introduces mutations into antibody variable (V) region genes at a high rate in many vertebrates, and is a major source of antibody diversity. The mechanism of this hypermutation process remains enigmatic, although retrospective studies and transgenic experiments have recently suggested a role for transcriptional regulatory elements. Here, we demonstrate that mouse heavy (H) chain loci in which the natural VH promoter has been replaced by a heterologous promoter undergo hypermutation. However, while the distribution of mutation in such loci appears normal, the frequency of mutation does not. Conversely, moving the VH promoter 750 bp upstream of its normal location results in a commensurate change in the site specificity of hypermutation in H chain loci, and the foreign DNA inserted into the VH leader intron to produce this promoter displacement is hypermutated in a manner indistinguishable from natural Ig DNA. These data establish a direct mechanistic link between the IgH transcription and hypermutation processes.

Show MeSH