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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 Here, we demonstrate that mouse heavy (H) chain loci in which the natural VH promoter has been replaced by a heterologous promoter undergo hypermutation.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 the enlarged leader intron hybrid loci.  Seven hybridomas from three  different mice (two of the CPM1  line and one of the CPM2 line)  representing the entire range of  VH mutation observed (see Fig. 2  and Table 2) were analyzed. The  mutation frequency was determined as follows: number of mutations in a 50-bp interval divided  by 350 (the total number of bases  in the interval sequenced from  all the hybridomas) × 100. The  first 50-bp interval began 8 bp  5′ of the purine rich motif in the  VH promoter. The solid lines under the D. melanogaster intron indicate those intervals that were  deleted from the hybrid loci in  hybridomas 4ACPM1-3F1 and  3ACPM2-23B2. A total of 350  bp were deleted from the  4ACPM1-3F1 hybrid locus; in  addition, this hybridoma also had  the most mutations in its V(D)J  coding sequence, 12, of any enlarged leader intron hybridoma  analyzed. *, 3′ flanking interval  in which two mutations were observed in the 4ACPM1-3F1 hybrid locus. A total of 470 bp were deleted from the 3ACPM2-23B2 hybrid locus. These  large deletions were scored as one mutation per interval deleted. The mutation frequency in the VDJ was calculated using either those mutations that do  not encode amino acid substitutions (silent mutations − stippled bars) or those mutations that encode amino acid changes (replacement mutations − diagonal striped bars).
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Figure 4: Mutation frequency distribution in the enlarged leader intron hybrid loci. Seven hybridomas from three different mice (two of the CPM1 line and one of the CPM2 line) representing the entire range of VH mutation observed (see Fig. 2 and Table 2) were analyzed. The mutation frequency was determined as follows: number of mutations in a 50-bp interval divided by 350 (the total number of bases in the interval sequenced from all the hybridomas) × 100. The first 50-bp interval began 8 bp 5′ of the purine rich motif in the VH promoter. The solid lines under the D. melanogaster intron indicate those intervals that were deleted from the hybrid loci in hybridomas 4ACPM1-3F1 and 3ACPM2-23B2. A total of 350 bp were deleted from the 4ACPM1-3F1 hybrid locus; in addition, this hybridoma also had the most mutations in its V(D)J coding sequence, 12, of any enlarged leader intron hybridoma analyzed. *, 3′ flanking interval in which two mutations were observed in the 4ACPM1-3F1 hybrid locus. A total of 470 bp were deleted from the 3ACPM2-23B2 hybrid locus. These large deletions were scored as one mutation per interval deleted. The mutation frequency in the VDJ was calculated using either those mutations that do not encode amino acid substitutions (silent mutations − stippled bars) or those mutations that encode amino acid changes (replacement mutations − diagonal striped bars).

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 the enlarged leader intron hybrid loci.  Seven hybridomas from three  different mice (two of the CPM1  line and one of the CPM2 line)  representing the entire range of  VH mutation observed (see Fig. 2  and Table 2) were analyzed. The  mutation frequency was determined as follows: number of mutations in a 50-bp interval divided  by 350 (the total number of bases  in the interval sequenced from  all the hybridomas) × 100. The  first 50-bp interval began 8 bp  5′ of the purine rich motif in the  VH promoter. The solid lines under the D. melanogaster intron indicate those intervals that were  deleted from the hybrid loci in  hybridomas 4ACPM1-3F1 and  3ACPM2-23B2. A total of 350  bp were deleted from the  4ACPM1-3F1 hybrid locus; in  addition, this hybridoma also had  the most mutations in its V(D)J  coding sequence, 12, of any enlarged leader intron hybridoma  analyzed. *, 3′ flanking interval  in which two mutations were observed in the 4ACPM1-3F1 hybrid locus. A total of 470 bp were deleted from the 3ACPM2-23B2 hybrid locus. These  large deletions were scored as one mutation per interval deleted. The mutation frequency in the VDJ was calculated using either those mutations that do  not encode amino acid substitutions (silent mutations − stippled bars) or those mutations that encode amino acid changes (replacement mutations − diagonal striped bars).
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Figure 4: Mutation frequency distribution in the enlarged leader intron hybrid loci. Seven hybridomas from three different mice (two of the CPM1 line and one of the CPM2 line) representing the entire range of VH mutation observed (see Fig. 2 and Table 2) were analyzed. The mutation frequency was determined as follows: number of mutations in a 50-bp interval divided by 350 (the total number of bases in the interval sequenced from all the hybridomas) × 100. The first 50-bp interval began 8 bp 5′ of the purine rich motif in the VH promoter. The solid lines under the D. melanogaster intron indicate those intervals that were deleted from the hybrid loci in hybridomas 4ACPM1-3F1 and 3ACPM2-23B2. A total of 350 bp were deleted from the 4ACPM1-3F1 hybrid locus; in addition, this hybridoma also had the most mutations in its V(D)J coding sequence, 12, of any enlarged leader intron hybridoma analyzed. *, 3′ flanking interval in which two mutations were observed in the 4ACPM1-3F1 hybrid locus. A total of 470 bp were deleted from the 3ACPM2-23B2 hybrid locus. These large deletions were scored as one mutation per interval deleted. The mutation frequency in the VDJ was calculated using either those mutations that do not encode amino acid substitutions (silent mutations − stippled bars) or those mutations that encode amino acid changes (replacement mutations − diagonal striped bars).
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