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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 Here, we demonstrate that mouse heavy (H) chain loci in which the natural VH promoter has been replaced by a heterologous promoter undergo hypermutation.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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Nucleotide sequences of VH genes in hybrid  loci expressed by hybridomas  from BP2 (B29 promoter) transgenic mice (above solid line) and  CPM (enlarged leader intron)  transgenic mice (below solid line).  Only those sequences containing at least one mutation are  shown. The reference sequence  is that of the unmutated 3665VH gene. Hybridoma names  are listed to the left of each sequence. Dashed lines indicate sequence identity. Mutations are  shown explicitly. Only those  codons in which mutations  were observed, as well as junctional codons, are shown. Recurrently observed nucleotide  changes at codons 58 and 59 are  indicated in bold. The rare junctional codons of the 36-65VH  transgene are underlined. Codons are numbered sequentially  from the mature amino terminus, and the location of complementarity determining regions (CDR) are shown. At codon 58, the recurrent mutation  (ACT→ ATT) results in a change from threonine to isoleucine, demonstrated to increase affinity for Ars two- to threefold (27). A variety of amino acid  substitutions have been recurrently observed at codon 59. A change from lysine to threonine (AAG→ ACG), observed in two enlarged leader intron hybrid loci, increases affinity for Ars two- to fourfold (27). The lysine to asparagine mutation (AGG→ AGC) observed at this position in 3 BP2 hybridomas  and 1 CPM hybridoma is recurrently observed, but its effect on Ars affinity has not been tested.
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Figure 3: Nucleotide sequences of VH genes in hybrid loci expressed by hybridomas from BP2 (B29 promoter) transgenic mice (above solid line) and CPM (enlarged leader intron) transgenic mice (below solid line). Only those sequences containing at least one mutation are shown. The reference sequence is that of the unmutated 3665VH gene. Hybridoma names are listed to the left of each sequence. Dashed lines indicate sequence identity. Mutations are shown explicitly. Only those codons in which mutations were observed, as well as junctional codons, are shown. Recurrently observed nucleotide changes at codons 58 and 59 are indicated in bold. The rare junctional codons of the 36-65VH transgene are underlined. Codons are numbered sequentially from the mature amino terminus, and the location of complementarity determining regions (CDR) are shown. At codon 58, the recurrent mutation (ACT→ ATT) results in a change from threonine to isoleucine, demonstrated to increase affinity for Ars two- to threefold (27). A variety of amino acid substitutions have been recurrently observed at codon 59. A change from lysine to threonine (AAG→ ACG), observed in two enlarged leader intron hybrid loci, increases affinity for Ars two- to fourfold (27). The lysine to asparagine mutation (AGG→ AGC) observed at this position in 3 BP2 hybridomas and 1 CPM hybridoma is recurrently observed, but its effect on Ars affinity has not been tested.

Mentions: The frequency of VH gene mutation in hybrid loci is not directly reflective of their intrinsic rate of mutation, as many mutations in coding region can be selected in vivo. Fig. 3 shows the sequence of the expressed VH genes in the B29 promoter-driven and enlarged leader intron hybrid loci that contain at least one somatic mutation. Of the eight VH genes in B29 promoter-driven hybrid loci, six contain somatic mutations at positions 58 and 59 in CDR2 that are recurrently observed among canonical VH genes. Two such mutations have been shown to result in increased affinity for Ars (27). In three such VH genes, these recurrent mutations are the only mutations observed, and in one VH gene, a recurrent mutation is one of only two mutations. Of these four VH genes (those expressed by hybridomas 4ABP2-16G6, AABP2-28aE5, 3ABP2-59D7, and 3ABP2-44H8), three are expressed by hybridomas isolated from secondary or tertiary responses. In wild-type 36-65VH genes in hybrid loci and endogenous canonical VH genes, particularly those expressed by secondary and tertiary hybridomas, such mutations are usually accompanied by many other “selectively neutral” (e.g., silent) mutations (18, 28). Of the nine mutated enlarged leader intron hybrid loci, five contain recurrent mutations. These data indicate that the frequency of VH coding region mutation is an overestimate of the reduced intrinsic mutation rate in this region in both types of modified hybrid loci, particularly those driven by the B29 promoter.


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

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

Nucleotide sequences of VH genes in hybrid  loci expressed by hybridomas  from BP2 (B29 promoter) transgenic mice (above solid line) and  CPM (enlarged leader intron)  transgenic mice (below solid line).  Only those sequences containing at least one mutation are  shown. The reference sequence  is that of the unmutated 3665VH gene. Hybridoma names  are listed to the left of each sequence. Dashed lines indicate sequence identity. Mutations are  shown explicitly. Only those  codons in which mutations  were observed, as well as junctional codons, are shown. Recurrently observed nucleotide  changes at codons 58 and 59 are  indicated in bold. The rare junctional codons of the 36-65VH  transgene are underlined. Codons are numbered sequentially  from the mature amino terminus, and the location of complementarity determining regions (CDR) are shown. At codon 58, the recurrent mutation  (ACT→ ATT) results in a change from threonine to isoleucine, demonstrated to increase affinity for Ars two- to threefold (27). A variety of amino acid  substitutions have been recurrently observed at codon 59. A change from lysine to threonine (AAG→ ACG), observed in two enlarged leader intron hybrid loci, increases affinity for Ars two- to fourfold (27). The lysine to asparagine mutation (AGG→ AGC) observed at this position in 3 BP2 hybridomas  and 1 CPM hybridoma is recurrently observed, but its effect on Ars affinity has not been tested.
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Figure 3: Nucleotide sequences of VH genes in hybrid loci expressed by hybridomas from BP2 (B29 promoter) transgenic mice (above solid line) and CPM (enlarged leader intron) transgenic mice (below solid line). Only those sequences containing at least one mutation are shown. The reference sequence is that of the unmutated 3665VH gene. Hybridoma names are listed to the left of each sequence. Dashed lines indicate sequence identity. Mutations are shown explicitly. Only those codons in which mutations were observed, as well as junctional codons, are shown. Recurrently observed nucleotide changes at codons 58 and 59 are indicated in bold. The rare junctional codons of the 36-65VH transgene are underlined. Codons are numbered sequentially from the mature amino terminus, and the location of complementarity determining regions (CDR) are shown. At codon 58, the recurrent mutation (ACT→ ATT) results in a change from threonine to isoleucine, demonstrated to increase affinity for Ars two- to threefold (27). A variety of amino acid substitutions have been recurrently observed at codon 59. A change from lysine to threonine (AAG→ ACG), observed in two enlarged leader intron hybrid loci, increases affinity for Ars two- to fourfold (27). The lysine to asparagine mutation (AGG→ AGC) observed at this position in 3 BP2 hybridomas and 1 CPM hybridoma is recurrently observed, but its effect on Ars affinity has not been tested.
Mentions: The frequency of VH gene mutation in hybrid loci is not directly reflective of their intrinsic rate of mutation, as many mutations in coding region can be selected in vivo. Fig. 3 shows the sequence of the expressed VH genes in the B29 promoter-driven and enlarged leader intron hybrid loci that contain at least one somatic mutation. Of the eight VH genes in B29 promoter-driven hybrid loci, six contain somatic mutations at positions 58 and 59 in CDR2 that are recurrently observed among canonical VH genes. Two such mutations have been shown to result in increased affinity for Ars (27). In three such VH genes, these recurrent mutations are the only mutations observed, and in one VH gene, a recurrent mutation is one of only two mutations. Of these four VH genes (those expressed by hybridomas 4ABP2-16G6, AABP2-28aE5, 3ABP2-59D7, and 3ABP2-44H8), three are expressed by hybridomas isolated from secondary or tertiary responses. In wild-type 36-65VH genes in hybrid loci and endogenous canonical VH genes, particularly those expressed by secondary and tertiary hybridomas, such mutations are usually accompanied by many other “selectively neutral” (e.g., silent) mutations (18, 28). Of the nine mutated enlarged leader intron hybrid loci, five contain recurrent mutations. These data indicate that the frequency of VH coding region mutation is an overestimate of the reduced intrinsic mutation rate in this region in both types of modified hybrid loci, particularly those driven by the B29 promoter.

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
Related in: MedlinePlus