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Expansion of stochastic expression repertoire by tandem duplication in mouse Protocadherin-α cluster.

Kaneko R, Abe M, Hirabayashi T, Uchimura A, Sakimura K, Yanagawa Y, Yagi T - Sci Rep (2014)

Bottom Line: Tandem duplications are concentrated within the Pcdh cluster throughout vertebrate evolution and as copy number variations (CNVs) in human populations, but the effects of tandem duplication in the Pcdh cluster remain elusive.Interestingly, the 5'-located duplicated Pcdh-αc2, which is constitutively expressed in the wild-type brain, shifted to stochastic expression accompanied by increased DNA methylation.These results demonstrate that tandem duplication in the Pcdh cluster expands the stochastic expression repertoire irrespective of sequence divergence.

View Article: PubMed Central - PubMed

Affiliation: Bioresource center, Gunma University Graduate School of Medicine.

ABSTRACT
Tandem duplications are concentrated within the Pcdh cluster throughout vertebrate evolution and as copy number variations (CNVs) in human populations, but the effects of tandem duplication in the Pcdh cluster remain elusive. To investigate the effects of tandem duplication in the Pcdh cluster, here we generated and analyzed a new line of the Pcdh cluster mutant mice. In the mutant allele, a 218-kb region containing the Pcdh-α2 to Pcdh-αc2 variable exons with their promoters was duplicated and the individual duplicated Pcdh isoforms can be disctinguished. The individual duplicated Pcdh-α isoforms showed diverse expression level with stochastic expression manner, even though those have an identical promoter sequence. Interestingly, the 5'-located duplicated Pcdh-αc2, which is constitutively expressed in the wild-type brain, shifted to stochastic expression accompanied by increased DNA methylation. These results demonstrate that tandem duplication in the Pcdh cluster expands the stochastic expression repertoire irrespective of sequence divergence.

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Tandem duplication increases DNA methylation of the Pcdh-α12 promoter, but not of Pcdh-α1 and Pcdh-α6.DNA methylation patterns on Pcdh-α1 (a), Pcdh-α6 (b), and Pcdh-α12 (c) in the cerebellum from 4-week-old wild-type and Pcdhαdup(2-c2)/dup(2-c2) mice. (Top) Schematic representations of Pcdh-α1, 6, and 12; positions of CpGs are shown to scale by vertical lines. (bottom) Results of bisulfite sequencing. Each circle represents a methylated (black) or unmethylated (white) CpG dinucleotide. Each row represents a single clone. A primer set was designed to amplify the region corresponding to the promoter (Pcdh-α1 and Pcdh-α12) or 5′ region of the exon (Pcdh-α6) containing the SNP. In addition, the 4-kb upstream region and 3′ region of Pcdh-α12 were analyzed. The percentage below each methylation pattern indicates the CpG methylation rate for each region. (d) Changes in DNA methylation levels during development. DNA methylation levels of the Pcdh-α1 promoter (top), 5′ region of the Pcdh-α6 exon (middle), and Pcdh-α12 promoter (bottom) were analyzed by bisulfite sequencing of sperm and of mice at E3.5, E7.5, E9.5, E12.5, 4-weeks old, and 3-months old. aP = 0.0071 compared with wt, P = 0.0002 compared with the 3′-located Pcdh-α12, bP < 0.0001 compared with wt, P < 0.0001 compared with the 3′-located Pcdh-α12, cP = 0.0005 compared with wt, P = 0.0264 compared with the 3′-located Pcdh-α12, dP = 0.0167 compared with wt, P = 0.0086 compared with the 3′-located Pcdh-α12. (e) DNA methylation levels in the liver and tail of 4-week-old mice. DNA methylation levels at the Pcdh-α1 promoter (top), 5′ region of the Pcdh-α6 exon (middle), and Pcdh-α12 promoter (bottom) were analyzed by bisulfite sequencing. eP = 0.0003 compared liver with tail of the 5′-located Pcdh-α12.
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f6: Tandem duplication increases DNA methylation of the Pcdh-α12 promoter, but not of Pcdh-α1 and Pcdh-α6.DNA methylation patterns on Pcdh-α1 (a), Pcdh-α6 (b), and Pcdh-α12 (c) in the cerebellum from 4-week-old wild-type and Pcdhαdup(2-c2)/dup(2-c2) mice. (Top) Schematic representations of Pcdh-α1, 6, and 12; positions of CpGs are shown to scale by vertical lines. (bottom) Results of bisulfite sequencing. Each circle represents a methylated (black) or unmethylated (white) CpG dinucleotide. Each row represents a single clone. A primer set was designed to amplify the region corresponding to the promoter (Pcdh-α1 and Pcdh-α12) or 5′ region of the exon (Pcdh-α6) containing the SNP. In addition, the 4-kb upstream region and 3′ region of Pcdh-α12 were analyzed. The percentage below each methylation pattern indicates the CpG methylation rate for each region. (d) Changes in DNA methylation levels during development. DNA methylation levels of the Pcdh-α1 promoter (top), 5′ region of the Pcdh-α6 exon (middle), and Pcdh-α12 promoter (bottom) were analyzed by bisulfite sequencing of sperm and of mice at E3.5, E7.5, E9.5, E12.5, 4-weeks old, and 3-months old. aP = 0.0071 compared with wt, P = 0.0002 compared with the 3′-located Pcdh-α12, bP < 0.0001 compared with wt, P < 0.0001 compared with the 3′-located Pcdh-α12, cP = 0.0005 compared with wt, P = 0.0264 compared with the 3′-located Pcdh-α12, dP = 0.0167 compared with wt, P = 0.0086 compared with the 3′-located Pcdh-α12. (e) DNA methylation levels in the liver and tail of 4-week-old mice. DNA methylation levels at the Pcdh-α1 promoter (top), 5′ region of the Pcdh-α6 exon (middle), and Pcdh-α12 promoter (bottom) were analyzed by bisulfite sequencing. eP = 0.0003 compared liver with tail of the 5′-located Pcdh-α12.

Mentions: We next extended the DNA methylation analysis to the stochastically expressed Pcdh-α isoforms. To distinguish the 5′-located from the 3′-located exons in the dup(2-c2) allele by SNP analysis after the bisulfite reaction, we focused on the Pcdh-α1 (promoter), Pcdh-α6 (exon 5′-region), and Pcdh-α12 (promoter) (Fig. 6a–6c). A mosaic methylation pattern was observed for all three, Pcdh-α1, Pcdh-α6, and Pcdh-α12, and similar DNA methylation levels, around 50%, were observed for Pcdh-α1 and Pcdh-α6 in the wild-type and the dup(2-c2) allele. However, in the promoter region of the 5′-located Pcdh-α12, we found a higher methylation level (50.0%) than in the wild-type (20.5%) or 3′-located Pcdh-α12 (30.6%).


Expansion of stochastic expression repertoire by tandem duplication in mouse Protocadherin-α cluster.

Kaneko R, Abe M, Hirabayashi T, Uchimura A, Sakimura K, Yanagawa Y, Yagi T - Sci Rep (2014)

Tandem duplication increases DNA methylation of the Pcdh-α12 promoter, but not of Pcdh-α1 and Pcdh-α6.DNA methylation patterns on Pcdh-α1 (a), Pcdh-α6 (b), and Pcdh-α12 (c) in the cerebellum from 4-week-old wild-type and Pcdhαdup(2-c2)/dup(2-c2) mice. (Top) Schematic representations of Pcdh-α1, 6, and 12; positions of CpGs are shown to scale by vertical lines. (bottom) Results of bisulfite sequencing. Each circle represents a methylated (black) or unmethylated (white) CpG dinucleotide. Each row represents a single clone. A primer set was designed to amplify the region corresponding to the promoter (Pcdh-α1 and Pcdh-α12) or 5′ region of the exon (Pcdh-α6) containing the SNP. In addition, the 4-kb upstream region and 3′ region of Pcdh-α12 were analyzed. The percentage below each methylation pattern indicates the CpG methylation rate for each region. (d) Changes in DNA methylation levels during development. DNA methylation levels of the Pcdh-α1 promoter (top), 5′ region of the Pcdh-α6 exon (middle), and Pcdh-α12 promoter (bottom) were analyzed by bisulfite sequencing of sperm and of mice at E3.5, E7.5, E9.5, E12.5, 4-weeks old, and 3-months old. aP = 0.0071 compared with wt, P = 0.0002 compared with the 3′-located Pcdh-α12, bP < 0.0001 compared with wt, P < 0.0001 compared with the 3′-located Pcdh-α12, cP = 0.0005 compared with wt, P = 0.0264 compared with the 3′-located Pcdh-α12, dP = 0.0167 compared with wt, P = 0.0086 compared with the 3′-located Pcdh-α12. (e) DNA methylation levels in the liver and tail of 4-week-old mice. DNA methylation levels at the Pcdh-α1 promoter (top), 5′ region of the Pcdh-α6 exon (middle), and Pcdh-α12 promoter (bottom) were analyzed by bisulfite sequencing. eP = 0.0003 compared liver with tail of the 5′-located Pcdh-α12.
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f6: Tandem duplication increases DNA methylation of the Pcdh-α12 promoter, but not of Pcdh-α1 and Pcdh-α6.DNA methylation patterns on Pcdh-α1 (a), Pcdh-α6 (b), and Pcdh-α12 (c) in the cerebellum from 4-week-old wild-type and Pcdhαdup(2-c2)/dup(2-c2) mice. (Top) Schematic representations of Pcdh-α1, 6, and 12; positions of CpGs are shown to scale by vertical lines. (bottom) Results of bisulfite sequencing. Each circle represents a methylated (black) or unmethylated (white) CpG dinucleotide. Each row represents a single clone. A primer set was designed to amplify the region corresponding to the promoter (Pcdh-α1 and Pcdh-α12) or 5′ region of the exon (Pcdh-α6) containing the SNP. In addition, the 4-kb upstream region and 3′ region of Pcdh-α12 were analyzed. The percentage below each methylation pattern indicates the CpG methylation rate for each region. (d) Changes in DNA methylation levels during development. DNA methylation levels of the Pcdh-α1 promoter (top), 5′ region of the Pcdh-α6 exon (middle), and Pcdh-α12 promoter (bottom) were analyzed by bisulfite sequencing of sperm and of mice at E3.5, E7.5, E9.5, E12.5, 4-weeks old, and 3-months old. aP = 0.0071 compared with wt, P = 0.0002 compared with the 3′-located Pcdh-α12, bP < 0.0001 compared with wt, P < 0.0001 compared with the 3′-located Pcdh-α12, cP = 0.0005 compared with wt, P = 0.0264 compared with the 3′-located Pcdh-α12, dP = 0.0167 compared with wt, P = 0.0086 compared with the 3′-located Pcdh-α12. (e) DNA methylation levels in the liver and tail of 4-week-old mice. DNA methylation levels at the Pcdh-α1 promoter (top), 5′ region of the Pcdh-α6 exon (middle), and Pcdh-α12 promoter (bottom) were analyzed by bisulfite sequencing. eP = 0.0003 compared liver with tail of the 5′-located Pcdh-α12.
Mentions: We next extended the DNA methylation analysis to the stochastically expressed Pcdh-α isoforms. To distinguish the 5′-located from the 3′-located exons in the dup(2-c2) allele by SNP analysis after the bisulfite reaction, we focused on the Pcdh-α1 (promoter), Pcdh-α6 (exon 5′-region), and Pcdh-α12 (promoter) (Fig. 6a–6c). A mosaic methylation pattern was observed for all three, Pcdh-α1, Pcdh-α6, and Pcdh-α12, and similar DNA methylation levels, around 50%, were observed for Pcdh-α1 and Pcdh-α6 in the wild-type and the dup(2-c2) allele. However, in the promoter region of the 5′-located Pcdh-α12, we found a higher methylation level (50.0%) than in the wild-type (20.5%) or 3′-located Pcdh-α12 (30.6%).

Bottom Line: Tandem duplications are concentrated within the Pcdh cluster throughout vertebrate evolution and as copy number variations (CNVs) in human populations, but the effects of tandem duplication in the Pcdh cluster remain elusive.Interestingly, the 5'-located duplicated Pcdh-αc2, which is constitutively expressed in the wild-type brain, shifted to stochastic expression accompanied by increased DNA methylation.These results demonstrate that tandem duplication in the Pcdh cluster expands the stochastic expression repertoire irrespective of sequence divergence.

View Article: PubMed Central - PubMed

Affiliation: Bioresource center, Gunma University Graduate School of Medicine.

ABSTRACT
Tandem duplications are concentrated within the Pcdh cluster throughout vertebrate evolution and as copy number variations (CNVs) in human populations, but the effects of tandem duplication in the Pcdh cluster remain elusive. To investigate the effects of tandem duplication in the Pcdh cluster, here we generated and analyzed a new line of the Pcdh cluster mutant mice. In the mutant allele, a 218-kb region containing the Pcdh-α2 to Pcdh-αc2 variable exons with their promoters was duplicated and the individual duplicated Pcdh isoforms can be disctinguished. The individual duplicated Pcdh-α isoforms showed diverse expression level with stochastic expression manner, even though those have an identical promoter sequence. Interestingly, the 5'-located duplicated Pcdh-αc2, which is constitutively expressed in the wild-type brain, shifted to stochastic expression accompanied by increased DNA methylation. These results demonstrate that tandem duplication in the Pcdh cluster expands the stochastic expression repertoire irrespective of sequence divergence.

Show MeSH