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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 the DNA methylation of Pcdh-αc2 and Pcdh-αc1.(a) Schematic representation of Pcdh-αc2; the positions of CpGs and HpaII/MspI sites are shown to scale by vertical lines. (b) 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 5′ region of the exon, which has the same sequence in the 5′- and 3′-located duplicated Pcdh-αc2 isoforms. The percentage below each methylation pattern indicates the CpG methylation rate for the region. C, D, Discrimination of the DNA methylation between the 5′- and 3′-located duplicated Pcdh-αc2 isoforms by HpaII digestion-mediated analysis. (c) Electrophoresis results. D, Example chromatograms showing that the HpaII-resistant fraction contained the region of the 5′-located duplicated Pcdh-αc2 exon. E, Schematic representation of Pcdh-αc1; positions of CpGs are shown to scale by vertical lines. F, Results of bisulfite sequencing. A primer set was designed to amplify the region corresponding to the Pcdh-αc1 promoter, which has the same sequence in the 5′- and 3′-located Pcdh-αc1 duplicates. The percentage below each methylation pattern indicates the CpG methylation rate for the region.
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f5: Tandem duplication increases the DNA methylation of Pcdh-αc2 and Pcdh-αc1.(a) Schematic representation of Pcdh-αc2; the positions of CpGs and HpaII/MspI sites are shown to scale by vertical lines. (b) 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 5′ region of the exon, which has the same sequence in the 5′- and 3′-located duplicated Pcdh-αc2 isoforms. The percentage below each methylation pattern indicates the CpG methylation rate for the region. C, D, Discrimination of the DNA methylation between the 5′- and 3′-located duplicated Pcdh-αc2 isoforms by HpaII digestion-mediated analysis. (c) Electrophoresis results. D, Example chromatograms showing that the HpaII-resistant fraction contained the region of the 5′-located duplicated Pcdh-αc2 exon. E, Schematic representation of Pcdh-αc1; positions of CpGs are shown to scale by vertical lines. F, Results of bisulfite sequencing. A primer set was designed to amplify the region corresponding to the Pcdh-αc1 promoter, which has the same sequence in the 5′- and 3′-located Pcdh-αc1 duplicates. The percentage below each methylation pattern indicates the CpG methylation rate for the region.

Mentions: The 5′-located Pcdh-αc2 was down-regulated in the Pcdhαdup(2-c2)/dup(2-c2) mouse cerebellum (Fig. 2) and acquired a stochastic expression pattern (Fig. 4), suggesting that the regulation was different between the 5′-located and the 3′-located Pcdh-αc2 in the dup(2-c2) allele. Since Pcdh-αc2 is extensively hypomethylated in the wild-type mouse brain, and higher mosaic DNA methylation levels are correlated with a lower transcription of stochastically expressed Pcdh genes34353637, we first examined the DNA methylation of Pcdh-αc2 in the Pcdhαdup(2-c2)/dup(2-c2) mouse cerebellum using bisulfite sequencing (Fig. 5a and 5b). The results clearly showed higher mosaic DNA methylation of Pcdh-αc2 in the Pcdhαdup(2-c2)/dup(2-c2) mouse (11.0%) compared with the wild-type mouse (0.3%).


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 the DNA methylation of Pcdh-αc2 and Pcdh-αc1.(a) Schematic representation of Pcdh-αc2; the positions of CpGs and HpaII/MspI sites are shown to scale by vertical lines. (b) 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 5′ region of the exon, which has the same sequence in the 5′- and 3′-located duplicated Pcdh-αc2 isoforms. The percentage below each methylation pattern indicates the CpG methylation rate for the region. C, D, Discrimination of the DNA methylation between the 5′- and 3′-located duplicated Pcdh-αc2 isoforms by HpaII digestion-mediated analysis. (c) Electrophoresis results. D, Example chromatograms showing that the HpaII-resistant fraction contained the region of the 5′-located duplicated Pcdh-αc2 exon. E, Schematic representation of Pcdh-αc1; positions of CpGs are shown to scale by vertical lines. F, Results of bisulfite sequencing. A primer set was designed to amplify the region corresponding to the Pcdh-αc1 promoter, which has the same sequence in the 5′- and 3′-located Pcdh-αc1 duplicates. The percentage below each methylation pattern indicates the CpG methylation rate for the region.
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f5: Tandem duplication increases the DNA methylation of Pcdh-αc2 and Pcdh-αc1.(a) Schematic representation of Pcdh-αc2; the positions of CpGs and HpaII/MspI sites are shown to scale by vertical lines. (b) 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 5′ region of the exon, which has the same sequence in the 5′- and 3′-located duplicated Pcdh-αc2 isoforms. The percentage below each methylation pattern indicates the CpG methylation rate for the region. C, D, Discrimination of the DNA methylation between the 5′- and 3′-located duplicated Pcdh-αc2 isoforms by HpaII digestion-mediated analysis. (c) Electrophoresis results. D, Example chromatograms showing that the HpaII-resistant fraction contained the region of the 5′-located duplicated Pcdh-αc2 exon. E, Schematic representation of Pcdh-αc1; positions of CpGs are shown to scale by vertical lines. F, Results of bisulfite sequencing. A primer set was designed to amplify the region corresponding to the Pcdh-αc1 promoter, which has the same sequence in the 5′- and 3′-located Pcdh-αc1 duplicates. The percentage below each methylation pattern indicates the CpG methylation rate for the region.
Mentions: The 5′-located Pcdh-αc2 was down-regulated in the Pcdhαdup(2-c2)/dup(2-c2) mouse cerebellum (Fig. 2) and acquired a stochastic expression pattern (Fig. 4), suggesting that the regulation was different between the 5′-located and the 3′-located Pcdh-αc2 in the dup(2-c2) allele. Since Pcdh-αc2 is extensively hypomethylated in the wild-type mouse brain, and higher mosaic DNA methylation levels are correlated with a lower transcription of stochastically expressed Pcdh genes34353637, we first examined the DNA methylation of Pcdh-αc2 in the Pcdhαdup(2-c2)/dup(2-c2) mouse cerebellum using bisulfite sequencing (Fig. 5a and 5b). The results clearly showed higher mosaic DNA methylation of Pcdh-αc2 in the Pcdhαdup(2-c2)/dup(2-c2) mouse (11.0%) compared with the wild-type mouse (0.3%).

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