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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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Stochastic expression of duplicated Pcdh-α3, Pcdh-α5, and Pcdh-α7 in single Purkinje cells.(a) Electrophoresis results of single-cell RT-PCR for the Pcdh-α3, Pcdh-α5, or Pcdh-α7 and Pcp-2 genes in individual Purkinje cells. The numbers #1-2 ~ #1-173 designate individual cells. 1–3, tubes into which the cDNA from an individual Purkinje cell was divided; independent PCRs were performed for each tube. This figure shows only the cells that gave PCR products for Pcdh-α3, Pcdh-α5, or Pcdh-α7. (b) SNP analysis to distinguish between Pcdh-α transcripts from the 5′-located (shown as dup-5′) and 3′-located (shown as dup-3′) isoforms on the dup(2-c2) allele and those from the wild-type allele (designated JF1). (c) Classification of Pcdh-α3, Pcdh-α5, or Pcdh-α7 expressed from the dup(2-c2) allele in individual Purkinje cells. Blue and purple bars indicate the number of Purkinje cells expressing the 5′-located and 3′-located isoforms on the dup(2-c2) allele, respectively. The cells expressing one or two of three transcripts were excluded from the analysis.
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f3: Stochastic expression of duplicated Pcdh-α3, Pcdh-α5, and Pcdh-α7 in single Purkinje cells.(a) Electrophoresis results of single-cell RT-PCR for the Pcdh-α3, Pcdh-α5, or Pcdh-α7 and Pcp-2 genes in individual Purkinje cells. The numbers #1-2 ~ #1-173 designate individual cells. 1–3, tubes into which the cDNA from an individual Purkinje cell was divided; independent PCRs were performed for each tube. This figure shows only the cells that gave PCR products for Pcdh-α3, Pcdh-α5, or Pcdh-α7. (b) SNP analysis to distinguish between Pcdh-α transcripts from the 5′-located (shown as dup-5′) and 3′-located (shown as dup-3′) isoforms on the dup(2-c2) allele and those from the wild-type allele (designated JF1). (c) Classification of Pcdh-α3, Pcdh-α5, or Pcdh-α7 expressed from the dup(2-c2) allele in individual Purkinje cells. Blue and purple bars indicate the number of Purkinje cells expressing the 5′-located and 3′-located isoforms on the dup(2-c2) allele, respectively. The cells expressing one or two of three transcripts were excluded from the analysis.

Mentions: To investigate whether the duplicated Pcdh-αs retained their stochastic expression at the single-neuron level, we performed single-cell RT-PCR and SNP analysis on Purkinje cells of the PcdhαJF1/dup(2-c2) mice. The PcdhαJF1/dup(2-c2) mice were F1 mice from a JF1 × Pcdhαdup(2-c2)/dup(2-c2) cross. To distinguish the 5′-located exons (B6) and 3′-located exons (CBA) in the dup(2-c2) allele, and exons in the wild-type allele (JF1) by SNP analysis, we focused on Pcdh-α3, Pcdh-α5, Pcdh-α7 (Fig. 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)

Stochastic expression of duplicated Pcdh-α3, Pcdh-α5, and Pcdh-α7 in single Purkinje cells.(a) Electrophoresis results of single-cell RT-PCR for the Pcdh-α3, Pcdh-α5, or Pcdh-α7 and Pcp-2 genes in individual Purkinje cells. The numbers #1-2 ~ #1-173 designate individual cells. 1–3, tubes into which the cDNA from an individual Purkinje cell was divided; independent PCRs were performed for each tube. This figure shows only the cells that gave PCR products for Pcdh-α3, Pcdh-α5, or Pcdh-α7. (b) SNP analysis to distinguish between Pcdh-α transcripts from the 5′-located (shown as dup-5′) and 3′-located (shown as dup-3′) isoforms on the dup(2-c2) allele and those from the wild-type allele (designated JF1). (c) Classification of Pcdh-α3, Pcdh-α5, or Pcdh-α7 expressed from the dup(2-c2) allele in individual Purkinje cells. Blue and purple bars indicate the number of Purkinje cells expressing the 5′-located and 3′-located isoforms on the dup(2-c2) allele, respectively. The cells expressing one or two of three transcripts were excluded from the analysis.
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Related In: Results  -  Collection

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f3: Stochastic expression of duplicated Pcdh-α3, Pcdh-α5, and Pcdh-α7 in single Purkinje cells.(a) Electrophoresis results of single-cell RT-PCR for the Pcdh-α3, Pcdh-α5, or Pcdh-α7 and Pcp-2 genes in individual Purkinje cells. The numbers #1-2 ~ #1-173 designate individual cells. 1–3, tubes into which the cDNA from an individual Purkinje cell was divided; independent PCRs were performed for each tube. This figure shows only the cells that gave PCR products for Pcdh-α3, Pcdh-α5, or Pcdh-α7. (b) SNP analysis to distinguish between Pcdh-α transcripts from the 5′-located (shown as dup-5′) and 3′-located (shown as dup-3′) isoforms on the dup(2-c2) allele and those from the wild-type allele (designated JF1). (c) Classification of Pcdh-α3, Pcdh-α5, or Pcdh-α7 expressed from the dup(2-c2) allele in individual Purkinje cells. Blue and purple bars indicate the number of Purkinje cells expressing the 5′-located and 3′-located isoforms on the dup(2-c2) allele, respectively. The cells expressing one or two of three transcripts were excluded from the analysis.
Mentions: To investigate whether the duplicated Pcdh-αs retained their stochastic expression at the single-neuron level, we performed single-cell RT-PCR and SNP analysis on Purkinje cells of the PcdhαJF1/dup(2-c2) mice. The PcdhαJF1/dup(2-c2) mice were F1 mice from a JF1 × Pcdhαdup(2-c2)/dup(2-c2) cross. To distinguish the 5′-located exons (B6) and 3′-located exons (CBA) in the dup(2-c2) allele, and exons in the wild-type allele (JF1) by SNP analysis, we focused on Pcdh-α3, Pcdh-α5, Pcdh-α7 (Fig. 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