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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 5′-located Pcdh-αc2, but constitutive expression of 3′-located Pcdh-αc2 in single Purkinje cells.(a) Electrophoresis results of the split single-cell RT-PCR for the Pcdh-αc2 and Pcp-2 genes in individual Purkinje cells. The numbers #2-1 ~ #2-16 designate individual cells. 1–3, tubes into which the cDNA from an individual Purkinje cell was divided; independent PCRs were performed for each tube. (b) Example chromatograms showing the absence (upper) and presence (lower) of the 5′-located duplicated Pcdh-αc2 transcript. (c) SNP analysis to distinguish between Pcdh-αc2 transcripts from the 5′-located (dup-5′) and 3′-located (dup-3′) isoforms on the dup(2-c2) allele and from the wild-type allele (JF1). (d) Number of Purkinje cells showing the 5′-located duplicated Pcdh-αc2 transcript in 0, 1, 2, or 3 tubes. (e) Comparison of the Pcdh-αc2 expression levels in individual Purkinje cells (cells #2-4 and #2-13). Yellow, green, and red bars represent the expression ratio from the 5′- and 3′-located Pcdh-αc2 exons on the dup(2-c2) allele, and the wild-type (JF1) exon, respectively. Error bars indicate ±S.E.M of the averages from 3 tubes.
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f4: Stochastic expression of 5′-located Pcdh-αc2, but constitutive expression of 3′-located Pcdh-αc2 in single Purkinje cells.(a) Electrophoresis results of the split single-cell RT-PCR for the Pcdh-αc2 and Pcp-2 genes in individual Purkinje cells. The numbers #2-1 ~ #2-16 designate individual cells. 1–3, tubes into which the cDNA from an individual Purkinje cell was divided; independent PCRs were performed for each tube. (b) Example chromatograms showing the absence (upper) and presence (lower) of the 5′-located duplicated Pcdh-αc2 transcript. (c) SNP analysis to distinguish between Pcdh-αc2 transcripts from the 5′-located (dup-5′) and 3′-located (dup-3′) isoforms on the dup(2-c2) allele and from the wild-type allele (JF1). (d) Number of Purkinje cells showing the 5′-located duplicated Pcdh-αc2 transcript in 0, 1, 2, or 3 tubes. (e) Comparison of the Pcdh-αc2 expression levels in individual Purkinje cells (cells #2-4 and #2-13). Yellow, green, and red bars represent the expression ratio from the 5′- and 3′-located Pcdh-αc2 exons on the dup(2-c2) allele, and the wild-type (JF1) exon, respectively. Error bars indicate ±S.E.M of the averages from 3 tubes.

Mentions: To investigate whether the 5′-located and 3′-located duplicated Pcdh-αc2 retained its constitutive expression at the single-neuron level, single-cell RT-PCR and SNP analysis was carried out (Fig. 4). Of the 16 single Purkinje cells analyzed, all showed the Pcdh-αc2 transcript from the wild-type (JF1) and 3′-located duplicated exon in all three tubes. They were also all positive for Pcp-2, confirming that they were differentiated Purkinje cells. These results suggested that the Pcdh-αc2 wild-type exon (JF1) and 3′-located duplicated exon were expressed constitutively in differentiated Purkinje cells.


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 5′-located Pcdh-αc2, but constitutive expression of 3′-located Pcdh-αc2 in single Purkinje cells.(a) Electrophoresis results of the split single-cell RT-PCR for the Pcdh-αc2 and Pcp-2 genes in individual Purkinje cells. The numbers #2-1 ~ #2-16 designate individual cells. 1–3, tubes into which the cDNA from an individual Purkinje cell was divided; independent PCRs were performed for each tube. (b) Example chromatograms showing the absence (upper) and presence (lower) of the 5′-located duplicated Pcdh-αc2 transcript. (c) SNP analysis to distinguish between Pcdh-αc2 transcripts from the 5′-located (dup-5′) and 3′-located (dup-3′) isoforms on the dup(2-c2) allele and from the wild-type allele (JF1). (d) Number of Purkinje cells showing the 5′-located duplicated Pcdh-αc2 transcript in 0, 1, 2, or 3 tubes. (e) Comparison of the Pcdh-αc2 expression levels in individual Purkinje cells (cells #2-4 and #2-13). Yellow, green, and red bars represent the expression ratio from the 5′- and 3′-located Pcdh-αc2 exons on the dup(2-c2) allele, and the wild-type (JF1) exon, respectively. Error bars indicate ±S.E.M of the averages from 3 tubes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4151104&req=5

f4: Stochastic expression of 5′-located Pcdh-αc2, but constitutive expression of 3′-located Pcdh-αc2 in single Purkinje cells.(a) Electrophoresis results of the split single-cell RT-PCR for the Pcdh-αc2 and Pcp-2 genes in individual Purkinje cells. The numbers #2-1 ~ #2-16 designate individual cells. 1–3, tubes into which the cDNA from an individual Purkinje cell was divided; independent PCRs were performed for each tube. (b) Example chromatograms showing the absence (upper) and presence (lower) of the 5′-located duplicated Pcdh-αc2 transcript. (c) SNP analysis to distinguish between Pcdh-αc2 transcripts from the 5′-located (dup-5′) and 3′-located (dup-3′) isoforms on the dup(2-c2) allele and from the wild-type allele (JF1). (d) Number of Purkinje cells showing the 5′-located duplicated Pcdh-αc2 transcript in 0, 1, 2, or 3 tubes. (e) Comparison of the Pcdh-αc2 expression levels in individual Purkinje cells (cells #2-4 and #2-13). Yellow, green, and red bars represent the expression ratio from the 5′- and 3′-located Pcdh-αc2 exons on the dup(2-c2) allele, and the wild-type (JF1) exon, respectively. Error bars indicate ±S.E.M of the averages from 3 tubes.
Mentions: To investigate whether the 5′-located and 3′-located duplicated Pcdh-αc2 retained its constitutive expression at the single-neuron level, single-cell RT-PCR and SNP analysis was carried out (Fig. 4). Of the 16 single Purkinje cells analyzed, all showed the Pcdh-αc2 transcript from the wild-type (JF1) and 3′-located duplicated exon in all three tubes. They were also all positive for Pcp-2, confirming that they were differentiated Purkinje cells. These results suggested that the Pcdh-αc2 wild-type exon (JF1) and 3′-located duplicated exon were expressed constitutively in differentiated Purkinje cells.

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
Related in: MedlinePlus