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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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Targeted tandem duplication in the Pcdh-α cluster.(a) Genomic structure of the Pcdh-α wild-type allele. The Pcdh-α allele consists of variable region exons (1–12, c1 and c2) and constant region exons (CR1–CR3). Each variable region exon is transcribed from its own promoter in a stochastic (black arrows) or constitutive (red arrows) manner. A Pcdh-α transcript is produced from one of the variable region exons and the set of constant region exons by splicing. (b) The G16Neo allele: a loxP site was inserted between Pcdh-α1 and Pcdh-α2. The loxP site is shown as a red triangle. (c) The SR allele: a loxP site was inserted between Pcdh-αc2 and CR1. (d) The dup(2-c2) allele: duplication of Pcdh-α2-Pcdh-αc2. The dup(2-c2) allele was produced by Cre-loxP-mediated trans-allelic meiotic recombination between the G16neo and SR alleles. The duplicated segments are shown under the position of the original segments. Importantly, each duplicate Pcdh-α3, Pcdh-α5, Pcdh-α6, Pcdh-α7, Pcdh-α9, Pcdh-α10, Pcdh-α12, and Pcdh-αc2 could be distinguished by SNP analysis; the 5′ genes were from B6 and the 3′ ones were from CBA. (e & f) Confirmation of the duplication allele by Southern blotting (e) and PCR (f). G, Histological analysis of the cerebellum of 4-week-old wild-type and Pcdhαdup(2-c2)/dup(2-c2) mice. Nissl staining (left), immunostaining for neurofilament (middle), and in situ hybridization using a Pcdh-α CR probe (right). Cb1-10, 1st – 10th lobule of the cerebellum; Gra, granule cell layer; Mol, molecular layer; Pur, Purkinje cell layer.
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f1: Targeted tandem duplication in the Pcdh-α cluster.(a) Genomic structure of the Pcdh-α wild-type allele. The Pcdh-α allele consists of variable region exons (1–12, c1 and c2) and constant region exons (CR1–CR3). Each variable region exon is transcribed from its own promoter in a stochastic (black arrows) or constitutive (red arrows) manner. A Pcdh-α transcript is produced from one of the variable region exons and the set of constant region exons by splicing. (b) The G16Neo allele: a loxP site was inserted between Pcdh-α1 and Pcdh-α2. The loxP site is shown as a red triangle. (c) The SR allele: a loxP site was inserted between Pcdh-αc2 and CR1. (d) The dup(2-c2) allele: duplication of Pcdh-α2-Pcdh-αc2. The dup(2-c2) allele was produced by Cre-loxP-mediated trans-allelic meiotic recombination between the G16neo and SR alleles. The duplicated segments are shown under the position of the original segments. Importantly, each duplicate Pcdh-α3, Pcdh-α5, Pcdh-α6, Pcdh-α7, Pcdh-α9, Pcdh-α10, Pcdh-α12, and Pcdh-αc2 could be distinguished by SNP analysis; the 5′ genes were from B6 and the 3′ ones were from CBA. (e & f) Confirmation of the duplication allele by Southern blotting (e) and PCR (f). G, Histological analysis of the cerebellum of 4-week-old wild-type and Pcdhαdup(2-c2)/dup(2-c2) mice. Nissl staining (left), immunostaining for neurofilament (middle), and in situ hybridization using a Pcdh-α CR probe (right). Cb1-10, 1st – 10th lobule of the cerebellum; Gra, granule cell layer; Mol, molecular layer; Pur, Purkinje cell layer.

Mentions: To study the consequences of tandem duplication in the Pcdh gene cluster, we generated a targeted tandem duplication in the mouse Pcdh-α cluster using the inter-strain targeted meiotic recombination (iTAMERE) system31. The wild-type mouse Pcdh-α cluster contains 14 large ‘variable’ exons, each of which encodes a cadherin-like type I membrane protein consisting of extracellular domains, a transmembrane domain, and a proximal cytoplasmic domain. Each variable exon is expressed from its own promoter and spliced to three short ‘constant’ exons, which encode a shared, 152-amino acid C-terminal domain (Fig. 1a)16. In the mutant allele [hereafter called dup(2-c2) or simply dup], a 218-kb region containing the Pcdh-α2 to Pcdh-αc2 variable exons with their promoters was duplicated; the 5′-located duplicate was derived from the C57BL/6J (B6) strain and the 3′-located one was derived from the CBA strain. We selected this particular region for duplication, because it contains both stochastically and constitutively expressed Pcdh-α isoforms, and because several of the isoforms involve a single nucleotide polymorphism (SNP) between the B6 and CBA strains in the coding region (Fig. 1b, c, d). The isoforms with a SNP between the B6 and CBA strains are Pcdh-α3, Pcdh-α5, Pcdh-α6, Pcdh-α7, Pcdh-α9, Pcdh-α10, Pcdh-α12, and Pcdh-αc2, and among these isoforms, only Pcdh-α12 has a polymorphism in its promoter region. Although the individual duplicated genes in the previous Pcdh-α duplication lines cannot be distinguished25, the individual duplicated genes derived from these eight Pcdh-αs in the dup(2-c2) allele can be distinguished from each other. Therefore, the dup(2-c2) mice for the first time enables the expressional analysis of the individual duplicated isoforms in the mouse Pcdh-α cluster.


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)

Targeted tandem duplication in the Pcdh-α cluster.(a) Genomic structure of the Pcdh-α wild-type allele. The Pcdh-α allele consists of variable region exons (1–12, c1 and c2) and constant region exons (CR1–CR3). Each variable region exon is transcribed from its own promoter in a stochastic (black arrows) or constitutive (red arrows) manner. A Pcdh-α transcript is produced from one of the variable region exons and the set of constant region exons by splicing. (b) The G16Neo allele: a loxP site was inserted between Pcdh-α1 and Pcdh-α2. The loxP site is shown as a red triangle. (c) The SR allele: a loxP site was inserted between Pcdh-αc2 and CR1. (d) The dup(2-c2) allele: duplication of Pcdh-α2-Pcdh-αc2. The dup(2-c2) allele was produced by Cre-loxP-mediated trans-allelic meiotic recombination between the G16neo and SR alleles. The duplicated segments are shown under the position of the original segments. Importantly, each duplicate Pcdh-α3, Pcdh-α5, Pcdh-α6, Pcdh-α7, Pcdh-α9, Pcdh-α10, Pcdh-α12, and Pcdh-αc2 could be distinguished by SNP analysis; the 5′ genes were from B6 and the 3′ ones were from CBA. (e & f) Confirmation of the duplication allele by Southern blotting (e) and PCR (f). G, Histological analysis of the cerebellum of 4-week-old wild-type and Pcdhαdup(2-c2)/dup(2-c2) mice. Nissl staining (left), immunostaining for neurofilament (middle), and in situ hybridization using a Pcdh-α CR probe (right). Cb1-10, 1st – 10th lobule of the cerebellum; Gra, granule cell layer; Mol, molecular layer; Pur, Purkinje cell layer.
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f1: Targeted tandem duplication in the Pcdh-α cluster.(a) Genomic structure of the Pcdh-α wild-type allele. The Pcdh-α allele consists of variable region exons (1–12, c1 and c2) and constant region exons (CR1–CR3). Each variable region exon is transcribed from its own promoter in a stochastic (black arrows) or constitutive (red arrows) manner. A Pcdh-α transcript is produced from one of the variable region exons and the set of constant region exons by splicing. (b) The G16Neo allele: a loxP site was inserted between Pcdh-α1 and Pcdh-α2. The loxP site is shown as a red triangle. (c) The SR allele: a loxP site was inserted between Pcdh-αc2 and CR1. (d) The dup(2-c2) allele: duplication of Pcdh-α2-Pcdh-αc2. The dup(2-c2) allele was produced by Cre-loxP-mediated trans-allelic meiotic recombination between the G16neo and SR alleles. The duplicated segments are shown under the position of the original segments. Importantly, each duplicate Pcdh-α3, Pcdh-α5, Pcdh-α6, Pcdh-α7, Pcdh-α9, Pcdh-α10, Pcdh-α12, and Pcdh-αc2 could be distinguished by SNP analysis; the 5′ genes were from B6 and the 3′ ones were from CBA. (e & f) Confirmation of the duplication allele by Southern blotting (e) and PCR (f). G, Histological analysis of the cerebellum of 4-week-old wild-type and Pcdhαdup(2-c2)/dup(2-c2) mice. Nissl staining (left), immunostaining for neurofilament (middle), and in situ hybridization using a Pcdh-α CR probe (right). Cb1-10, 1st – 10th lobule of the cerebellum; Gra, granule cell layer; Mol, molecular layer; Pur, Purkinje cell layer.
Mentions: To study the consequences of tandem duplication in the Pcdh gene cluster, we generated a targeted tandem duplication in the mouse Pcdh-α cluster using the inter-strain targeted meiotic recombination (iTAMERE) system31. The wild-type mouse Pcdh-α cluster contains 14 large ‘variable’ exons, each of which encodes a cadherin-like type I membrane protein consisting of extracellular domains, a transmembrane domain, and a proximal cytoplasmic domain. Each variable exon is expressed from its own promoter and spliced to three short ‘constant’ exons, which encode a shared, 152-amino acid C-terminal domain (Fig. 1a)16. In the mutant allele [hereafter called dup(2-c2) or simply dup], a 218-kb region containing the Pcdh-α2 to Pcdh-αc2 variable exons with their promoters was duplicated; the 5′-located duplicate was derived from the C57BL/6J (B6) strain and the 3′-located one was derived from the CBA strain. We selected this particular region for duplication, because it contains both stochastically and constitutively expressed Pcdh-α isoforms, and because several of the isoforms involve a single nucleotide polymorphism (SNP) between the B6 and CBA strains in the coding region (Fig. 1b, c, d). The isoforms with a SNP between the B6 and CBA strains are Pcdh-α3, Pcdh-α5, Pcdh-α6, Pcdh-α7, Pcdh-α9, Pcdh-α10, Pcdh-α12, and Pcdh-αc2, and among these isoforms, only Pcdh-α12 has a polymorphism in its promoter region. Although the individual duplicated genes in the previous Pcdh-α duplication lines cannot be distinguished25, the individual duplicated genes derived from these eight Pcdh-αs in the dup(2-c2) allele can be distinguished from each other. Therefore, the dup(2-c2) mice for the first time enables the expressional analysis of the individual duplicated isoforms in the mouse Pcdh-α cluster.

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