Total Expression and Dual Gene-regulatory Mechanisms Maintained in Deletions and Duplications of the Pcdha Cluster

Abstract

The clustered protocadherin-␣ (Pcdha) genes, which are expressed in the vertebrate brain, encode diverse membrane proteins whose functions are involved in axonal projection and in learning and memory. The Pcdha cluster consists of 14 tandemly arranged genes (Pcdha1-Pcdha12, Pcdhac1, and Pcdhac2, from 5 to 3). Each first exon (the variable exons) is transcribed from its own promoter, and spliced to the constant exons, which are common to all the Pcdha genes. Cerebellar Purkinje cells show dual expression patterns for Pcdha. In individual Purkinje cells, different sets of the 5 genes in the cluster, Pcdha1-12, are randomly expressed, whereas both 3 genes, Pcdhac1 and Pcdhac2, are expressed constitutively. To elucidate the relationship between the genomic structure of the Pcdha cluster and their expression in Purkinje cells, we deleted or duplicated multiple variable exons and analyzed the expression of Pcdha genes in the mouse brain. In all mutant mice, transcript levels of the constant exons and the dual expression patterns were maintained. In the deletion mutants, the missing genes were flexibly compensated by the remaining variable exons. On the other hand, in duplication mutants, the levels of the duplicated genes were trimmed. These results indicate that the Pcdha genes are comprehensively regulated as a cluster unit, and that the regulators that randomly and constitutively drive Pcdha gene expression are intact in the deleted or duplicated mutant alleles. These dual regulatory mechanisms may play important roles in the diversity and fundamental functions of neurons.

The complex genetics of hypoplastic left heart syndrome

“…Embryos were fixed in 4% paraformaldehyde–DEPC-treated PBS at 4 °C overnight and processed as described previously 43 . Plasmid for Pcdha9 in situ probes was obtained from T. Yagi (Osaka University) and has been described previously 43 .…”

“…Plasmid for Pcdha9 in situ probes was obtained from T. Yagi (Osaka University) and has been described previously 43 . After in situ hybridization, embryos were postfixed in 4% paraformaldehyde at 4 °C overnight, then embedded in O.C.T (Tissue-Tek) and cryosectioned at 20 µm.…”

“…Mice with deletion of Pcdha2 through Pcdha11 (ref. 43), although viable, have not been examined for cardiac phenotype. We found that Pcdha9 mutants with BAV were adult viable.…”

Identification of the Cluster Control Region for the Protocadherin-β Genes Located beyond the Protocadherin-γ Cluster

“…Briefly, the sections were fixed with 4% paraformaldehyde for 10 min at room temperature, followed by acetylation for 10 min. The sections were hybridized with digoxigenin-labeled antisense probes to Pcdh-␣CR, and Pcdh-␣ isoforms mRNAs (13,18), at 72°C. The Pcdh-␤ isoform-specific probes were designed based on mouse Pcdh-␤16 (nucleotides 277-1293; GenBank TM accession number NM_053141) and Pcdh-␤22 (nucleotides 213-1146; GenBank TM accession number NM_053147).…”

“…In many regions of the brain, a few scattered neurons are labeled by in situ hybridization of probes for each clustered Pcdh isoform (3,7,9,(15)(16)(17)(18). By the single-cell RT-PCR analysis of Purkinje cells, a single neuron expresses one or more isoforms from each clustered Pcdh subfamily, with the exception of the Pcdh-␣ and -␥ C-type isoforms (16,17).…”