Teruyoshi Hirayama

Monoallelic yet combinatorial expression of variable exons of the protocadherin-alpha gene cluster in single neurons.

Diverse protocadherin-α genes (Pcdha, also called cadherin-related neuronal receptor or CNR) are expressed in the vertebrate brain. Their genomic organization involves multiple variable exons and a set of constant exons, similar to the immunoglobulin (Ig) and T-cell receptor (TCR) genes. This diversity can be used to distinguish neurons. Using polymorphisms that distinguish the C57BL/6 and MSM mouse strains, we analyzed the allelic expression of the Pcdha gene cluster in individual neurons. Single-cell analysis of Purkinje cells using multiple RT-PCR reactions showed the monoallelic and combinatorial expression of each variable exon in the Pcdha genes. This report is the first description to our knowledge of the allelic expression of a diversified receptor family in the central nervous system. The allelic and combinatorial expression of distinct variable exons of the Pcdha genes is a potential mechanism for specifying neuron identity in the brain.

CTCF Is Required for Neural Development and Stochastic Expression of Clustered Pcdh Genes in Neurons

The CCCTC-binding factor (CTCF) is a key molecule for chromatin conformational changes that promote cellular diversity, but nothing is known about its role in neurons. Here, we produced mice with a conditional knockout (cKO) of CTCF in postmitotic projection neurons, mostly in the dorsal telencephalon. The CTCF-cKO mice exhibited postnatal growth retardation and abnormal behavior and had defects in functional somatosensory mapping in the brain. In terms of gene expression, 390 transcripts were expressed at significantly different levels between CTCF-deficient and control cortex and hippocampus. In particular, the levels of 53 isoforms of the clustered protocadherin (Pcdh) genes, which are stochastically expressed in each neuron, declined markedly. Each CTCF-deficient neuron showed defects in dendritic arborization and spine density during brain development. Their excitatory postsynaptic currents showed normal amplitude but occurred with low frequency. Our results indicate that CTCF regulates functional neural development and neuronal diversity by controlling clustered Pcdh expression.

Relationship between DNA Methylation States and Transcription of Individual Isoforms Encoded by the Protocadherin-α Gene Cluster

The protocadherin-α (Pcdh-α) gene encodes diverse transmembrane proteins that are differentially expressed in individual neurons in the vertebrate central nervous system. The Pcdh-α genomic structure contains variable first exons, each regulated by its own promoter. Here, we investigated the effect of DNA methylation on gene regulation in the Pcdh-α gene cluster. We studied two mouse cell lines, C1300 and M3, that expressed different combinations of Pcdh-α isoforms and found that 1) the transcription of specific Pcdh-α isoforms correlated significantly with the methylation state of the promoter and the 5′ (but not the 3′) region of the first exon and 2) mosaic or mixed methylation states of the promoters were associated with both active and inactive transcription. Demethylation of C1300 cells up-regulated all of the Pcdh-α isoforms, and, in a promoter assay, hypermethylation of the promoters repressed their transcriptional activity. Cell lines subcloned from the demethylated C1300 cells transcribed different combinations of Pcdh-α isoforms than the parental, nondemethylated cells, and the promoters showed differential mosaic or mixed methylation patterns. In vivo, the promoter and 5′-regions of the Pcdh-αC1 and αC2 exons, which are transcribed in all neurons, were extensively hypomethylated. In contrast, the promoters of the Pcdh-α1 to -α12 isoforms, which are transcribed differentially by individual Purkinje cells, exhibited mosaic methylation patterns. Overall, our results demonstrated that mosaic or mixed DNA methylation states in the promoter and 5′-region of the first exon may help regulate differential Pcdh-α transcription and that hypermethylation is sufficient to repress transcription.

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

The clustered protocadherins (Pcdhs), Pcdh-α, -β, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-β cluster control region (CCR) containing six HS sites (HS16, 17, 17′, 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-β gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-β genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-β expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

The role and expression of the protocadherin-alpha clusters in the CNS.

The clustered protocadherins comprise the largest subfamily of the cadherin superfamily and are predominantly expressed in the nervous system. The family of clustered protocadherins (clustered Pcdh family) is substructured into three distinct gene arrays in mammals: Pcdh-alpha, Pcdh-beta, and Pcdh-gamma. These are regulated by multiple promoters and cis-alternative splicing without DNA recombination. Pcdh-alpha proteins interact with beta1-integrin to promote cell adhesion. They also form oligomers with Pcdh-gamma proteins at the same membrane sites. During neuronal maturation, Pcdh-alpha expression is dramatically downregulated by myelination. The clustered Pcdh family has multiple variable exons that differ somewhat in number and sequence across vertebrate species. At the single-cell level, Pcdh-alpha mRNAs are regulated monoallelically, resulting in the combinatorial expression of distinct variable exons from each allele. These findings support the idea that diversified Pcdh molecules contribute to neural circuit development and provide individual cells with their specific identity.

Somatic mutations of synaptic cadherin(CNR family)transcripts in the nervous system

Cadherin‐related neuronal receptor (CNR) family genes have been identified in the nervous system by screening molecules bound to Fyn‐tyrosine kinase. The CNR family is comprised of diverse synaptic cadherins. The genomic organization of the CNR genes, composed of variable and constant regions, is similar to that of the immunoglobulin gene cluster. The nervous system is characterized by the acquisition of diverse function. This feature is similar to the immune system. In the immune system, the generation and selection of immunoglobulin gene mutants is the underlying basis for acquired immunity. We therefore examined somatic regulation of the CNR family genes in the nervous system to determine whether a similar mechanism controls nervous system development.

Clustered protocadherins and neuronal diversity.

Neuronal diversity is a fundamental requirement for complex neuronal networks and brain function. The clustered protocadherin (Pcdh) family possesses several characteristic features that are important for the molecular basis of neuronal diversity. Clustered Pcdhs are expressed predominantly in the central nervous system, in neurites, growth cones, and synapses. They consist of about 60 isoforms, and their expression is stochastically and combinatorially regulated in individual neurons. The multiple clustered Pcdhs expressed in individual neurons form heteromultimeric protein complexes that exhibit homophilic adhesion properties. Theoretically, the clustered Pcdhs could generate more than 3×10(10) possible variations in each neuron and 12,720 types of cis-tetramers per neuron. The clustered Pcdhs are important for normal neuronal development. The clustered Pcdh genes have also attracted attention as a target for epigenetic regulation.

The methyltransferase SETDB1 regulates a large neuron-specific topological chromatin domain

We report locus-specific disintegration of megabase-scale chromosomal conformations in brain after neuronal ablation of Setdb1 (also known as Kmt1e; encodes a histone H3 lysine 9 methyltransferase), including a large topologically associated 1.2-Mb domain conserved in humans and mice that encompasses >70 genes at the clustered protocadherin locus (hereafter referred to as cPcdh). The cPcdh topologically associated domain (TADcPcdh) in neurons from mutant mice showed abnormal accumulation of the transcriptional regulator and three-dimensional (3D) genome organizer CTCF at cryptic binding sites, in conjunction with DNA cytosine hypomethylation, histone hyperacetylation and upregulated expression. Genes encoding stochastically expressed protocadherins were transcribed by increased numbers of cortical neurons, indicating relaxation of single-cell constraint. SETDB1-dependent loop formations bypassed 0.2–1 Mb of linear genome and radiated from the TADcPcdh fringes toward cis-regulatory sequences within the cPcdh locus, counterbalanced shorter-range facilitative promoter–enhancer contacts and carried loop-bound polymorphisms that were associated with genetic risk for schizophrenia. We show that the SETDB1 repressor complex, which involves multiple KRAB zinc finger proteins, shields neuronal genomes from excess CTCF binding and is critically required for structural maintenance of TADcPcdh.

Distinct and Cooperative Functions for the Protocadherin-α, -β and -γ Clusters in Neuronal Survival and Axon Targeting

The clustered protocadherin (Pcdh) genes are divided into the Pcdhα, Pcdhβ, and Pcdhγ clusters. Gene-disruption analyses in mice have revealed the in vivo functions of the Pcdhα and Pcdhγ clusters. However, all Pcdh protein isoforms form combinatorial cis-hetero dimers and enter trans-homophilic interactions. Here we addressed distinct and cooperative functions in the Pcdh clusters by generating six cluster-deletion mutants (Δα, Δβ, Δγ, Δαβ, Δβγ, and Δαβγ) and comparing their phenotypes: Δα, Δβ, and Δαβ mutants were viable and fertile; Δγ mutants lived less than 12 h; and Δβγ and Δαβγ mutants died shortly after birth. The Pcdhα, Pcdhβ, and Pcdhγ clusters were individually and cooperatively important in olfactory-axon targeting and spinal-cord neuron survival. Neurodegeneration was most severe in Δαβγ mutants, indicating that Pcdhα and Pcdhβ function cooperatively for neuronal survival. The Pcdhα, Pcdhβ, and Pcdhγ clusters share roles in olfactory-axon targeting and neuronal survival, although to different degrees.

Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins

BackgroundThe specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections.ResultsWe show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity.ConclusionsOur results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.