The role and expression of the protocadherin-alpha clusters in the CNS Teruyoshi Hirayama1,2 and Takeshi Yagi1,2

The clustered protocadherins comprise the largest subfamily of within the protocadherin family because of the unusual the superfamily and are predominantly expressed in organization of their genes into clusters, including a the nervous system. The family of clustered protocadherins tandem array of variable exons and one set of constant (clustered Pcdh family) is substructured into three distinct gene exons [1,2]. Of the clustered Pcdhs, the Pcdh-a cluster was arrays in mammals: Pcdh-a, Pcdh-b, and Pcdh-g. These are originally found in mouse brain using the yeast-two regulated by multiple promoters and cis-alternative splicing hybrid system with the Src homology domain3 (SH3) without DNA recombination. Pcdh-a proteins interact with region of Fyn as bait, and was named the cadherin-related b1- to promote . They also form oligomers neuronal receptor (CNR). with Pcdh-g proteins at the same membrane sites. During neuronal maturation, Pcdh-a expression is dramatically When first discovered, the Pcdh-a and Pcdh-g genes downregulated by myelination. The clustered Pcdh family has attracted immediate interest, because the striking simila- multiple variable exons that differ somewhat in number and rities between the Pcdh-a and the T-cell receptor (TCR)or sequence across vertebrate species. At the single-cell level, immunoglobulin (Ig) genes suggested a model of somatic Pcdh-a mRNAs are regulated monoallelically, resulting in the DNA gene rearrangement and mutation [3–5]. This is combinatorial expression of distinct variable exons from each notable because in lymphocytes, acquired immunity is allele. These findings support the idea that diversified Pcdh based on DNA rearrangement and somatic mutations. At molecules contribute to neural circuit development and provide about the same time as the Pcdh-a and -g genes were individual cells with their specific identity. discovered, mice with mutations in XRCC4 or DNA Addresses ligase IV, both of which are key molecules for the 1 KOKORO-Biology Group, Laboratories for Integrated Biology, DNA rearrangement in TCR and Ig genes, were found Graduate School of Frontier Biosciences, Osaka University, 1–3 to have abnormal brain development [6]. The genomic Yamadaoka, Suita, Osaka, 565-0871, Japan structures of the clustered Pcdh family rendered them 2 CREST, Japan Science and Technology Agency (JST), Japan attractive candidates for somatic DNA rearrangement in Corresponding author: Yagi, Takeshi ([email protected]) the brain [7]. Key unresolved questions in the nervous system are how the diversified neuronal network is gen- erated and what the molecular basis of this process is. Current Opinion in Neurobiology 2006, 16:336–342

This review comes from a themed issue on To investigate the somatic DNA rearrangement of Pcdh-a Signalling mechanisms in the mouse brain, a genomic library was screened and an Edited by Erin M Schuman and Peter H Seeburg unusual intron-less sequence of Pcdh-a6 DNA was iso- lated. Interestingly, the intron-less Pcdh-a6 was inserted Available online 11th May 2006 into the 28S ribosomal DNA gene locus (see glossary), 0959-4388/$ – see front matter and both edges of the integration site had an additional 5 # 2006 Elsevier Ltd. All rights reserved. base pair (bp) duplicated sequence [8]. This suggested DOI 10.1016/j.conb.2006.05.003 that this intron-less section of DNA was derived from a reverse-transcription event (see glossary) rather than from DNA rearrangement. In fact, retrotransposition events (see glossary), including reverse transcription (RT), geno- Introduction mic DNA breaks, and integration reactions, do occur Cell adhesion molecules are important not only in the during neuronal development [9]. The possibility of process of development but also in the formation of somatic substitutions of Pcdh-a transcripts was investi- functional neuronal circuits. The cadherin superfamily gated using reverse transcriptase–polymerase chain reac- consists of calcium-dependent cell adhesion molecules tion (RT-PCR) and complementary DNA (cDNA) that have several distinct ectodomains (ECs). The pro- screening. Although the RT-PCR method indicated tocadherin family is the largest in the cadherin super- the possibility of somatic substitutions, the cDNA family; its members are characterized by their screening method revealed that almost all of the substitu- extracellular domain, which contains five or six ECs. tions identified in the RT-PCR were caused by PCR Currently, within the protocadherin family, about 60 errors, except those at postnatal day (P) 1 [10,11]. These different protocadherins have been identified and almost results indicated that DNA rearrangement and somatic all of these are predominantly expressed in the nervous substitutions do not, or rarely, occur in the clustered Pcdh system. The clustered Pcdh family forms a subfamily family.

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then dramatically decreases at P10, as myelin associated Glossary 28S ribosomal DNA gene locus:: 28S ribosomal RNA encoded gene protein (MAG) and O1 (an oligodendrocyte cell-surface locus. In a eukaryote cell ribosomes are comprise two subunits, a 60S marker) immunoreactivity increase. In support of this large subunit and a 40S small subunit. The large subunit contains finding, in the shiverer mutant mouse, in which the gene three (28S, 5.8S, 5S) ribosomal RNAs. encoding myelin basic protein is deleted, the expression cis-alternative splicing:: A process in a single pre-mRNA during which introns are removed, and exons are joined. of Pcdh-a proteins does not decrease at P19. These Retrotransposition:: Transposition through an RNA intermediate. results suggest that the Pcdh-a family contributes to Reverse-transcription event:: Complementary DNA synthesis from neural circuit maturation during development. Interest- an RNA template by reverse transcriptase. ingly, a recent report demonstrated that the formation of Transactivate:: Stimulation of transcription activity by a trans appropriate neuronal circuits in the visual cortex is ter- activator. Transposition:: DNA segments move from one gene position to minated by myelination [19]. another. trans-splicing:: Splicing that occurs among separate pre-mRNAs. Most of the cadherin superfamily proteins show calcium- dependent homophilic cell adhesion activity. However, little homophilic cell adhesion activity has been found Three other models have been proposed for regulation of with the Pcdh-a proteins. By contrast, heterophilic cell clustered Pcdh gene expression: cis-alternative splicing adhesion activity has been reported between the Pcdh-a with a single promoter, cis-alternative splicing with multi- 4 and the b1 integrin in an in vitro cell aggregation assay in ple promoters, and trans-splicing with multiple promoters the HEK293T cell line (Figure 1). This heterophilic cell (see glossary) [3]. Molecular and genetic analyses demon- adhesion activity exhibits Ca2+-dependency. The RGD strated that clustered Pcdh transcripts are predominantly (Arg-Gly-Asp) motif, which is an essential residue for generated by cis-alternative splicing with multiple pro- integrin-dependent cell adhesion activity, is found among moters [12,13]. mammalian Pcdh-a family members [20]. recognize the RGD motif of their ligands, including In this review, we focus on recent studies of Pcdh-a, first fibronectin, vitronectin, fibrinogen, von Willebrand factor discussing the expression pattern of its transcripts and the and many other large glycoproteins [21]. known molecules that interact with the Pcdh-a proteins. We then summarize the phylogenetic analyses and what Immunoelectron microscopy and subcellular fractiona- is known about the molecular evolution of the clustered tion showed that both Pcdh-a and Pcdh-g proteins loca- Pcdh family. Finally, we discuss the remarkable, recently lize to the postsynaptic density (PSD) [14,22,23], and published result that the expression of each Pcdh-a allele double-label immunostaining showed that their expres- is regulated independently. sion patterns partially overlap. Interestingly, although only small amounts of the Pcdh-a proteins are reportedly Expression and interaction expressed on the cell surface, when Pcdh-a and Pcdh-g In-situ hybridization analysis revealed that the Pcdh-a were coexpressed in HEK293T cells there was a more transcripts are mainly expressed in the olfactory bulb, than 5-fold increase in the cell-surface expression of cerebellum, hippocampus and cerebral cortex. Interest- Pcdh-a. These results indicated that Pcdh-a and Pcdh- ingly, double in-situ hybridization showed that different g might form a hetero-protein complex. Confirmation of neurons expressed different sets of Pcdh-a genes [14]. Pcdh-a and Pcdh-g interaction was obtained by coimmu- The same pattern was demonstrated for Pcdh-g genes noprecipitation using extracts of mouse brain and neuro- using a single-cell RT-PCR method [12]. Identifying the blastoma (Figure 1). This combinatorial expression of the mechanisms underlying the expression of different sets of Pcdh-a and Pcdh-g proteins enables individual neurons Pcdh-a and Pcdh-g genes in individual cells may illumi- to express more diverse codes, perhaps establishing their nate how neuronal cell identity is specified. neuronal cell identity [22].

Pcdh-a proteins were found in the marginal zone, sub- Last year, two studies demonstrated that Pcdh-g proteins plate and intermediate zone during neocortical develop- undergo proteolytic processing by metalloproteinases ment. Subplate and intermediate neurons are important and g-secretases (Figure 1)[24,25]. In E-cadherin and for the development of the correct corticofugal and tha- N-cadherin, of the classical cadherin family, the intracel- lamocortical connections [15,16]. A recent immunohisto- lular domains are cleaved by g-secretase [26,27]. The chemical study showed that Pcdh-a proteins are fragments released into the cytosol bind to CREB (cyclic expressed in the developing corticofugal and thalamocor- AMP response element binding protein) binding protein tical axon pathways [17]. Another study showed that (CBP) released from the nucleus, and their complex is myelination functions as a trigger for the decline of degraded by a proteasome. As a result of this degradation, Pcdh-a protein expression [18]. In the optic nerve, CBP and CREB mediated transcription is downregulated. one of the most-studied axonal tracts, the expression of By contrast, the Pcdh-g intracellular fragments localize to Pcdh-a proteins increases during nerve development and the nucleus, presumably because they contain a bipartite www.sciencedirect.com Current Opinion in Neurobiology 2006, 16:336–342 338 Signalling mechanisms

Figure 1

Schematic overview of the Pcdh–a and Pcdh-g protein interactions. Pcdh-a (yellow) and Pcdh–g (green) co-expression greatly enhances their transportation from the endoplasmic reticulum to the cell surface compared with the expression of Pcdh–a alone. A portion of the Pcdh-a protein forms a heterocomplex with Pcdh-g. b1-Integrin interacts with EC1 of mouse Pcdh-a. The Pcdh-g cytoplasmic domain is cleaved by g-secretase after the ectodomain is cleaved by an unknown metalloproteinase. The C-terminal fragments are transported into the nucleus and directly or indirectly transactivate the Pcdh-g genes. Abbreviations: a, Pcdh-a; g, Pcdh-g; EC, ectodomain; TM, transmembrane domain; CP, cytoplasmic domain; ER, endoplasmic reticulum.

nuclear transport signal [28]. The Pcdh-g cytoplasmic mouse, chicken, zebrafish and coelacanth [3,4,29– domain can transactivate (see glossary) all the Pcdh-g 31,32,33](Figure 2). These genes are predominantly promoters [24]. Similar to Pcdh-g, the Pcdh-a cytoplas- expressed in the central nervous system. By contrast, mic domain has a lysine-rich sequence that resembles a the clustered Pcdh family genes are not found in the nuclear transport signal; however, it is not clear whether invertebrate species Drosophila melanogaster and Caenor- Pcdh-a is a target of g-secretase. habditis elegans.InDrosophila, about 38,000 isoforms of the DSCAM cell adhesion molecules are generated by Molecular evolution alternative splicing [34]. One possible explanation for this The clustered Pcdh family genes have been identified is that the clustered Pcdh family genes were generated in several vertebrate species: human, chimpanzee, rat, in the genome of a common vertebrate ancestor and

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Figure 2

Comparison of the human, chimpanzee, rat, mouse, chicken, coelacanth and zebrafish Pcdh clusters. Paralogous relationships in each gene cluster are indicated by the vertical colored boxes. Orthologous relationships in different clusters are indicated by the colored bars connected with a line. The variable- and constant- region exons of Pcdh-a (a), Pcdh-b (b), and Pcdh-g (g), are shown at the top. The Pcdh-b cluster is absent in zebrafish. The z genes (indicated at left just above the coelacanth sequence) and o genes (indicated at the bottom left of the zebrafish cluster 2 sequence) are absent from the mammalian genomes. The coelacanth and zebrafish paralogous subgroups are different from the others. The chicken Pcdh clusters, except Pcdh-a, have not been completely sequenced. Black vertical boxes represent relic or pseudogenes. diversified during brain evolution. The RGD motif in the to cytosine (GC) content at the third codon [36]. More- extracellular domain 1 (EC1) of the mouse Pcdh-a family, over, there is substantial evidence indicating that gene the crucial motif for interaction with b1 integrin, is highly conversion events occurred in the clustered Pcdh family conserved in mammals, but other species do not have the [35,37,38]. Taken together, these results indicate that RGD motif. Therefore, the interaction between the clustered Pcdh gene evolution is driven by a combination Pcdh-a family and the b1 integrin might determine of lineage-specific duplication of variable exons, region- certain neural features that are characteristic of mammals. restricted gene conversion within species and adaptive variation [35]. In humans, the Pcdh-a gene cluster is Phylogenetic analyses of the clustered Pcdh variable located at 5q31 and has many single nucleotide poly- exons have demonstrated orthologous relationships morphisms (SNPs), including amino acid exchanges and among human, rat and mouse [29,35]. However, such extensive linkage disequilibrium and deletion [38,39]. orthologous relationships were not found among the These features of molecular diversity in the human clustered Pcdh variable exons of chicken, zebrafish and population suggest that the clustered Pcdh family is an coelacanth [31,32,33]. In contrast to the variable exons, interesting candidate for a role in psychiatric diseases and the putative promoter elements and constant exons are in determining the functional characteristics of the brain. highly conserved across species. The highly conserved ECs, such as EC5, have paralogous rather than ortholo- A new type of monoallelic regulation: gous relationships across species. Paralogous relationships independently regulated alleles resulting from gene conversion have been demonstrated Double labeling in-situ hybridization analysis revealed in several multigene families that have increased guanine that individual neurons express different sets of Pcdh-a www.sciencedirect.com Current Opinion in Neurobiology 2006, 16:336–342 340 Signalling mechanisms

Figure 3

Monoallelic and combinatorial expression of the Pcdh-a gene cluster. The expression level of Pcdh-a transcripts is regulated independently by either the paternal or the maternal allele. Maternal and paternal genes are shown in red and blue boxes with the exon number above. Arrows indicate strongly expressed variable exons. The differential expression in individual neurons could specify neuronal identity in the brain. isoforms [14]. Furthermore, Pcdh-g isoforms are also dif- expresses mainly a single OR gene derived from one ferentially expressed in single neurons [12]. Using poly- allele [44,45]. OR gene expression is rigidly regulated in morphisms in the C57BL/6 and Mishima (MSM) mouse individual OSNs, but the details of monoallelic gene strains to track strain-specific alleles, the allelic expres- regulation remain unexplained [46,47,48,49]. The reg- sion patterns in individual neurons were investigated in ulation of Pcdh-a genes also differs from the previously F1 mice from a cross between C57BL/6 females and reported monoallelic regulation mechanisms, in that the MSM males. Single-cell analysis of Purkinje cells using expression is regulated by combinatory monoallelic multiple RT-PCR reactions revealed that the Pcdh-a expression, but the details mostly remain unclear. None- isoforms have monoallelic and combinatorial expression theless, similar to the OR genes, and unlike the Ig and in individual neurons (Figure 3)[40]. Thus, the allelic TCR genes, the Pcdh-a genes are expressed without DNA expression patterns are regulated differently from pre- recombination events. Comparison of the upstream viously reported mechanisms used by diverse receptor sequence for each clustered Pcdh gene shows a highly families. conserved sequence element (CSE) with a common core sequence, CGCT, in the proximal regions of all the Monoallelic gene expression can be classified into three variable exons [50]. In a reporter assay, nucleotide sub- main groups: first, random X-chromosome inactivation in stitutions within the CSE completely abolished the repor- female cells; second, imprinting genes in autosomes from ter activity [13]. Therefore, the CSE is considered to be a only the maternal or the paternal allele; third, allelic basic promoter element of clustered Pcdh genes. Inter- exclusion in autosomes, including Ig, TCR, natural estingly, the CSE is not absolutely required for the killer-cell receptors, odorant receptors (ORs) and phero- transactivation activity of the Pcdh-g cytoplasmic mone receptors. However, the regulation of the Pcdh-a domain, although the promoter activity of a CSE deletion genes is different from the above examples: the different mutant dropped 20 fold [24]. The transactivation sets of isoforms are expressed from each allele and are activity of the Pcdh-g cytoplasmic domain suggests that monoallelically regulated in individual cells [14]. a feedback mechanism exists in the regulation of the Pcdh-g genes. Indirect feedback mechanisms have been The underlying mechanisms of this form of monoallelic demonstrated for the Ig, TCR and OR genes. The expres- regulation are completely different from those governing sion of a functional receptor prevents the additional gene other allelic regulation aspects [41]. Somatic DNA recombination events in the Ig and TCR genes and, in the recombination events are essential for the regulation of case of OR genes, the expression of other OR receptors functional Ig and TCR expression as single genes in [51–54]. Interestingly, these gene families share the fea- single cells [42]. It is still uncertain whether these allelic ture that they encode diverse cell-surface proteins and exclusions are regulated deterministically or stochasti- provide individual cells with a specific identity. The cally [43]. In ORs, an olfactory sensory neuron (OSN) allelic and combinatorial expression of distinct variable

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a 12. Wang X, Su H, Bradley A: Molecular mechanisms governing exons of the Pcdh- genes might be key to specifying Pcdh-gamma gene expression: evidence for a multiple neuronal cell identity in the brain. promoter and cis-alternative splicing model. Genes Dev 2002, 16:1890-1905. Conclusions 13. Tasic B, Nabholz CE, Baldwin KK, Kim Y, Rueckert EH, Ribich SA, At first, studies of the clustered Pcdh gene family mainly Cramer P, Wu Q, Axel R, Maniatis T: Promoter choice determines splice site selection in protocadherin alpha and focused on whether a DNA recombination mechanism, gamma pre-mRNA splicing. Mol Cell 2002, 10:21-33. analogous to that of the Ig and TCR genes, underlies 14. Kohmura N, Senzaki K, Hamada S, Kai N, Yasuda R, Watanabe M, clustered Pcdh gene expression. At present, we know that Ishii H, Yasuda M, Mishina M, Yagi T: Diversity revealed by a clustered Pcdh gene expression is regulated through mul- novel family of expressed in neurons at a synaptic complex. 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