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

The clustered protocadherins (Pcdhs), Pcdh-alpha, -beta, and -gamma, 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-alpha regulatory element in vitro, affects especially the expression of specific Pcdh-alpha isoforms in vivo. We also identified a Pcdh-beta cluster control region (CCR) containing six HS sites (HS16, 17, 17', 18, 19, and 20) downstream of the Pcdh-gamma cluster. This CCR comprehensively activates the expression of the Pcdh-beta gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR non-uniformly down-regulates some Pcdh-gamma isoforms and does not affect Pcdh-alpha expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-gamma cluster to activate the upstream Pcdh-beta genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-beta 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.