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

Shinnichi Yokota,Teruyoshi Hirayama,Keizo Hirano,Ryosuke Kaneko,Shunsuke Toyoda,Yoshimi Kawamura,Masumi Hirabayashi,Takahiro Hirabayashi,Takeshi Yagi

doi:10.1074/jbc.m111.245605

Abstract

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.

Highlights

The brain contains enormous numbers of neurons, which are assembled into functional neural circuits and express diverse molecules that mediate cell-cell interactions
The expression levels of the Pcdh-␤ genes distal from HS5-1 and the Pcdh-␥ genes were unchanged by the HS5-1 deletion (Fig. 1, C and D)
The expression levels of some Pcdh-␤ genes, the ones closest to HS5-1, increased significantly in the HS5-1-deleted mice (Fig. 1C). These findings suggested that HS5-1 is necessary for the appropriate expression of the Pcdh-␣ and -␤ genes

Summary

The abbreviations used are

Protocadherin; B6, C57BL/6; CCR, protocadherin-␤ cluster control region; CR, constant region; CTCF, CCCTC-binding factor; E, embryonic day; HS, DNase I hypersensitive sites; LCR, locus control region; P, postnatal day; qPCR, quantitative PCR; X-gal, 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside. We identified a Pcdh-␤ cluster control region (CCR) containing six HS sites (HS16, 17, 17Ј, 18, 19, and 20) in a region 320 kb downstream of the Pcdh-␤22 gene. This CCR comprehensively activated the Pcdh-␤ genes. The deletion of only HS19 –20, which had strong enhancer activity, decreased the Pcdh-␤ gene expression by half, and this effect was exerted from beyond the Pcdh-␥ cluster. The CCR containing these six HS sites functions primarily as a regulatory unit for the Pcdh-␤ genes These results support the idea that each Pcdh cluster is regulated by distinct regulatory elements

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION