Extraordinary single-cell diversity is generated in the vertebrate nervous 
system by the combinatorial expression of the clustered protocadherin genes 
(Pcdhα, -β, and -γ). This diversity is generated by a combination of stochastic 
promoter choice and alternative pre-mRNA splicing. Here we show that both the 
insulator-binding protein CTCF and the cohesin complex subunit Rad21 bind to two 
highly conserved DNA sequences, the first within and the second downstream of 
transcriptionally active Pcdhα promoters. Both CTCF and Rad21 bind to these 
sites in vitro and in vivo, this binding directly correlates with alternative 
isoform expression, and knocking down CTCF expression reduces alternative 
isoform expression. Remarkably, a similarly spaced pair of CTCF/Rad21 binding 
sites was identified within a distant enhancer element (HS5-1), which is 
required for normal levels of alternative isoform expression. We also identify 
an additional, unique regulatory role for cohesin, as Rad21 binds to another 
enhancer (HS7) independently of CTCF, and knockdown of Rad21 reduces expression 
of the constitutive, biallelically expressed Pcdhα isoforms αc1 and αc2. We 
propose that CTCF and the cohesin complex initiate and maintain Pcdhα promoter 
choice by mediating interactions between Pcdhα promoters and enhancers.