Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of Protocadherin-α gene expression

Extraordinary single-cell diversity is generated in the vertebrate nervous system by the combinatorial expression of the clustered protocadherin genes (Pcdh alpha, -beta, and -gamma). 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 Pcdh alpha 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 Pcdh alpha isoforms alpha c1 and alpha c2. We propose that CTCF and the cohesin complex initiate and maintain Pcdh alpha promoter choice by mediating interactions between Pcdh alpha promoters and enhancers.