1. Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-α gene expression.
- Author
-
Monahan K, Rudnick ND, Kehayova PD, Pauli F, Newberry KM, Myers RM, and Maniatis T
- Subjects
- Alternative Splicing genetics, Animals, Base Sequence, CCCTC-Binding Factor, Cell Line, Chromatin Immunoprecipitation, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Enhancer Elements, Genetic genetics, Gene Knockdown Techniques, Humans, Mice, Molecular Sequence Data, Nuclear Proteins genetics, Phosphoproteins genetics, Protein Isoforms metabolism, RNA, Small Interfering genetics, Reverse Transcriptase Polymerase Chain Reaction, Sequence Alignment, Sequence Analysis, DNA, Cohesins, Cadherins genetics, Cadherins metabolism, Cell Cycle Proteins metabolism, Chromosomal Proteins, Non-Histone metabolism, Gene Expression Regulation genetics, Nuclear Proteins metabolism, Phosphoproteins metabolism, Repressor Proteins metabolism
- Abstract
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.
- Published
- 2012
- Full Text
- View/download PDF