title: Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-α gene expression.
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.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Monahan
authors.forename: Noam D
authors.initials: ND
authors.lastname: Rudnick
authors.forename: Polina D
authors.initials: PD
authors.lastname: Kehayova
authors.forename: Florencia
authors.initials: F
authors.lastname: Pauli
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Newberry
authors.forename: Richard M
authors.initials: RM
authors.lastname: Myers
authors.forename: Tom
authors.initials: T
authors.lastname: Maniatis
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D000076246:CCCTC-Binding Factor; D015820:Cadherins / Q000235:genetics* / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D000097722:Cohesins
chemical_list: D000076246:CCCTC-Binding Factor; D015820:Cadherins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000097722:Cohesins; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; C465862:Rad21 protein, mouse