CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function

Cell. 2015 Aug 13;162(4):900-10. doi: 10.1016/j.cell.2015.07.038.

Abstract

CTCF and the associated cohesin complex play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context, the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. These findings reveal how 3D chromosome architecture can be encoded by linear genome sequences.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • CCCTC-Binding Factor
  • Cadherins / genetics
  • Cell Cycle Proteins / metabolism
  • Chromosomal Proteins, Non-Histone / metabolism
  • Chromosomes / chemistry
  • Chromosomes / metabolism*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA / chemistry
  • Enhancer Elements, Genetic
  • Gene Expression
  • Genetic Techniques*
  • Genome, Human
  • Humans
  • K562 Cells
  • Mice
  • Promoter Regions, Genetic
  • Repressor Proteins / metabolism*
  • beta-Globins / genetics

Substances

  • CCCTC-Binding Factor
  • CTCF protein, human
  • Cadherins
  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • Ctcf protein, mouse
  • Repressor Proteins
  • beta-Globins
  • cohesins
  • DNA

Associated data

  • GEO/GSE71072
  • GEO/GSE71275