LADL: light-activated dynamic looping for endogenous gene expression control

Nat Methods. 2019 Jul;16(7):633-639. doi: 10.1038/s41592-019-0436-5. Epub 2019 Jun 24.

Abstract

Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA-FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.

Publication types

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

MeSH terms

  • Animals
  • Carrier Proteins / genetics
  • Cells, Cultured
  • DNA-Binding Proteins
  • Gene Expression Regulation*
  • Kruppel-Like Transcription Factors / genetics
  • Light
  • Male
  • Mice
  • Nerve Tissue Proteins / genetics
  • Promoter Regions, Genetic
  • Protein Engineering*
  • RNA, Guide / genetics

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • GKLF protein
  • Kruppel-Like Transcription Factors
  • Nerve Tissue Proteins
  • RNA, Guide
  • Zfp462 protein, mouse