Epigenome editing strategies for the functional annotation of CTCF insulators

Daniel R Tarjan,Bradley E Bernstein,William A Flavahan

doi:10.1038/s41467-019-12166-w

Abstract

The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs). Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing. Finally, we apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.

Highlights

The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs)
We show that fusions of dCas[9] to transcriptional repressors and DNA methyltransferases can be used to selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif
We apply these strategies to activate PDGFRA expression in glioblastoma stem cells, simulating an insulator loss mechanism implicated in brain tumorigenesis

Summary

Introduction

The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs). We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing We apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. The consequence of CTCF loss in a given cell type will depend on the ensuing topological alteration, as well as the state of nearby genes, enhancers and sequence elements Adding to this complexity, TAD boundaries often contain multiple CTCF binding sites[1]. We further demonstrate that stable, partially heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing with dCas9-KRAB and dCas9-DNMT3A3L We apply these strategies to activate PDGFRA expression in glioblastoma stem cells, simulating an insulator loss mechanism implicated in brain tumorigenesis

Methods

Results

Conclusion