Computer Designed PRC2 Inhibitor, EBdCas9, Reveals Functional TATA boxes in Distal Promoter Regions

Abstract

SummaryThe critical process in development, bifurcation of cellular fates, requires epigenetic H3K27me3 marks propagated by PRC2 complex. However, the precise chromatin loci of functional H3K27me3 marks are not yet known. Here we identify critical PRC2 functional sites at a single nucleosome resolution. We fused a computationally designed protein, EED binder (EB) that competes with EZH2 and thereby disrupts PRC2 function, to dCas9 (EBdCas9) to direct PRC2 inhibition at a precise locus using gRNA. We targeted EBdCas9 to 4 different genes (TBX18, p16, CDX2 and GATA3) and observed epigenetic remodeling at a single nucleosome resolution resulting in gene activation. Remarkably, while traditional TATA box is located 30bp upstream of TSS, we identified a functional TATA box, >500bp of TSS, normally repressed by PRC2 complex. Deletion of this TATA box eliminates EBdCas9 dependent TBP recruitment and transcriptional activation. Targeting EBdCas9 to CDX2 and GATA3 results in trophoblast trans-differentiation. EBdCas9 technology is broadly applicable for epigenetic regulation at a single locus to control gene expression.Graphical Abstract

Deciphering Plant Chromatin Regulation via CRISPR/dCas9-Based Epigenome Engineering

“…A genetically encoded PRC2 inhibitor named EED-binder was used to compete with EZH2 activity in vivo, likely through the titration of EED, another core subunit of PRC2 [ 65 ]. When targeted to different loci regulated by PRC2 in induced pluripotent stem cells, inhibition of PRC2 via the EED-binder–dCas9 fusion led to their upregulation and an increase in DNA accessibility as demonstrated by ATAC-seq analysis [ 66 ]. These targeted approaches aimed at locally modulating H3K27 methylation could also be useful to assess whether the impact of H3K27-methylated regions can act from a distance, forming loops with transcriptional repression activity [ 67 ].…”