Search

Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18 , p16 , CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation. [Display omitted] • EBdCas9 inhibits PRC2 function in precise genomic locations • EBdCas9 upregulates TBX18 , p16 , CDX2 , and GATA3 by de-repression • EBdCas9 identifies PRC2-controlled, active TATA box >500 bp upstream of TBX18 TSS • EBdCas9 is sufficient to induce transdifferentiation and repress cancer cell cycle Levy et al. fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9). EBdCas9 represses PRC2 action in precise loci, remodels epigenomic marks, exposes transcriptional elements, and induces transdifferentiation. [ABSTRACT FROM AUTHOR]