RNA LEVER mediates long-range regulation of e-globin by keeping PRC2 in check

EZH2, a core component of polycomb repressive complex 2 (PRC2), is a histone H3 methyltransferase required for gene silencing during embryonic development. The regulatory function of promiscuous binding of RNA to EZH2 is not known, although evidences suggest that binding of RNA inactivates EZH2 in vitro. Here we present a seesaw mechanism for the regulation of e-globin through inactivating EZH2 by an upstream RNA (LEVER) in K562 cells. We show that LEVER, identified by RNA immune precipitation sequencing (RIP-seq) and NANOPORE sequencing, binds EZH2 and thereby preventing the accumulation of H3K27 methylation along the axis where LEVER emerges. The opened chromatin within LEVER in turn competes for the DNA looping between the locus control region (LCR) and the promoter of e-globin. Hence, LEVER downregulates e-globin by inhibiting EZH2. Overall design: EZH2 associated RNA in wild type K562 cells is profiled by RIP-seq. Subcellular distribution of EZH2 associated RNA is characterized through profiling K562 cytoplasmic, nuclear, nuclear poly A+ or nuclear polyA- RNA by RNA-seq. LEVER RNA structure is determined by NANOPORE sequencing on K562 nuclear RNA or total polyA+ RNA. LEVER is either knockout or knockdown in K562 cells by CRISPR-Cas9 or -dCas9 strategy. H3K27 histones marks in K562 NT-gRNA (control) or LEVER gRNA KD cells are profiled by ChIP-seq. Intra-chromatin interaction at e-globin promoter or locus control region (LCR) in NT-gRNA (control) or LEVER KO cells is analysed through 4C-seq.