CRISPR gene editing of AtRING1 unravels a critical role of RAWUL domain in PRC1 repression of transcription.

Polycomb Group (PcG) proteins, including members of Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2), regulate many key developmental processes through transcriptional gene repression. While the molecular mechanisms of PRC2 and its histone methyltransferase involved in depositing histone 3 lysine 27 trimethylation (H3K27me3) are well understood, the components and E3 ubiquitin ligase functions of PRC1 in plants remain largely elusive. In Arabidopsis, AtRING1 is a key PRC1 component, containing an N-terminal RING-finger domain and a C-terminal RAWUL domain. Previous studies have relied on T-DNA insertion mutants in the investigation of AtRING1 function. By editing AtRING1A using CRISPR/Cas9 technology in the atring1b-1 background, here we have generated and characterized one N-terminal stop mutant atring1(ko) and two C-terminal deletion mutants atring1(▵C-terminal) lacking the RAWUL domain. We show evidence that atring1(ko) represents the strongest loss-of-function mutant, exhibiting embryonic callus-like structures, demonstrating the essential role of AtRING1 in cell differentiation. Remarkably, the atring1(▵C-terminal) mutants exhibit mild developmental defects, suggesting that the RING domain alone retains partial function, while the RAWUL domain fine-tunes PRC1 activity. Our molecular analyses support a model in which AtRING1/PRC1-mediated H2A monoubiquitination (H2Aub1) often precedes PRC2-mediated H3K27me3 deposition at some target loci. Strikingly, the RAWUL domain is required for efficient H2Aub1 enrichment and influences H3K27me3 deposition in a locus-specific manner. Taken together, our study provides new insights into the molecular mechanism underlying PRC1 E3 ligase activity, supporting that PRC1 function facilitates PRC2 activity in epigenetic gene silencing.