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 atring1ko and two C-terminal deletion mutants atring1▵C-terminal lacking the RAWUL domain. We show evidence that atring1ko 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.
Wang et al. (2026) studied this question.