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

## TL;DR

This study uses CRISPR to show that the RAWUL domain of AtRING1 is crucial for PRC1's role in gene repression in Arabidopsis.

## Contribution

The study reveals the RAWUL domain's role in PRC1 activity and its facilitation of PRC2 function in epigenetic silencing.

## Key findings

- The atring1ko mutant shows embryonic callus-like structures, indicating AtRING1's essential role in cell differentiation.
- The RAWUL domain fine-tunes PRC1 activity and influences H2Aub1 and H3K27me3 deposition at specific loci.

## Abstract

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.

The mechanism of how AtRING1, a key PRC1 component, regulates Arabidopsis development is far from clear. The new atring1

ko
mutant generated by using CRIPSR/Cas9 exhibits embryonic callus phenotype, demonstrating an essential role of AtRING1 in cell differentiation. Its C‐terminal RAWUL domain fine‐tunes PRC1 activity in facilitating H2Aub1 deposition, which precedes PRC2‐mediated H3K27me3 deposition and influences its accumulation at some specific gene loci.