Capture of regulatory factors via CRISPR-dCas9 for mechanistic analysis of fine-tuned SERRATE expression in Arabidopsis.

ABSTRACT

SERRATE (SE) plays an important role in many biological processes and under biotic stress resistance. However, little about the control of SE has been clarified. Here we present a method named native chromatin-associated proteome affinity by CRISPR-dCas9 (CASPA-dCas9) to holistically capture native regulators of the SE locus. Several key regulatory factors including PHYTOCHROME RAPIDLY REGULATED 2 (PAR2), WRKY DNA-binding protein 19 (WRKY19) and the MYB-family protein MYB27 of SE are identified. MYB27 recruits the long non-coding RNA-PRC2 (SEAIR-PRC2) complex for H3K27me3 deposition on exon 1 of SE and subsequently represses SE expression, while PAR2-MYB27 interaction inhibits both the binding of MYB27 on the SE promoter and the recruitment of SEAIR-PRC2 by MYB27. The interaction between PAR2 and MYB27 fine-tunes the SE expression level at different developmental stages. In addition, PAR2 and WRKY19 synergistically promote SE expression for pathogen resistance. Collectively, our results demonstrate an efficient method to capture key regulators of target genes and uncover the precise regulatory mechanism for SE.