Capture of regulatory factors via CRISPR/dCas9 for mechanistic analysis of fine-tuned SERRATE expression in Arabidopsis
SERRATE (SE) plays an important role in many biological processes and under biotic stress resistance in Arabidopsis. However, little about the control of SE expression has been clarified. Here, we present a method named native chromatin-associated proteome affinity by CRISPR/dCas9 (CASPA-dCas9) to holistically capture native regulators on SE locus. Several key regulatory factors including PHYTOCHROME RAPIDLY REGULATED 2 (PAR2), WRKY DNA-binding protein 19 (WRKY19) and the MYB27 family protein MYB27 of SE are identified. MYB27 functions to recruits the lncRNA-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 SE promoter and the recruitment of SEAIR-PRC2 by MYB27. The dosage-dependent interaction between PAR2 and MYB27 fine-tunes SE expression level at different developmental stages. Besides, PAR2 and WRKY19 synergistically promote SE expression for pathogen resistance. Collectively, our results demonstrate an efficient method CASPA-dCas9 to capture key regulators of target genes and uncovered the precise regulatory mechanism on SE expression controlled by the identified key factors in Arabidopsis.