The CCR4-NOT complex component NOT1 regulates RNA-directed DNA methylation and transcriptional silencing by facilitating Pol IV-dependent siRNA production [Bisulfite-seq]

Small interfering RNAs (siRNAs) are required for establishment and maintenance of DNA methylation through RNA-directed DNA methylation (RdDM) pathway in plants. While the siRNA biogenesis is well known, relatively little is known about how the process is regulated. By a forward genetic screen in Arabidopsis thaliana, we identified a mutant defective in NOT1 and demonstrated that NOT1 is required for transcriptional silencing at RdDM target genomic loci. Furthermore, we revealed that NOT1 is required for Pol IV-dependent siRNA accumulation and DNA methylation at a subset of RdDM target loci. By immunoprecipitation combined with mass spectrometry, we showed that NOT1 is a constituent of a multi-subunit CCR4-NOT deadenylase complex. We demonstrated that the CCR4-NOT components can function as a whole to mediate chromatin silencing. Our work thus establishes that the CCR4-NOT complex regulates the production of Pol IV-dependent siRNAs and thereby facilitates DNA methylation and transcriptional silencing in Arabidopsis. Overall design: Examination of the DNA methylation level in Arabidopsis