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The CCR4‐NOT Complex Component NOT1 Regulates RNA‐Directed DNA

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The CCR4‐NOT Complex Component NOT1 Regulates RNA‐Directed DNA The Plant Journal (2020) doi: 10.1111/tpj.14818 The CCR4-NOT complex component NOT1 regulates RNA-directed DNA methylation and transcriptional silencing by facilitating Pol IV-dependent siRNA production Hao-Ran Zhou1, Rong-Nan Lin1, Huan-Wei Huang1, Lin Li1, Tao Cai1, Jian-Kang Zhu2, She Chen1 and Xin-Jian He1,3,* 1National Institute of Biological Sciences, Beijing 102206, China, 2Shanghai Center for Plant Stress Biology and Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 201602, China, and 3Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China Received 3 March 2020; revised 29 April 2020; accepted 7 May 2020. *For correspondence (e-mail [email protected]). SUMMARY Small interfering RNAs (siRNAs) are responsible for establishing and maintaining DNA methylation through the RNA-directed DNA methylation (RdDM) pathway in plants. Although siRNA biogenesis is well known, it is relatively unclear 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 silenc- ing at RdDM target genomic loci. We demonstrated that NOT1 is required for Pol IV-dependent siRNA accu- mulation and DNA methylation at a subset of RdDM target genomic loci. Furthermore, we revealed that NOT1 is a constituent of a multi-subunit CCR4-NOT deadenylase complex by immunoprecipitation com- bined with mass spectrometry and demonstrated that the CCR4-NOT components can function as a whole to mediate chromatin silencing. Therefore, our work establishes that the CCR4-NOT complex regulates the biogenesis of Pol IV-dependent siRNAs, and hence facilitates DNA methylation and transcriptional silencing in Arabidopsis. Keywords: NOT1, CCR4, pol IV, RNA, DNA methylation, transcriptional silencing. coding RNAs, which are then converted to double-stranded INTRODUCTION RNAs (dsRNAs) by RNA-DIRECTED RNA POLYMERASE 2 As an important epigenetic mark, DNA methylation at cyto- (RDR2) (Haag et al., 2012). Then, these dsRNAs are cleaved sine is required for transposon silencing, genome stability into 24-nucleotide (24-nt) small interfering RNAs (siRNAs) and regulation of gene expression in eukaryotes (He et al., by DICER LIKE 3 (DCL3) and loaded on to ARGONAUTE 4 2011; Borges and Martienssen, 2013). In plants, DNA (AGO4) (Xie et al., 2004; Pontes et al., 2006; Ye et al., 2012). methylation occurs in the symmetric CG and CHG sites It is believed that the interactions among AGO4-siRNA and the asymmetric CHH sites, where H represents A, T or complex, Pol V transcripts and KTF1 recruit the DNA C (Law and Jacobsen, 2010; Matzke and Mosher, 2014; methyltransferase DRM2 to the target loci, by which DNA Zhang et al., 2018). CG, CHG and CHH methylation are cat- methylation is finally established (Bohmdorfer et al., 2014; alyzed and maintained by METHYLTRANSFERASE1 Zhong et al., 2014). (MET1), CHROMOMETHYLASE3 (CMT3) and CMT2, respec- Given that Pol IV-dependent siRNAs are responsible for tively (Stroud et al., 2013; Zemach et al., 2013; Du et al., the establishment of DNA methylation, investigation of 2015). The initiation and maintenance of DNA methylation whether and how the production of Pol IV-dependent siR- at CHH sites and to a smaller degree at CG and CHG sites NAs is regulated could contribute towards understanding is also mediated via the RNA-directed DNA methylation the mechanism of the establishment of DNA methylation. (RdDM) pathway (Law and Jacobsen, 2010; He et al., 2014; SHH1/DTF1 interacts with Pol IV and is responsible for the Matzke and Mosher, 2014), in which two plant-specific recruitment of Pol IV to genomic loci with histone H3K9 RNA polymerases, Pol IV and Pol V, are involved (Herr methylation; the production of Pol IV-dependent siRNAs is et al., 2005; Zhang et al., 2007; Wierzbicki et al., 2008; Haag strikingly reduced in the shh1/dtf1 mutant (Law et al., 2013; and Pikaard, 2011). Pol IV generates single-stranded non- Zhang et al., 2013b). The chromatin-remodeling regulators © 2020 Society for Experimental Biology and John Wiley & Sons Ltd 1 2 Hao-Ran Zhou et al. CLSY1-4 interact with Pol IV and facilitate siRNA produc- silencing (Braun et al., 2011; Mathys et al., 2014). For tion and DNA methylation in a locus-specific manner (Yang instance, GW182, a component of the miRNA-induced et al., 2018; Zhou et al., 2018). Reduction of Pol IV-depen- silencing complex, interacts with Negative on TATA-less 1 dent siRNAs was also observed in the Pol V mutant nrpe1 (NOT1) and functions to recruit the CCR4-NOT complex to (Mosher et al., 2008), which is indirectly attributed to a its target genomic loci in animal cells (Braun et al., 2011). self-reinforcing loop between Pol IV-dependent siRNA pro- In Arabidopsis, NOT2 was shown to regulate the produc- duction and DNA methylation in the RdDM pathway (Law tion of primary miRNA transcripts and the location of et al., 2013; Zhang et al., 2013b; Johnson et al., 2014; Liu DCL1, thereby facilitating miRNA-induced silencing (Wang et al., 2014). RNA processing factors, including some pre- et al., 2013). These studies reveal the function of the mRNA splicing factors and the exosome subunit RRP6L1, CCR4-NOT complex in miRNA-induced silencing at the were reported to regulate siRNA production, Pol V tran- post-transcriptional level. However, it is unknown whether scription, DNA methylation and transcriptional silencing at the CCR4-NOT complex affects chromatin modification RdDM target loci in Arabidopsis (Ausin et al., 2012; Dou and transcriptional silencing. et al., 2013; Zhang et al., 2013a; Zhang et al., 2014; Ye Here, we identified NOT1 involved in chromatin silenc- et al., 2016). Involvement of pre-mRNA splicing factors and ing in Arabidopsis by a forward genetic screen. Mutation exosome regulators in siRNA-mediated chromatin silenc- of NOT1 released the silencing state of a transgene and ing was also observed in other eukaryotes (Bayne et al., several endogenous RdDM target loci. Pol IV-dependent 2008; Tabach et al., 2013; Yamanaka et al., 2013), suggest- 24-nt siRNA production and DNA methylation were ing that different eukaryotic organisms regulate siRNA reduced in the not1 mutant, suggesting that NOT1 regu- accumulation and chromatin silencing through conserved lates chromatin silencing through RNA-directed DNA mechanisms. However, it is unknown whether there are methylation. Moreover, components of the Arabidopsis any other conserved mechanisms involved in the regula- CCR4-NOT complex were deciphered in our study. We tion of siRNA-mediated chromatin silencing. demonstrate that components of the CCR4-NOT complex The CCR4-NOT complex is a conserved multi-subunit function as a whole to mediate transcriptional silencing. protein complex in yeast and animals (Nasertorabi et al., 2011). Not1 is the largest subunit in the CCR4-NOT com- RESULTS plex and integrates the other components of the complex Identification of not1 as a mutant with a released as a scaffold protein (Collart and Panasenko, 2012). The transcriptional silencing phenotype CCR4-NOT complex is involved in mRNA deadenylation as it contains the deadenylases Ccr4 and Caf1 (Collart and We performed a genetic screen to discover new compo- Panasenko, 2012; Wahle and Winkler, 2013). Deadenyla- nents taking part in chromatin silencing. In this genetic tion of mRNAs is responsible for initiation of mRNA decay screening system, the wild-type plants (C24 ecotype) har- (van Hoof and Parker, 2002; Chen and Shyu, 2011). Addi- bor a luciferase transgene driven by the RD29A promoter tionally, the CCR4-NOT complex regulates cellular pro- (RD29A-LUC) and an NPTII transgene driven by the 35S cesses such as transcriptional initiation and elongation, promoter (35S-NPTII) that enable the plants, respectively, RNA export, translation repression and protein ubiquitina- to emit strong luminescence in stress conditions and to tion (Collart and Panasenko, 2012; Inada and Makino, grow normally on medium with kanamycin (Figure 1a). 2014). Components of the CCR4-NOT complex are also Both RD29A-LUC and 35S-NPTII were silenced in a null known to participate in microRNA (miRNA)-induced gene mutant of ROS1, which is a 5-mC glycosylase initiating (a) (b) Figure 1. Silencing of RD29A-LUC transgene and Luminescence endogenous loci is affected by not1. (a) Expression of RD29A-LUC and 35S-NPTII trans- genes in wild-type (WT), ros1, ros1/#88 and ros1nr- AtGP1 pd1 was detected by luminescence imaging and by growing them on MS medium with 150 mg/L kana- solo LTR mycin, respectively. (b) Transcript levels of endogenous loci in WT and SDC the indicated mutants were determined by semi- MS + Kanamycin quantitative reverse transcription–polymerase chain ERT7 reaction. ACTIN7 gene was amplified as an internal control. WT ros1 ERT14 ACTIN7 ros1/ ros1/ #88 nrpd1 no RT © 2020 Society for Experimental Biology and John Wiley & Sons Ltd, The Plant Journal, (2020), doi: 10.1111/tpj.14818 NOT1 regulates RNA-directed DNA methylation 3 DNA demethylation (Figure 1a) (Gong et al., 2002). In responsible for the defect in the silencing of both the screening for ros1 suppressors, we have previously identi- RD29A-LUC transgene and the endogenous RdDM target fied several mutants involved in DNA methylation and loci. transcriptional silencing (Dataset S1) (Liu et al., 2011; Dou NOT1 is involved in silencing at the transcriptional level et al., 2013; Zhou et al., 2013; Zhang et al., 2013a; Han et al., 2014). Here, we identified a previously uncharacter- In yeast and animals, the CCR4-NOT complex was reported ized ros1 suppressor mutant, #88, in which the silencing of to act at different cellular processes to regulate gene RD29A-LUC but not 35S-NPTII is released (Figure 1a). The expression, including transcription, mRNA degradation different effects on the silencing of RD29A-LUC and 35S- and protein quality control (Collart and Panasenko, 2012; NPTII were also detected in the mutants of RdDM compo- Miller and Reese, 2012).
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