Co-targeting RNA Polymerases IV and V Promotes Efficient De Novo DNA Methylation in Arabidopsis

Soo Young Park,Basudev Ghoshal,Jason Gardiner,Steven E. Jacobsen,Jenny Miao-Chi Zhao,Suhua Feng,Wanlu Liu,Joanne Chory,Javier Gallego-Bartolomé,Peggy Hsuanyu Kuo

doi:10.1016/j.cell.2019.01.029

Abstract

SummaryThe RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation.

Highlights

Cytosine DNA methylation is a key epigenetic mark involved in the silencing of transposable elements (TEs) and genes in eukaryotes
polymerase IV (Pol IV) accessory proteins include the CLASSY SNF2-related putative chromatin remodeler family (CLSY) involved in global Pol IV recruitment (Zhou et al, 2018), and SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), which binds the repressive histone mark H3K9 methylation associated with DNA methylation and is required for Pol IV recruitment at a subset of RNA-directed DNA methylation (RdDM) sites (Law et al, 2013). small interfering RNAs (siRNAs) for RdDM can be alternatively generated from other RNAs, like viral or Pol-II dependent, in ‘‘non-canonical RdDM’’ (Cuerda-Gil and Slotkin, 2016)
Novel zinc finger (ZF) Fusion Proteins that Promote FLOWERING WAGENINGEN (FWA) Methylation We utilized the targeting approach previously described in Johnson et al (2014) to test RdDM components for their ability to promote DNA methylation when fused to ZF

Summary

Introduction

Cytosine DNA methylation is a key epigenetic mark involved in the silencing of transposable elements (TEs) and genes in eukaryotes. In the RdDM ‘‘arm 1,’’ RNA polymerase IV (Pol IV) generates transcripts (P4-RNAs) that are converted into double-stranded RNAs (dsRNA) by RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and subsequently processed into 24-nt siRNAs by DICER-LIKE 3 (DCL3) (Kuo et al, 2017; Matzke et al, 2015) (Figure S1A). SiRNAs for RdDM can be alternatively generated from other RNAs, like viral or Pol-II dependent, in ‘‘non-canonical RdDM’’ (Cuerda-Gil and Slotkin, 2016). These RNAs can be processed into dsRNAs by RDR1 and RDR6 and subsequently cleaved into siRNAs by different DCL proteins (Cuerda-Gil and Slotkin, 2016). SiRNAs are loaded into ARGONAUTE 4 (AGO4) or its homologs, AGO6 and AGO9 (Matzke et al, 2015)

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Discussion

Conclusion