Repression of CHROMOMETHYLASE 3 Prevents Epigenetic Collateral Damage in Arabidopsis

Abstract

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Moreover, CMT3-induced ectopic methylation of genes undergoing transcriptional activation can reduce their corresponding transcript levels. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.

Conditional GWAS of non-CG transposon methylation inArabidopsis thalianareveals major polymorphisms in five genes

“…Knock-outs of MIR823A in several backgrounds affected CMT3 expression and mCHG in a manner consistent with this regulatory model (Papareddy et al, 2021), although the effects on methylation were very weak (Fig. 4D, E).…”

“…1). Consistent with this, one of the five major associations from GWAS of mCHG |mCHH pinpoints CMT3 and another MIR823a , a gene encoding a microRNA that directly down-regulates CMT3 by cleaving its transcripts during early embryogenesis (Papareddy et al, 2021). The most significant peak for mCHG |mCHH on RdDM-targeted transposons was located 196 bp downstream of MIR823a , while the peak corresponding to CMT3 was located 50 bp upstream of the gene (Fig.…”

“…For example, at an FDR of 20%, univariate GWAS of mCHG identifies only AGO1 and NRPE1 for RdDM-targeted transposons, and nothing for CMT2-targeted transposons (Table S2), whereas conditional GWAS of mCHG |mCHH generates a long list of known regulators of mCHG (Table S2). For RdDM-targeted transposons, we find four a priori genes above or near genome-wide significance: the previously mentioned CMT2 and CMT3 , plus MIR823a , which encodes a microRNA targeting CMT3 (Rajagopalan et al, 2006; Papareddy et al, 2021), and MULTICOPY SUPPRESSOR OF IRA1 ( MSI1 ), likely a component of chromatin assembly factor (CAF-1) responsible for proper heterochromatin formation with FASCIATA1 ( FAS1 ) and FASCIATA2 ( FAS2 ) (Hennig et al, 2003, 2005). At lower significance levels, we find REPRESSOR OF SILENCING 3 ( ROS3 ) and DNA METHYLTRANSFERASE 2 ( DNMT2 ).…”

“…Siliques were collected five days after pollination when the embryos reached an early torpedo stage according to the previous report (Papareddy et al, 2021). Using the tungsten needles, seeds were dissected from siliques and collected in 1.5 ml tubes, and washed 3-4 times with fresh 10% RNA later solution (Thermo Fisher Scientific).…”

MethylScore, a pipeline for accurate and context-aware identification of differentially methylated regions from population-scale plant whole-genome bisulfite sequencing data

“…In plants, as in most eukaryotes, DNA methylation is part of an epigenetic mechanism involved in transposon silencing (Miura et al, 2001), heterochromatin formation (Lippman et al, 2004) and gene regulation (Jaenisch & Bird, 2003). It also plays a role in genome organization (Huff & Zilberman, 2014; Zemach et al, 2013), regulation of development (Finnegan et al, 1996; Papareddy et al, 2021; Ronemus et al, 1996) and imprinting (Gehring et al, 2006; Jullien et al, 2008; Pignatta et al, 2018). DNA methylation can also be a source of phenotypic variation in natural populations (Eichten et al, 2011; Heyn et al, 2013; Schmitz et al, 2013).…”