SIZ1-mediated SUMOylation of ROS1 Enhances Its Stability and Positively Regulates Active DNA Demethylation inArabidopsis

Abstract

The 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1)-mediated active DNA demethylation is critical for shaping the genomic DNA methylation landscape in Arabidopsis. Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown. Using a methylation-sensitive PCR (CHOP-PCR)-based forward genetic screen for Arabidopsis DNA hypermethylation mutants, we identified the SUMO E3 ligase SIZ1 as a critical regulator of active DNA demethylation. Dysfunction of SIZ1 leads to hyper-methylation at approximately 1,400 genomic regions. SIZ1 physically interacts with ROS1 and mediates the SUMOylation of ROS1. The SUMOylation of ROS1 is reduced in siz1 mutant plants. Compared to that in wild type plants, the protein level of ROS1 is significantly decreased, even though there is an increased level of ROS1 transcripts in siz1 mutant plants. Our results suggest that SIZ1 positively regulates active DNA demethylation by promoting the stability of ROS1 protein through SUMOylation.catalyze histone acetylations, which in turn attact the SWR1 complex for the deposition of H2A.Z required for the direct recruitment of ROS1 (Lang et al., 2015; Li et al., 2015; Li et al., 2012; Nie et al., 2019; Qian et al., 2012; Wang et al., 2015). ROS1 belongs to a small family of enzymes that also includes DME, DML2 and DML3 (Zhu, 2009), which are iron-sulfur proteins requiring Fe-S clusters as cofactors for enzymatic activities (Balk and Pilon, 2011; Netz et al., 2014). A cytosolic iron-sulfur assembly (CIA) pathway component, MET18, is required for delivery of Fe-S clusters to ROS1, and is thus important for the enzymatic activity of ROS1 (Duan et al., 2015; Wang et al., 2016). Little is known about whether and how ROS1 may be regulated by post-translational modifications.Mechanistically similar to ubiquitination, SUMOylation occurs through an enzyme cascade (Seeler and Dejean, 2003). A maturation process is needed for the nascently synthesized SUMO protein, where the precursor protein is cleaved by a family of SUMO proteases SENPs/Ulps to exposure the C-terminal Gly-Gly motif that is indispensable for SUMO conjugation to its substrates (Mukhopadhyay and Dasso, 2007). In Arabidopsis, the mature forms of SUMOs are subsequently activated by a heterodimeric E1 activating enzyme (SAE1/SAE2) in an ATP-dependent way, then transferred to an E2 conjugating enzyme (SCE1), and finally covalently conjugated to the specific lysine residues of substrate proteins (Augustine and Vierstra, 2018). The SUMO E3 ligase can facilitate SUMO conjugation by E2 to the substrates, and can increase the substrate specificity (Gareau and Lima, 2010; Johnson, 2004). In yeast and metazoans, SUMOylation has been observed to regulate innate immunity, chromatin stability, gene transcription, DNA base excision repair and subcellular localization of target proteins (Gill, 2005; Johnson, 2004). In Arabidopsis, the dysfunction of SUMO E3 ligase SIZ1 is reported to affect ABA responses, cold tolerance, flowering time, basal ...

Somatic mutations during rapid clonal domestication of Populus alba var. pyramidalis

“…In addition, we found another mutation in Chr10.1859 , which is homologous to AT2G36490 ( REPRESSOR OF SILENCING1 , ROS1 ) in Arabidopsis . AT2G36490 is associated with several important functions, including heat stress resistance and DNA demethylation regulation (Kim, Kidokoro, et al, 2020; Kong et al, 2020; Malabarba et al, 2021). Such mutations in genes with important functions may enhance an individual's defensive capabilities and its adaptability to a changing environment.…”

Interplay of phytohormones and epigenetic regulation: A recipe for plant development and plasticity

“…In addition, ROS1‐associated WD40 domain‐containing protein (RWD40), ROS1‐associated methyl‐DNA binding protein 1 (RMB1), and ROS1‐associated homeodomain protein 1 (RHD1) form a protein complex with ROS1, which is required for ROS1‐dependent DNA demethylation (Liu et al, 2021). The stability of ROS1 is regulated by the SUMO E3 ligase SIZ1 (SAP AND MIZ1 DOMAIN‐CONTAINING LIGASE 1) via a physical interaction with ROS1 and by SUMOylation (Kong et al, 2020). These key players could also serve as targets for drug screening.…”

Active DNA demethylation in plants: 20 years of discovery and beyond

“…Heat stress significantly increases protein SUMOylation (Yoo et al, 2006). The DNA glycosylase domain of ROS1 can be SU-MOylated in vitro and directly interacts with SUMO1 in vivo (Kong et al, 2020). ROS1 protein levels are significantly reduced in the SUMOylation E3 ligase mutant siz1, which contains over 1,000 hyper-DMRs that strongly overlap with those of ros1 (Kong et al, 2020).…”

“…The DNA glycosylase domain of ROS1 can be SUMOylated in vitro and directly interacts with SUMO1 in vivo (Kong et al, 2020). ROS1 protein levels are significantly reduced in the SUMOylation E3 ligase mutant siz1 , which contains over 1,000 hyper‐DMRs that strongly overlap with those of ros1 (Kong et al, 2020). Whether heat stress‐induced DNA demethylation depends on ROS1 is currently unknown.…”