SunTag-NOVA robustly installed DNA methylation and repressed transcription at the endogenous FWA, FT and TMM genes with minimal genome-wide off-target consequences, and establishes SunTag-NOVA as a specific epigenome-editing platform for plants.
Abstract
Precise installation of DNA methylation at selected loci offers a powerful strategy for regulating gene expression without altering DNA sequence, but existing plant epigenome editors are constrained by limited efficiency, locus dependence, and genome-wide off-target methylation. Here, we developed SunTag-MQ1v variants incorporating TRBIP1, which promotes removal of the antagonistic H3K4me3 mark, and CHLAMY, an oligomerizing alpha crystalline domain protein from Chlamydomonas reinhardtii. TRBIP1 enhanced methylation and silencing at the Arabidopsis FWA promoter but caused widespread off-target methylation and severe developmental defects. Adding CHLAMY produced SunTag-CHLAMY-TRBIP1-MQ1v (designated as SunTag-NOVA), which successfully overcame the lethality and widespread off-target effects associated with direct TRBIP1-MQ1v fusions. We demonstrate that CHLAMY drives higher-order oligomerization of the editing complex, which enhances target specificity and mitigates off-target accumulation. SunTag-NOVA robustly installed DNA methylation and repressed transcription at the endogenous FWA, FT and TMM genes with minimal genome-wide off-target consequences. These results show that combining local chromatin modification with controlled effector assembly can improve targeted DNA methylation, and establish SunTag-NOVA as a specific epigenome-editing platform for plants.
Mammalian genomes encode multiple transposable element (TE) silencing pathways that distinguish their targets through different molecular features, with KRAB zinc finger proteins recognizing DNA sequence, the HUSH complex sensing intronless transcripts, and the PIWI–piRNA pathway using small RNA guides. How chromatin state itself contributes to TE recognition remains less defined. Here we identify Spindlin1 (SPIN1), a three-Tudor-domain histone reader implicated in germline piRNA-directed DNA methylation, as a transcriptional repressor of evolutionarily young TEs in mouse embryonic stem cells. SPIN1 selectively binds LINE and ERV loci carrying H3K4me3 and H3K9me3, a chromatin signature enriched at young, transcription-permissive elements, and recognition of these marks by Tudor domains 1 and 2 is required for TE targeting. SPIN1 engages SPINDOC as a Tudor 1 and 3-dependent cofactor whose loss phenocopies SPIN1 depletion, and associates with the H3K9 methyltransferases SETDB1 and G9a. SPIN1 loss reduces H3K9me3 and is accompanied by increased chromatin accessibility, without altering DNA methylation. Thus, SPIN1 uses a histone-state-based mechanism to identify and repress young TEs in pluripotent cells, mechanistically distinct from its germline mode in which SPIN1 cooperates with the PIWI–piRNA pathway to promote DNA methylation, illustrating how a single histone reader engages distinct silencing machineries across cellular contexts.
DNA methylation, a conserved epigenetic modification, influences several crucial functions, including gene expression, silencing of transposable elements, genomic stability, and growth and development. While advances in whole-genome bisulfite sequencing enable the identification of numerous DNA methylation sites in plants, their utility depends on characterizing DNA methylation marks for specific traits and applying them effectively in crop improvement. Central to both goals is the ability to investigate and manipulate DNA methylation at specific loci. This review focuses on recent breakthroughs in site-specific DNA methylation and demethylation in plants. Methods for detecting DNA methylation in crop plants, highlighting their respective advantages and limitations are also evaluated. We further examine the recruitment of different DNA-binding proteins to target regions. By synthesizing current research on site-specific DNA methylation and demethylation in plants, this review offers guidance on selecting the most effective strategies for site-specific DNA methylation editing. These advances hold considerable potential for crop breeding by unlocking phenotypic variation from the same genetic material, without altering the underlying DNA sequence, a transformative approach for agricultural improvement.
Anita Choudhary, Vijay Gahlaut, V. Jaiswal· Journal of Experimental Bota...· 0 citations
This review comprehensively discusses the molecular mechanisms underlying epigenetic regulation, key epigenome editing tools, their modes of action, and the emerging applications of epigenome editing in plant biology, highlighting it as a potential tool for sustainable agriculture and precision breeding.
Marifa Gulzar, Gazala Nazir, F. Mushtaq et al.· Saudi Journal of Food Securi...· 0 citations
The data show the RdDM pathway is a major regulatory contributor to generative development and heat stress responses in barley and demonstrates that rdr2 mutant shows reduced heat stress memory capacity rendering the mutant plants more vulnerable to high temperature.
Auwalu Abdu, H. Szaker, András Kis et al.· bioRxiv· 0 citations
Abstract RNA-guided obligate mobile element guided activity systems derived from transposable elements have emerged as compact genome-editing tools that may replace clustered regularly interspaced short palindromic repeats platforms. We established a dual-mode genome regulation platform using ISDge10 TnpB effectors and engineered ωRNAs, in which modulation of the ωRNA guide length enables switching between programmable transcriptional activation and genome editing. TnpB programmed with a 10-nt guide region of the ωRNA engages target DNA without inducing double-strand breaks. Fusion of transcriptional activators with Sso7d (DNA-binding protein from Sulfolobus solfataricus) enables specific transcriptional upregulation across endogenous loci. Restoring the ωRNA guide length to 20 nt triggers DNA cleavage, thereby supporting homology-directed repair-mediated sequence correction. A catalytically inactivated TnpB-based adenine base editor enabled A-to-G base conversion at genomic targets. TnpB shows strict ωRNA-dependent mismatch sensitivity with low off-target effects, suggesting its potential as a high-fidelity genome regulation platform. Compact ISDge10 TnpB facilitates co-packaging of effector and ωRNA in a single adeno-associated virus vector and co-expression of large functional domains. Thus, this study expands RNA-guided genome-editing capabilities.
Yeounsun Oh, Se‑Been Jeon, Lee Wha Gwon et al.· Nucleic Acids Research· 0 citations
Fiber-seq simultaneously profiles chromatin accessibility, DNA methylation, protein footprints, and genetic variation on single molecules at near—base-pair resolution, revealing how genetic and epigenetic features interact to regulate gene expression and provides a powerful new framework for dissecting immune cell function and disease mechanisms.
Emily A Madden, James T. Anderson, M. Cowles et al.· Journal of Immunology· 0 citations
