Context-dependent crosstalk between DNA methylation and histone modifications shapes durable CRISPR/dCas9-based epigenetic editing

Context-dependent crosstalk between DNA methylation and histone modifications shapes durable CRISPR/dCas9-based epigenetic editing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Context-dependent crosstalk between DNA methylation and histone modifications shapes durable CRISPR/dCas9-based epigenetic editing Goran Josipović, Nika Foglar, Ana Vujić, Fran Lukšić, Qiong Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9042636/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Understanding how histone modifications and DNA methylation cooperate within defined chromatin contexts to shape long-term transcriptional outcomes remains a major challenge in epigenetic engineering. Here, we dissect locus-specific crosstalk between DNMT3A-dCas9 and distinct dCas9-fused histone modifiers co-targeted to the CpG islands of ZEB1 and SNAI1, two promoters with similar basal activity but distinct chromatin architectures in HepG2 cells. Deposition of H3K9me2/3 or removal of H3K4 methylation enhanced DNMT3A-mediated cytosine methylation in an effector- and locus-dependent manner. DNMT3A combined with G9a or G9a-me3 induced robust and durable DNA methylation at SNAI1, resulting in long-term repression, but failed to synergize at ZEB1. Interestingly, cooperative DNMT3A–G9a activity at SNAI1 occurred independently of histone acetylation and was correlated with locus-specific enrichment of H2A.Z and PRC2/EZH2-associated factors BCLAF1 and THRAP3, as shown by protein profiling. In contrast, the distribution of active marks throughout the large ZEB1 CpG island likely restricted stable G9a-mediated H3K9 methylation, possibly through antagonism with preexisting H3K4me3, which prevented additional DNA methylation. Synergy of DNMT3A with LSD1 or RIOX1 further demonstrated that a fully demethylated H3K4 state, rather than deacetylation, is critical for the effectors cooperation. Collectively, our findings show that chromatin context dictates the compatibility and efficacy of combinatorial epigenetic editors, providing a framework for designing stable CRISPR-based transcriptional reprogramming strategies. Biological sciences/Genetics/Epigenetics Biological sciences/Genetics/Gene regulation Biological sciences/Genetics/Gene expression CRISPR/dCas9 combinatorial epigeneƟc ediƟng DNA methylaƟon H3K9me2/3 H3K4me epigeneƟc crosstalk chromaƟn context Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementMaterial.pdf Supplement Material Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9042636","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":618495136,"identity":"0f377450-7e1f-48c4-997f-154bceacf39d","order_by":0,"name":"Goran Josipović","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3RsQqCQBjA8U+Ea7loPSj0FYwDIRCkN9HFrXe46ZqstV5EHIUPavEBGgshGpUmh6BDwdFzDLr/4nH447vjAEymn6wAiNSHgF30G2w6IdFUMkS9acTdItaPPIgPs/L9avIghGV6HyXr4pKwuExiSXfZ5qwWYnX1xokofYglcsLmGZ9LjIAl4wdThNc9oU/+kRhqiQulxxRxFLErS6IldMSD/i4OoYlvpVJdSjvlhNi0eUDdPVZ1K4NwoZ1yKwAs0a1J9yJkHKgpRzEQu9b9bTKZTP/ZF0rgRl5kYiy/AAAAAElFTkSuQmCC","orcid":"","institution":"Genos Glycoscience Research Laboratory","correspondingAuthor":true,"prefix":"","firstName":"Goran","middleName":"","lastName":"Josipović","suffix":""},{"id":618495137,"identity":"9b979185-8c2c-4a2b-a30c-3011a3a06a67","order_by":1,"name":"Nika Foglar","email":"","orcid":"","institution":"Genos Glycoscience Research Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Nika","middleName":"","lastName":"Foglar","suffix":""},{"id":618495138,"identity":"a9b918e7-94a1-4742-8861-7cbc48ebebc5","order_by":2,"name":"Ana Vujić","email":"","orcid":"","institution":"Department of Biology, Faculty of Science, University of Zagreb","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"","lastName":"Vujić","suffix":""},{"id":618495139,"identity":"c8f3e6a7-f843-44e0-a17a-8839fcd7d203","order_by":3,"name":"Fran Lukšić","email":"","orcid":"","institution":"Genos Glycoscience Research Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Fran","middleName":"","lastName":"Lukšić","suffix":""},{"id":618495140,"identity":"d7212418-2ce9-461e-9b60-39a134d49654","order_by":4,"name":"Qiong Wang","email":"","orcid":"","institution":"Faculty of Life and Environmental Sciences, Biomedical Center, University of Iceland","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Wang","suffix":""},{"id":618495141,"identity":"03f33484-21dd-43a3-8047-544ca4ba5c43","order_by":5,"name":"Vedrana Vičić Bočkor","email":"","orcid":"","institution":"Genos Glycoscience Research Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Vedrana","middleName":"Vičić","lastName":"Bočkor","suffix":""},{"id":618495142,"identity":"1aa8aa32-f843-4a7b-aa8b-6a98cbb6c1ce","order_by":6,"name":"Marija Klasić","email":"","orcid":"","institution":"Department of Biology, Faculty of Science, University of Zagreb","correspondingAuthor":false,"prefix":"","firstName":"Marija","middleName":"","lastName":"Klasić","suffix":""},{"id":618495143,"identity":"a252fac4-5583-4c83-9a71-a6be2e5e6cc3","order_by":7,"name":"Siniša Habazin","email":"","orcid":"","institution":"Genos Glycoscience Research Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Siniša","middleName":"","lastName":"Habazin","suffix":""},{"id":618495144,"identity":"f0fe0dcb-9f56-4aa3-a3c6-368a4f7153d2","order_by":8,"name":"Anika Mijakovac","email":"","orcid":"","institution":"Department of Biology, Faculty of Science, University of Zagreb; 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