Epigenetic control of PDX1 and NGN3 by a computationally designed PRC2 inhibitor enforces pancreatic endocrine differentiation from pluripotent stem cells

Epigenetic control of PDX1 and NGN3 by a computationally designed PRC2 inhibitor enforces pancreatic endocrine differentiation from pluripotent stem cells | 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 Epigenetic control of PDX1 and NGN3 by a computationally designed PRC2 inhibitor enforces pancreatic endocrine differentiation from pluripotent stem cells Vincenzo Cirulli, Laura Crisa, shiri levy, Daniel Mar, Karol Bomsztyk, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9115136/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 Directed differentiation of pluripotent stem cells (PSCs) into pancreatic islets is a cornerstone strategy for diabetes cell therapy. This process relies on growth factor–driven activation of core transcriptional regulators, notably PDX1 and NGN3, to restrict the multi-lineage potential of definitive endoderm to pancreatic progenitors and endocrine cell types. Yet differentiation efficiency and lineage fidelity vary markedly across PSC lines. Here, we demonstrate that a dominant constraint is persistent Polycomb Repressive Complex 2 (PRC2)–mediated epigenetic repression at the PDX1 and NGN3 loci, limiting endocrine specification despite inductive signaling. To directly test whether chromatin states at the PDX1 and NGN3 loci gate developmental competence, we deployed a computationally engineered epigenetic effector (EBdCas9) to transiently and sequentially remove H3K27me3 at those loci during defined developmental windows. Targeted epigenetic resolution robustly enhanced endocrine lineage commitment and accelerated β-cell differentiation across genetically diverse PSC lines. In contrast, direct transcriptional activation with VP64dCas9 increased PDX1 and NGN3 expression but did not improve differentiation outcomes. Integrated cell population studies and genome-wide chromatin and transcriptomic analyses reveal that PRC2-targeted remodeling preferentially activates endocrine gene networks while limiting progenitor expansion and lineage-inappropriate programs. These findings establish that gene-targeted manipulation of PRC2-mediated repression at PDX1 and NGN3 can be used to control cell lineage competence. Collectively, our study reframes variability in PSC differentiation as a failure of epigenetic resolution rather than transcriptional insufficiency and introduces locus-specific chromatin remodeling as a generalizable strategy to enforce developmental fidelity. Biological sciences/Developmental biology/Stem cells/Stem-cell differentiation Biological sciences/Stem cells/Stem-cell differentiation Full Text Additional Declarations There is NO Competing Interest. Supplementary Files TableS8.xlsx Supplemental Table 8 TableS9.xlsx Supplemental Table 9 TableS11AD.pdf Supplemental Table 11-A-D TableS10.pdf Supplemental Table 10 TableS7.xlsx Supplemental Table 7 TableS6.xlsx Supplemental Table 6 TableS3.xlsx Supplemental Table 3 TableS4.xlsx Supplemental Table 4 TableS2.xlsx Supplemental Table 2 TableS5.xlsx Supplemental Table 5 TableS1.xlsx Supplemental Table 1 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. 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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-9115136","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":611580948,"identity":"08373f22-7726-41c1-a0be-ad8eda17c85a","order_by":0,"name":"Vincenzo 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This process relies on growth factor–driven activation of core transcriptional regulators, notably PDX1 and NGN3, to restrict the multi-lineage potential of definitive endoderm to pancreatic progenitors and endocrine cell types. Yet differentiation efficiency and lineage fidelity vary markedly across PSC lines. Here, we demonstrate that a dominant constraint is persistent Polycomb Repressive Complex 2 (PRC2)–mediated epigenetic repression at the PDX1 and NGN3 loci, limiting endocrine specification despite inductive signaling. \r\nTo directly test whether chromatin states at the PDX1 and NGN3 loci gate developmental competence, we deployed a computationally engineered epigenetic effector (EBdCas9) to transiently and sequentially remove H3K27me3 at those loci during defined developmental windows. Targeted epigenetic resolution robustly enhanced endocrine lineage commitment and accelerated β-cell differentiation across genetically diverse PSC lines. In contrast, direct transcriptional activation with VP64dCas9 increased PDX1 and NGN3 expression but did not improve differentiation outcomes. Integrated cell population studies and genome-wide chromatin and transcriptomic analyses reveal that PRC2-targeted remodeling preferentially activates endocrine gene networks while limiting progenitor expansion and lineage-inappropriate programs. \r\nThese findings establish that gene-targeted manipulation of PRC2-mediated repression at PDX1 and NGN3 can be used to control cell lineage competence. 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