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However, fully reversing DNA methylation abnormalities requires lowering DNMT1 below a deep threshold, posing major drug discovery challenges. Here, we demonstrated that a similarly stringent threshold applied to UHRF1 in maintaining hypermethylated TSG suppression. Genetically reducing redundancy in DNMT1 or UHRF1 markedly lowered these thresholds, sensitizing cells to their inhibition. These findings were translated into engineered recombinant reporter systems tied to endogenous hypermethylated promoters in hypomorphic cells, substantially improving sensitivity and dynamic range for monitoring HMA\u2010induced TSG reactivation. Pilot high\u2010throughput screening validated assay robustness and specificity for DNMT1 and UHRF1 inhibitors. Additionally, these platforms enabled optimization of timing and combination strategies pairing DNMT1\/UHRF1 inhibitors with other epigenetic agents to maximize TSG re\u2010expression. Our study supports the concept of maintenance methylation thresholds and provides versatile tools for discovering novel HMAs and designing epigenetic combination therapies.<\/jats:p>","DOI":"10.1002\/advs.202519080","type":"journal-article","created":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T12:32:28Z","timestamp":1774528348000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Reduced Maintenance DNA Methylation Thresholds Enable Sensitive Reporter Assays for UHRF1 and DNMT1 Inhibition"],"prefix":"10.1002","volume":"13","author":[{"given":"Cuicui","family":"Xia","sequence":"first","affiliation":[{"name":"Division of Life Sciences and Medicine University of Science and Technology of China  Hefei China"},{"name":"Institute of Drug Discovery Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine China\u2010New Zealand Joint Laboratory on Biomedicine and Health Guangzhou Institutes of 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