Abstract Aberrant DNA methylation, in regions such as gene promoters, is considered an epigenetic hallmark of many cancers, contributing to tumorigenesis by means of transcriptionally silencing tumor suppressor genes. However, DNA methylation and mRNA expression correlations are often presented with inconsistent evidence supporting causal regulation. We hypothesized that causal regulatory methylation elements would exhibit heightened demethylation sensitivity. To investigate, we analyzed 21 whole-genomic bisulfite sequenced samples before and after demethylation and identified narrow-width elements within a short plateau, termed Methylation Mesa (MM). MM signatures are conserved across primary samples as well as human and mouse cell lines and are largely independent of CpG islands. Intriguingly, when comparing demethylation–expression correlations across promoter CpG islands, ±1 kb promoter regions, and MMs near transcription start sites, a significant correlation is observed exclusively for MM demethylation. To further assess causality, we developed CRISPR-DiR, wherein DNMT1-interacting RNAs (DiRs) were coupled to CRISPR to introduce targeted demethylation with superior resolution. We demonstrated that demethylation of a Mesa triggers locus and distal chromatin rewiring events that initiate mRNA expression greater than promoter-CpG island targeting. Thus, we report narrow-width genome-wide regulatory elements capable of initiating gene transcription and provide a fine-resolution technology for their targeted and precise demethylation for both validation and therapeutic purposes. Citation Format: Yanjing V. Liu, Jeremiah Suryatenggara, Hannan Wong, Jing Ping Tang, Hong Kee Tan, Junsu Kwon, Qiling Zhou, Simone Ummarino, Alexander Ebralidze, John Doench, Li Chai, Touati Benoukraf, Devendra Hiwase, Daniel Thomas, Annalisa Di Ruscio, Daniel G. Tenen, Mahmoud A. Bassal. Methylation Mesa Define Functional Regulatory Elements for Targeted Gene Demethylation and Activation abstract. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86 (13Suppl): Abstract nr P64.
Liu et al. (2026) studied this question.