RNA m6A Modifications and their Impact on Different Stages of Beige Adipogenesis

Abstract

N⁶-Methyladenosine (m6A) is the most abundant internal mRNA modification and plays a pivotal role in post-transcriptional gene regulation, including mRNA splicing, translation, and degradation. Dysregulation of m6A has been linked to metabolic abnormalities such as type 2 diabetes mellitus and obesity. The m6A landscape is governed by a dynamic interplay between methyltransferase "writers" (METTL3, METTL14, WTAP), demethylase "erasers" (ALKBH5, FTO), and "readers" that interpret methylation marks. Although variations in m6A levels have been observed during adipogenesis in animal models, human-specific data, particularly in beige adipocyte differentiation, remain limited.
To address this gap, this work employs a transgene-free protocol to reprogram human fibroblasts (HDFa and HDFn) into induced pluripotent stem cells (iPSCs), followed by their directed differentiation into beige adipocytes. The regulatory influence of m6A modifiers using pharmacological and genetic approaches was investigated. Early-stage treatment with small-molecule inhibitors targeting METTL3 (STM2457 and UZH2) and ALKBH5 (IOX1) revealed lineage and stage-specific sensitivity to m6A disruption. Treatment with STM2457 revealed cell source-specific vulnerabilities. Differentiation of HDFa-iPSCs failed to exit pluripotency and initiate adipogenesis, while HDFn-iPSCs displayed partial early differentiation but impaired maturation. In contrast, UZH2 treatment paradoxically increased m6A levels in HDFa-iPSCs differentiation and led to persistent adipogenic defects, suggesting compensatory feedback in methylation control and disrupted epigenetic balance. IOX1 impaired thermogenic gene induction and adipogenic stability in HDFn-iPSCs, while HDFa-iPSCs showed partial resilience.
To further comprehend the m6A writer complex, CRISPR/Cas9-mediated knockdown of WTAP and ZC3H13 was performed. Both knockdowns achieved 30-33% efficiency and revealed distinct roles in modulating thermogenic gene expression and lineage commitment. WTAP knockdown caused transient m6A reduction and partial suppression of beige identity, whereas ZC3H13 knockdown resulted in sustained m6A loss and failure of adipogenic progression.
Together, the results highlight the context-dependent, temporal regulation of m6A during beige adipogenesis and underscore the influence of donor cell origin and epigenetic memory on m6A dynamics. This work provides the first human iPSC-based framework to study m6A-mediated adipogenic regulation and offers insight into potential epitranscriptomic targets for obesity therapies and metabolic disease modeling.

Date of Award	2025
Original language	American English
Supervisor	Abdulrahim Sajini (Supervisor)