Functional characterization of UHRF1 variants in facilitating DNA methylation

Ubiquitin-like with plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1 (UHRF1) is essential for DNA methylation inheritance. However, the functional impacts of several natural and engineered UHRF1 variants are either insufficiently characterized or obscured by conflicting results, with some discrepancies likely stemming from cellular toxicity and adaptive responses induced by DNA methylation changes. In this study, we utilized mouse embryonic stem cells (mESCs)-which uniquely tolerate the complete loss of DNA methylation-to evaluate the functional consequences of clinical mutations, isoform variation, and epitope tagging. Using rescue experiments in Uhrf1-deficient mESCs, we characterized two UHRF1 mutations identified in a patient with immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, demonstrating that the R618X nonsense mutation creates a null allele, while the R296W missense mutation is hypomorphic. Furthermore, we confirmed that N-terminal tagging abolishes UHRF1 function, whereas human UHRF1 (hUHRF1) isoform 2, featuring a 13-residue N-terminal extension, is functionally inactive. AlphaFold3 structural predictions suggest that these additional residues at the N terminus disrupt essential inter-domain interactions. Collectively, our results define the activity of UHRF1 variants and resolve existing inconsistencies in the field.