SUMOylation enhances DNMT1 function to repress mega-intergenic RNAs and viral mimicry.

DNA methyltransferase 1 (DNMT1) is essential for mammalian development and is frequently dysregulated in cancer. While its recruitment to hemimethylated DNA by UHRF1 is well established, its broader chromatin occupancy and regulation remain unclear. We show that DNMT1 is enriched at unmethylated CpG islands of actively transcribed genes largely by its CXXC domain. Upon selective catalytic inhibition, DNMT1 redistributes to partially methylated, inaccessible regions in a UHRF1-dependent and RFTS-dependent manner. Although coding gene expression is largely unchanged, sustained DNMT1 inhibition and subsequent global DNA hypomethylation trigger reactivation of endogenous viral elements, including an uncharacterized class of double-stranded RNA mega-intergenic transcripts (mintRNAs) driving a cell-intrinsic viral mimicry response. We identify RNF4 as a major regulator of DNMT1 catalytic function that, beyond its canonical role in DNA-protein crosslink resolution, controls the levels of SUMOylated DNMT1, establishing SUMOylation as a rheostat that regulates DNMT1 activity to maintain DNA methylation and safeguard against viral mimicry.