ABSTRACTWhile epigenetic mechanisms such as DNA methylation and histone modification are known to be important for gene suppression, relatively little is still understood about the interplay between these systems. The UHRF1 protein can interact with both DNA methylation and repressive chromatin marks, but its primary function in humans has been unclear. To determine what that was, we first established stable UHRF1 knockdowns (KD) in normal, immortalized human fibroblasts using targeting shRNA, since CRISPR knockouts (KO) were lethal. Although these showed a loss of DNA methylation across the whole genome, transcriptional changes were dominated by the activation of genes involved in innate immune signalling, consistent with the presence of viral RNA from retrotransposable elements (REs). We confirmed using mechanistic approaches that 1) REs were demethylated and transcriptionally activated; 2) this was accompanied by activation of interferons and interferon-stimulated genes and 3) the pathway was conserved across other adult cell types. Restoring UHRF1 in either transient or stable KD systems could abrogate RE reactivation and the interferon response. Notably, UHRF1 itself could also re-impose RE suppression independent of DNA methylation, but not if the protein contained point mutations affecting histone 3 with trimethylated lysine 9 (H3K9me3) binding. Our results therefore show for the first time that UHRF1 can act as a key regulator of retrotransposon silencing independent of DNA methylation.