Heterochromatin and RNAi act independently to ensure genome stability in Mucorales human fungal pathogens

Chromatin modifications play a fundamental role in controlling transcription and genome stability and yet despite their importance, are poorly understood in early-diverging fungi. We present a comprehensive study of histone lysine and DNA methyltransferases across the Mucoromycota, emphasizing heterochromatin formation pathways that rely on the Clr4 complex involved in H3K9-methylation, the Polycomb-repressive complex 2 driving H3K27-methylation, or DNMT1-like methyltransferases that catalyze 5mC DNA methylation. Our analysis uncovered H3K9-methylated heterochromatin as the major chromatin modification repressing transcription in these fungi, which lack both Polycomb silencing and cytosine methylation. Although small RNAs generated by RNA interference (RNAi) pathways facilitate the formation of heterochromatin in many eukaryotic organisms, we show that RNAi is not required to maintain either genomic or centromeric heterochromatin in . H3K9-methylation and RNAi act independently to control centromeric regions, suggesting a functional subspecialization. Whereas the H3K9 methyltransferase Clr4 and heterochromatin formation are essential for cell viability, RNAi is dispensable for viability yet acts as the main epigenetic, regulatory force repressing transposition of centromeric GremLINE1 elements. Mutations inactivating canonical RNAi lead to rampant transposition and insertional inactivation of targets resulting in antimicrobial drug resistance. This fine-tuned, Rdrp2-dependent RNAi activity is critical for genome stability, restricting GremLINE1 retroelements to the centromeres where they occupy long heterochromatic islands. Taken together, our results suggest that RNAi and heterochromatin formation are independent genome defense and regulatory mechanisms in the Mucorales, contributing to a paradigm shift from the cotranscriptional gene silencing observed in fission yeasts to models in which heterochromatin and RNAi operate independently in early-diverging fungi.