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 the first comprehensive study of histone-lysine and DNA methyltransferases across the Mucoromycota, uncovering H3K9-methylated heterochromatin as the major chromatin modification repressing transcription in these fungi, which lack both Polycomb silencing and cytosine methylation. Although RNA interference (RNAi) facilitates the formation of heterochromatin in many eukaryotic organisms, we show that RNAi is not required to maintain either genomic or centromeric heterochromatin in Mucor. H3K9-methylation and RNAi act independently to control centromeric regions, suggesting a functional sub-specialization. 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 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 transposable elements 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 co-transcriptional gene silencing observed in fission yeasts to models in which heterochromatin and RNAi operate independently in early-diverging basal fungi. Significance The Mucorales are a group of basal fungi known to cause mucormycosis, a fungal infection with high mortality rates and for which treatment options are limited. Understanding key regulatory mechanisms that drive adaptation to the host, influence virulence traits, and control genome stability will provide insights to overcome the challenges posed by these fungal pathogens. Our research establishes constitutive heterochromatin and RNAi as two central epigenetic, regulatory forces that act independently to repress transposable elements in the Mucorales. Modulating these regulatory mechanisms may lead to reduced transposition and subsequently to less frequent drug target disruption that could be exploited to combat antimicrobial drug resistance.