Lax, C.; Osorio-Concepcion, M.; Nicolas-Munoz, N.; Tahiri, G.; Mondo, S. J.; Ng, V.; Navarro, E.; Grigoriev, I. V.; Meza-Carmen, V.; Nicolas, F. E.; Garre, V.

Epigenetic mechanisms provide sophisticated regulatory layers that modulate gene expression across diverse organisms, yet their organization and crosstalk remain poorly understood in non-dikarya fungi (NDF). Here, we characterize the genome-wide landscape of chromatin organization in the fungus Rhizopus microsporus, revealing a compartmentalized architecture where active histone modifications (H3K4me1, H3K4me3, H3K27ac) define transcriptionally active euchromatin distinct from H3K9me3-marked constitutive heterochromatin. Through comprehensive ChIP-seq analysis, we demonstrate that these modifications exhibit distinct distribution patterns over gene bodies and co-localize with 6-methyladenine (6mA) clusters (MACs), an essential epigenetic mark that is associated with transcription in this fungus. We identified functional specialization among H3K4 methyltransferase Set1 paralogs, where Set1a primarily deposits H3K4me3 and Set1b deposits H3K4me1. In contrast, both Gcn5 paralogs function redundantly in H3K27 acetylation. Knockout analysis reveals that these enzymes are critical for sporulation, stress resistance, and pathogenesis. Importantly, we uncover an epigenetic crosstalk in which active histone modifications restrict off-target 6mA deposition, regulate methylation cluster stability, and buffer transcriptional variation. Our findings reveal conserved principles of epigenetic crosstalk between active histone modifications and the essential DNA modification 6mA that may represent a fundamental mechanism of chromatin regulation in eukaryotes.