Loss of Lysine-Specific Demethylase 1 (LSD1) Drives Aberrant Heterochromatin Formation in Neurospora crassa

Both H3K9me3 and DNA methylation are subject to spreading mechanisms to effectively cover incipient chromatin across heterochromatin domains. Boundary elements and associated limiting factors are necessary to prevent heterochromatin from spreading into neighboring, gene-rich heterochromatin. LSD1 was identified to be one such factor, given previous studies in other models and high conservation throughout eukaryotes. This study identifies the LSD complex in Neurospora and characterizes the heterochromatin spreading defect in Neurospora crassa ∆lsd1 strains. We found ∆lsd1 strains to possess variable extents of excessive heterochromatin spreading, and that this is dependent on the presence of DNA methylation, unlike at canonical heterochromatin domains where loss of DNA methylation has no effect on the presence of other heterochromatin marks (H3K9me3 and HP1-binding). Our findings provide insight of LSD1 function in heterochromatin regulation. Bisulfite-seq: We analyzed 5mC distribution in asexually propagated ∆lsd1 Neurospora strains, as determined by bisulfite treatment followed by whole genome sequencing. ChIP-seq: We analyzed H3K4me3 in WT and ∆lsd1 strains as well as H3K9me3 in ∆lsd1;∆hda-1 and lsd1 cat null strains by chromatin immunoprecipitation. Strains were grown, crosslinked, lysed, modified nucleosomes were immunopurified, and associated DNA was sequenced. RNA-seq: We analyzed gene expression changes in ∆lsd1 Neurospora crassa strains by poly-A+ mRNA-sequencing performed in duplicate. A wild type strain (N3752) serves as the reference strain (deposited in GSE82222: GSM2186738 and GSM2186742).