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David Immanuel Schumacher, Ramona Lütkenhaus, Florian Altegoer, Ines Teichert, Ulrich Kück, Minou Nowrousian

• \(\bf Background:\) Fungal fruiting bodies are complex three-dimensional structures that are formed to protect and disperse the sexual spores. Their morphogenesis requires the concerted action of numerous genes; however, at the molecular level, the spatio-temporal sequence of events leading to the mature fruiting body is largely unknown. In previous studies, the transcription factor gene pro44 and the histone chaperone gene asf1 were shown to be essential for fruiting body formation in the ascomycete Sordaria macrospora. Both PRO44 and ASF1 are predicted to act on the regulation of gene expression in the nucleus, and mutants in both genes are blocked at the same stage of development. Thus, we hypothesized that PRO44 and ASF1 might be involved in similar aspects of transcriptional regulation. In this study, we characterized their roles in fruiting body development in more detail. \(\bf Results:\) The PRO44 protein forms homodimers, localizes to the nucleus, and is strongly expressed in the outer layers of the developing young fruiting body. Analysis of single and double mutants of asf1 and three other chromatin modifier genes, cac2, crc1, and rtt106, showed that only asf1 is essential for fruiting body formation whereas cac2 and rtt106 might have redundant functions in this process. RNA-seq analysis revealed distinct roles for asf1 and pro44 in sexual development, with asf1 acting as a suppressor of weakly expressed genes during morphogenesis. This is most likely not due to global mislocalization of nucleosomes as micrococcal nuclease-sequencing did not reveal differences in nucleosome spacing and positioning around transcriptional start sites between \(\Delta\)asf1 and the wild type. However, bisulfite sequencing revealed a decrease in DNA methylation in \(\Delta\)asf1, which might be a reason for the observed changes in gene expression. Transcriptome analysis of gene expression in young fruiting bodies showed that pro44 is required for correct expression of genes involved in extracellular metabolism. Deletion of the putative transcription factor gene asm2, which is downregulated in young fruiting bodies of Δpro44, results in defects during ascospore maturation. \(\bf Conclusions:\) In summary, the results indicate distinct roles for the transcription factor PRO44 and the histone chaperone ASF1 in the regulation of sexual development in fungi.