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Loss-of-function Aspergillus nidulans CclA, a Bre2 ortholog involved in histone 3 lysine 4 methylation, activated the expression of cryptic secondary metabolite (SM) clusters in A. nidulans. One novel cluster generated monodictyphenone, emodin and emodin derivatives while a second encoded two anti-osteoporosis polyketides, F9775A and F9775B. Modification of the chromatin landscape in fungal SM clusters allows for a simple technological means to express silent fungal secondary metabolite gene clusters.Aspergilli are ubiquitous filamentous fungi whose members include human and plant pathogens and industrial fungi with tremendous medical, agricultural and biotechnological importance. Although demonstrating synteny along large tracks of their sequenced genomes, * Corresponding authors: phone: (323) 442-1670; fax: (323) 442-1390, clayw@usc.edu, phone: (608) 262-9795; fax: (608) (2) clusters 3 . Yet the expression of most SM clusters and their concomitant products remain veiled. Two approaches for activating otherwise silent clusters were recently described. One strategy, utilizing the knowledge that many SM clusters contain a pathway specific transcription factor, fused an inducible promoter to a cluster transcription factor leading to the production of hybrid polyketide-nonribosomal peptide metabolites, the cytotoxic aspyridones A (3) and B (4) 4 . A second approach, based on genomic mining of microarrays generated from mutants of the global regulator of secondary metabolism LaeA 5, 6, 7 , led to the identification of the anti-tumor compound terrequinone A (5) 8 . Efforts to uncover the regulatory role of LaeA revealed that some subtelomeric SM clusters were located in heterochromatic regions of the genome where suppression was relieved by deletion of a key histone deacetylase 9 . The importance of histone modifications in SM clusters was further reflected in the initiation and spread of histone H4 acetylation concurrent with transcriptional activation of the subtelomeric A. parasiticus aflatoxin (6) gene cluster 10 .A consideration of the accruing evidence linking chromatin modifications with SM cluster regulation led us to examine the hypothesis that additional chromatin modifying proteins were important in SM cluster regulation. In particular, we examined a member of the COMPASS (complex associated with Set1) complex for possible regulatory roles in SM silencing. The COMPASS complex is a conserved eukaryotic transcriptional effector both facilitating and repressing chromatin-mediated processes through methylation of lysine 4 of histone 3 (H3K4) 11,12 . While H3K4me2 and H3K4me3 are found predominantly on active loci 12 , the COMPASS complex also regulates homothallic mating silencing, ribosomal DNA silencing, telomere length, and subtelomeric gene expression in yeast [13][14][15] .A critical member of the COMPASS complex is the SPRY domain protein designated Bre2 in Saccharomyces cerevisiae 11 . Analysis of the A. nidulans genome revealed a putative ortholog, here named CclA. Extracts of cclA delet...

Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM-gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de-repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein-1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM-specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.