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.

Sequence analyses of fungal genomes have revealed that the potential of fungi to produce secondary metabolites is greatly underestimated. In fact, most gene clusters coding for the biosynthesis of antibiotics, toxins, or pigments are silent under standard laboratory conditions. Hence, it is one of the major challenges in microbiology to uncover the mechanisms required for pathway activation. Recently, we discovered that intimate physical interaction of the important model fungus Aspergillus nidulans with the soil-dwelling bacterium Streptomyces rapamycinicus specifically activated silent fungal secondary metabolism genes, resulting in the production of the archetypal polyketide orsellinic acid and its derivatives. Here, we report that the streptomycete triggers modification of fungal histones. Deletion analysis of 36 of 40 acetyltransferases, including histone acetyltransferases (HATs) of A. nidulans, demonstrated that the Saga/Ada complex containing the HAT GcnE and the AdaB protein is required for induction of the orsellinic acid gene cluster by the bacterium. We also showed that Saga/Ada plays a major role for specific induction of other biosynthesis gene clusters, such as sterigmatocystin, terrequinone, and penicillin. Chromatin immunoprecipitation showed that the Saga/Ada-dependent increase of histone 3 acetylation at lysine 9 and 14 occurs during interaction of fungus and bacterium. Furthermore, the production of secondary metabolites in A. nidulans is accompanied by a global increase in H3K14 acetylation. Increased H3K9 acetylation, however, was only found within gene clusters. This report provides previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes.

GALl, GAL4 and GALIO transcription [5,6]. The sequence 5'-SYGGRG-Y been proposed as a consensus for CreA-binding [7]. Unlike MIG1, however, CreA contains an additional domain downstream of the zinc-finger, which has been reported to bear high similarity to S. cerevisiae RGR1 [8,9], and whose function is unknown. Since its cloning and sequencing, molecular evidence has been presented for an involvement of CreA in the catabolite repression of transcription of genes involved in proline utilization [7], ethanol metabolism [10,11] and polysaccharide hydrolysis [12] in A. nidulans.Nothing is known as yet on the mechanism of carbon catabolite repression in other fungi. The filamentous fungus Trichoderma reesei is an industrially important producer of several extracellular enzymes, including a highly active cellulase [13] and hemicellulase enzyme system [14]. The formation of some of these enzymes (e.g. cellobiohydrolase I; endo-fl-l,4-xylanase I) is repressed by glucose [15,16]. It has been reported that the 5'-upstream nt-sequence of the T. reesei gene encoding cellobiohydrolase I (cbhl) shows consensus sequences for binding of a potential CreA-homologue [17]. Deletion of these sequences resulted in glucose derepressed transcription of cbhl [17]. It is therefore possible that carbon catabolite repression in T. reesei occurs by a mechanism similar to that existant in Aspergillus. However, the presence of a DNA-binding protein in T. reesei similar to CreA has not yet been published. As a first step towards understanding the mechanisms and cloning of the genes involved in carbon catabolite repression in T. reesei, we demonstrate here the presence of a creA homologue in T. reesei Crel --and provide evidence that the native gene product is a DNA-binding protein, thereby showing that the mechanisms of carbon catabolite repression have been basically conserved in the ascomycetous classes of Pyrenomycetes and Plectomycetes.Carbon catabolite repression in microorganisms is a means I~) control the synthesis of a range of enzymes required for the t~tilization of less favoured carbon sources when more readily t~tilized carbon sources are present in the medium. Several genes participating in this process have been identified in Sactzaromyces cerevisiae [1,2]. In the multicellular fungi, the creA gene cloned from Aspergillus nidulans [3] and A. niger [4] is the ~nly hitherto regulatory gene known to mediate carbon cat~l bolite repression. It encodes a DNA-binding protein containi Mlg a two-zinc-finger domain of the C2H2 class, which mediates ,~4% similarity to MIG1 from S. cerevisiae, which is also *Corresponding author. Fax: (43)(1) 581-6266. l-mail: jos@eichow.tuwien.ac.at Experimental Strain, cloning vector and plasmidTrichoderma reesei strain QM 9414 (ATCC 26921 ) was used throughout this study and maintained on malt agar. Bluescript II/SK+ (Stratagene, La Jolla, CA) and E. coli LC 137 (Pharmacia-LKB, Uppsala, Sweden) were used as cloning and plasmid vectors, respectively. Cloning of the T. reesei crel geneFungal genomic DNA...