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ible regulatory roles in SM silencing. The COMPASS complex is a conserved eukaryotic transcriptional Anastrozole effector both facilitating and repressing chromatin mediated processes through methylation of lysine 4 of histone 3 11, 12. Whilst H3K4me2 and H3K4me3 are discovered predominantly on active loci 12, the COMPASS complex also regulates homothallic mating silencing, ribosomal DNA silencing, telomere length, and subtelomeric Anastrozole gene expression in yeast 13 15. A vital member in the COMPASS complex would be the SPRY domain protein designated Bre2 in Saccharomyces cerevisiae11. Analysis in the A. nidulans genome revealed a putative ortholog, here named CclA. Extracts of cclA deletants , deficient in H3K4 di and trimethylation , presented an altered chemical landscape as depicted by thin layer chromatography .
Previous function has shown the key SM made by A. nidulans would be the polyketide sterigmatocystin . To reduce ST and ST precursor backgrounds, stcJ encoding a fatty acid synthase essential for ST production16, was also deleted, generating a double stcJ JZL184 , cclA mutant. HPLC profiles of stcJ showed the production of two known metabolites of A. nidulans austinol and dehydroaustinol and the absence of ST . Analysis in the stcJ , cclA double mutant yielded a minimum of six further aromatic compounds. Full oneand two dimensional NMR analysis revealed the compounds as monodictyphenone , emodin and four other emodin analogs . Monodictyphenone has been previously isolated from a marine fungus Monodictys putredinis17 too as an engineered strain of A. nidulans18. This strain of A.
nidulans expressed the Glarea lozoyensis polyketide synthase gene encoding for 6 methylsalicylic acid and the authors could not ascertain whether the monodictyphenone HSP made in addition to 6 methylsalicylic acid was due to the heterologous gene or expression of an endogenous A. nidulans PKS218. Our data clearly shows that monodictyphenone is a item of A. nidulans and not derived from the heterologously expressed gene. Monodictyphenone , a metabolite with antimicrobial properties, shares structural similarity to a known A. terreus metabolite sulochrin that is derived from the anthraquinone emodin 19. Emodin , not known until now to be made by A. nidulans, is an active anthraquinone constituent demonstrating anti mutagenic, anti cancer, vasorelaxant, immunosuppressive anti inflammation and anti apoptosis activities20.
Monodictyphenone and emodin and its derivatives , share a similar aromatic polyketide structure suggesting that a single non reduced polyketide synthase is involved in their biosynthesis. JZL184 We selected ten in the twelve non reduced polyketide synthases inside a. nidulans for disruption. The two known NR PKSs not targeted had been the ST PKS and the wA PKS . Metabolite analysis in the ten PKS mutant strains identified a single PKS responsible for production of all compounds 9 14 . AN0150 is located 0.5 Mb from the correct telomere of 5 Mb chromosome VIII and is surrounded by many genes with high homologies to genes discovered within the ST and aflatoxin clusters . A single of these genes, AN0148, showed similarity to AflR, a Zn2Cys6 binuclear transcription element essential for expression of enzymatic genes within the ST cluster21.
Replacement in the promoter region of AN0148 with all the Anastrozole alcA inducible promoter allowed induction of compounds 9 14 and developed an HPLC profile similar to the cclA , stcJ double mutant . We next determined if improved production of compounds 9 14 was reflected in gene expression in cclA . Figure 1c shows up regulated gene expression in cclA from AN10021 through AN10023, two exception becoming AN0147 and AN10035 that had been expressed equally well within the control strain. An examination of histone H3 methylation and acetylation levels in cclA by chromatin immunoprecipitation of two cluster genes and 1 flanking gene indicated a robust reduction of H3K4me2 and H3K4me3 in all three genes confirming the role in the putative COMPASS complex JZL184 member CclA in lysine 4 methylation of H3 .
Interestingly, reduced levels of H3K4me2 3 also resulted in low levels of H3K9me2 3, a chromatin mark associated with gene silencing and heterochromatin formation, within the two genes belonging to the cluster, but not within the flanking, non expressed gene in cclA . JZL184 Thus, strongly reduced levels of H3K4me2 3 too as H3K9me2 3 at the 5 end of cluster genes are essential for derepression in the course of secondary metabolism. We hypothesize that this cluster of genes encodes enzymes or regulatory proteins essential for monodictyphenone and emodin production and name them mdpA mdpL. AN10039 and AN0153 may represent boundaries of this gene cluster allowing us to propose a likely pathway . Lastly we asked if CclA regulation extended to other clusters. Interestingly, two antiosteoporosis yellow polyketides, F9775B and F9775A , isolated from Paecilomyces carneus22 could also be detected from the cclA strain grown on YAG solid medium immediately after acidified extraction . Disruption in the NR PKS AN790