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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. Although 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 on the COMPASS complex will be the SPRY domain protein designated Bre2 in Saccharomyces cerevisiae11. Analysis on 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 work has shown the key SM produced by A. nidulans will be the polyketide sterigmatocystin . To reduce ST and ST precursor backgrounds, stcJ encoding a fatty acid synthase needed for ST production16, was also deleted, generating a double stcJ JZL184 , cclA mutant. HPLC profiles of stcJ showed the production of two recognized metabolites of A. nidulans austinol and dehydroaustinol as well as the absence of ST . Analysis on the stcJ , cclA double mutant yielded at least 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 as well as an engineered strain of A. nidulans18. This strain of A.
nidulans expressed the Glarea lozoyensis polyketide synthase gene encoding for 6 methylsalicylic acid as well as the authors could not ascertain no matter if the monodictyphenone HSP produced in addition to 6 methylsalicylic acid was on account of 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 recognized A. terreus metabolite sulochrin that is derived from the anthraquinone emodin 19. Emodin , not recognized until now to be produced 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 comparable aromatic polyketide structure suggesting that a single non reduced polyketide synthase is involved in their biosynthesis. JZL184 We selected ten on the twelve non reduced polyketide synthases in a. nidulans for disruption. The two recognized NR PKSs not targeted had been the ST PKS as well as the wA PKS . Metabolite analysis on 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 various genes with high homologies to genes discovered within the ST and aflatoxin clusters . One of these genes, AN0148, showed similarity to AflR, a Zn2Cys6 binuclear transcription element needed for expression of enzymatic genes within the ST cluster21.
Replacement on the promoter region of AN0148 using the Anastrozole alcA inducible promoter allowed induction of compounds 9 14 and designed an HPLC profile comparable towards the cclA , stcJ double mutant . We next determined if elevated 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 strong reduction of H3K4me2 and H3K4me3 in all three genes confirming the function on 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 related with gene silencing and heterochromatin formation, within the two genes belonging towards the cluster, but not within the flanking, non expressed gene in cclA . JZL184 Therefore, strongly reduced levels of H3K4me2 3 as well as H3K9me2 3 at the 5 end of cluster genes are needed for derepression for the duration of secondary metabolism. We hypothesize that this cluster of genes encodes enzymes or regulatory proteins needed for monodictyphenone and emodin production and name them mdpA mdpL. AN10039 and AN0153 may well 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 right after acidified extraction . Disruption on the NR PKS AN790