Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans

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.     

Increased conidiation associated with progression along the sterigmatocystin biosynthetic pathway

The Aspergillus nidulans sterigmatocystin (ST) gene cluster contains both regulatory (aflR) and biosynthetic genes (stc genes) required for ST production. A total of 26 genes are in the cluster, 13 of which have been assigned a known function in the biosynthetic pathway. This complex secondary pathway represents a physiological cost to the fungus. We tested the amount of asexual spore production using a series of isogenic lines of A. nidulans, differing only in a mutation in aflR (resulting in a strain containing no ST intermediates) or a mutation in three stc genes that produced either no ST intermediates (ΔstcJ), an early ST intermediate, norsoloroinic acid (ΔstcE) or a late ST intermediate, versicolorin A (ΔstcU). In two independently replicated experiments we compared the numbers of conidia produced by each of these mutant strains and a wild type ST producer in a neutral (growth media) and a host (corn seed) environment. A stepwise increase in asexual spore production was observed with each progressive step in the ST pathway. Thus, the data suggest that recruitment or loss of these secondary metabolite pathway genes has a selective advantage apart from the physiological activity of the metabolite.     

丝状真菌新型全局性调控因子LaeA

全局调控在丝状真菌次级代谢调控及其生长发育过程中有着重要的作用。LaeA是2004年首次在构巢曲霉中被发现的第一个丝状真菌全局性调控因子,继而在烟曲霉、黄曲霉、产黄青霉、橘青霉中相继被报道。LaeA能够全局性调控抗生素和真菌毒素等次级代谢产物的合成,影响真菌形态分化,另外还通过影响沉默基因的表达从而调控未知代谢产物的产生,因而能为真菌中天然产物的开发提供新的重要途径。本文就其在丝状真菌中的发现、功能、作用机制及其应用等方面进行综述。     

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.     

Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>: cross-talk regulation of secondary metabolite pathways

Penicillium chrysogenum is an excellent model fungus to study the molecular mechanisms of control of expression of secondary metabolite genes. A key global regulator of the biosynthesis of secondary metabolites is the LaeA protein that interacts with other components of the velvet complex (VelA, VelB, VelC, VosA). These components interact with LaeA and regulate expression of penicillin and PR-toxin biosynthetic genes in P. chrysogenum. Both LaeA and VelA are positive regulators of the penicillin and PR-toxin biosynthesis, whereas VelB acts as antagonist of the effect of LaeA and VelA. Silencing or deletion of the laeA gene has a strong negative effect on penicillin biosynthesis and overexpression of laeA increases penicillin production. Expression of the laeA gene is enhanced by the P. chrysogenum autoinducers 1,3 diaminopropane and spermidine. The PR-toxin gene cluster is very poorly expressed in P. chrysogenum under penicillin-production conditions (i.e. it is a near-silent gene cluster). Interestingly, the downregulation of expression of the PR-toxin gene cluster in the high producing strain P. chrysogenum DS17690 was associated with mutations in both the laeA and velA genes. Analysis of the laeA and velA encoding genes in this high penicillin producing strain revealed that both laeA and velA acquired important mutations during the strain improvement programs thus altering the ratio of different secondary metabolites (e.g. pigments, PR-toxin) synthesized in the high penicillin producing mutants when compared to the parental wild type strain. Cross-talk of different secondary metabolite pathways has also been found in various Penicillium spp.: P. chrysogenum mutants lacking the penicillin gene cluster produce increasing amounts of PR-toxin, and mutants of P. roqueforti silenced in the PR-toxin genes produce large amounts of mycophenolic acid. The LaeA-velvet complex mediated regulation and the pathway cross-talk phenomenon has great relevance for improving the production of novel secondary metabolites, particularly of those secondary metabolites which are produced in trace amounts encoded by silent or near-silent gene clusters.     

Fungus-Specific Sirtuin HstD Coordinates Secondary Metabolism and Development through Control of LaeA

The sirtuins are members of the NAD⁺-dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.