Distinct amino acids of histone H3 control secondary metabolism in Aspergillus nidulans

Abstract

Chromatin remodelling events play an important role in the secondary metabolism of filamentous fungi. Previously, we showed that a bacterium, Streptomyces rapamycinicus, is able to reprogram the histone-modifying Spt-Ada-Gcn5-acetyltransferase/ADA (SAGA/ADA) complex of the model fungus Aspergillus nidulans. Consequently, the histone H3 amino acids lysine 9 and lysine 14 at distinct secondary metabolism genes were specifically acetylated during the bacterial fungal interaction, which, furthermore, was associated with the activation of the otherwise silent orsellinic acid gene cluster. To investigate the importance of the histone modifications for distinct gene expression profiles in fungal secondary metabolism, we exchanged several amino acids of histone H3 of A. nidulans. These amino acids included lysine residues 9, 14, 18, and 23 as well as serine 10 and threonine 11. Lysine residues were replaced by arginine or glutamine residues, and serine/threonine residues were replaced by alanine. All generated mutant strains were viable, allowing direct analysis of the consequences of missing posttranslational histone modifications. In the mutant strains, major changes in the expression patterns at both the transcriptional and metabolite levels of the penicillin, sterigmatocystin, and orsellinic acid biosynthesis gene clusters were detected. These effects were due mainly to the substitution of the acetylatable lysine 14 of histone H3 and were enhanced in a lysine 14/lysine 9 double mutant of histone H3. Taken together, our findings show a causal linkage between the acetylation of lysine residue 14 of histone H3 and the transcription and product formation of secondary metabolite gene clusters.

Fig 1

Histone H3 substitution strains of A. nidulans. (A) Schematic overview of the amino acid substitutions present in histone H3 mutant proteins. Indicated in gray is the histone H3 protein; amino acid residues marked in black were not replaced. The exchange of amino acids to arginine, glutamine, and alanine is marked in blue, red, and green, respectively. (B) Generation of histone H3 substitution strains of A. nidulans. Schematic representation of the genomic H3 locus of wild-type and mutant strains. Arrows mark genes, and the dashed line indicates the region of H3 in which substitutions were introduced. Black boxes mark ∼1,500-bp-long regions serving for homologous recombination.

Fig 2

Colony phenotype on agar plates, number of conidia, and biomass formation in experimental cultures of histone H3 mutant strains of A. nidulans. (A) Growth phenotype. The mutant strains were grown on AMM agar plates for 3 days at 37°C. (B) Number of conidia produced by the different strains. Fresh conidia were plated on AMM agar plates, and conidia were harvested after 3 days of incubation at 37°C. Statistical significance of data is given by the P value (**, P < 0.01; ***, P < 0.001). (C) Biomass formation in experimental cultures after 24 h of cultivation at 37°C in 20 ml of AMM.

Fig 3

Penicillin titers and mRNA steady-state levels of penicillin biosynthesis genes of histone H3 mutant strains. Wild-type and histone mutant strains were grown in fermentation media and harvested. (A) Penicillin titers measured after 36 h are given as relative values, with the amount measured for the wild type set at 100%. Statistical significance of data is given by the P value (**, P < 0.01; ***, P < 0.001). (B) Fold difference of the mRNA steady-state level of penicillin biosynthesis genes ipnA and aatA and the nonrelated gene ANIA_2647 measured by qRT-PCR after 12 h of incubation of strains. The β-actin gene of A. nidulans was used as an internal standard. Statistical significance of data is given by the P value (*, P < 0.05; **, P < 0.01).

Fig 4

Sterigmatocystin titer and mRNA steady-state levels of sterigmatocystin cluster genes of histone H3 mutant strains grown in AMM. Sterigmatocystin titers are given as relative values, with the amount measured for the wild type set at 100%. Statistical significance of data is given by the P value (**, P < 0.01; *, P < 0.05; ***, P < 0.001). (A) Sterigmatocystin titers after 48 h. (B) Sterigmatocystin titers after 36 h. (C) qRT-PCR analysis of sterigmatocystin cluster genes aflR and stcA, the gene ANIA_7830, located next to the sterigmatocystin cluster, and the β-actin-encoding acn gene used as an internal standard. Gene expression was measured after 36 h of cultivation of strains.

Fig 5

(A) Orsellinic acid production during interaction of S. rapamycinicus with the histone H3 substitution strains of A. nidulans after 24 h of coincubation. (B) qRT-PCR analysis of ors genes orsA, orsB, and orsC and of the nonrelated gene AN7908. Fold difference is given according to the 2^−ΔΔCT method. Expression levels were calculated with the A. nidulans β-actin gene as an internal standard. Mycelia for RNA isolation were taken after 3 h of coincubation of A. nidulans strains with S. rapamycinicus.