Yazmid Reyes-Dominguez

Chromatin-level regulation of biosynthetic gene clusters

Loss-of-function Aspergillus nidulans CclA, a Bre2 ortholog involved in histone H3 lysine 4 methylation, activated the expression of cryptic secondary metabolite clusters in A. nidulans. One new cluster generated monodictyphenone, emodin and emodin derivatives, whereas a second encoded two anti-osteoporosis polyketides, F9775A and F9775B. Modification of the chromatin landscape in fungal secondary metabolite clusters allows for a simple technological means to express silent fungal secondary metabolite gene clusters.

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation

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.

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.

Dissecting individual steps of nitrogen transcription factor cooperation in the Aspergillus nidulans nitrate cluster

In the ascomycete fungus Aspergillus nidulans, the transcriptional activation of nitrate assimilating genes (niiA, niaD) depends on the cooperativity between a general nitrogen status‐sensing regulator (the GATA factor AreA) and a pathway‐specific activator (the Zn‐cluster regulator NirA). Because nitrate assimilation leads to intracellular ammonium formation, it is difficult to determine the individual contributions of NirA and AreA in this complex activation/inactivation process. In an attempt to find a suitable marker for the nitrogen status sensed by AreA, we determined the intracellular free amino acid levels on different nitrogen growth conditions. We show that the amount of glutamine (Gln) inversely correlates with all known AreA activities. We find that AreA mediates chromatin remodelling by increasing histone H3 acetylation, a process triggered by transcriptional activation and, independently of transcription, by nitrogen starvation. NirA also participates in the chromatin opening process during nitrate induction but its function is not related to histone acetylation. This chromatin remodelling function of NirA is dispensable only in nitrogen‐starved cells, conditions that lead to elevated AreA chromatin occupancy and histone H3 hyperacetylation. Continuous nitrate assimilation leads to self‐nitrogen metabolite repression but nitrate‐activated NirA is partially compensating for lowered AreA activities under these conditions.

Overexpression of the Aspergillus nidulans histone 4 acetyltransferase EsaA increases activation of secondary metabolite production

Regulation of secondary metabolite (SM) gene clusters in Aspergillus nidulans has been shown to occur through cluster‐specific transcription factors or through global regulators of chromatin structure such as histone methyltransferases, histone deacetylases, or the putative methyltransferase LaeA. A multicopy suppressor screen for genes capable of returning SM production to the SM deficient ΔlaeA mutant resulted in identification of the essential histone acetyltransferase EsaA, able to complement an esa1 deletion in Saccharomyces cereviseae. Here we report that EsaA plays a novel role in SM cluster activation through histone 4 lysine 12 (H4K12) acetylation in four examined SM gene clusters (sterigmatocystin, penicillin, terrequinone and orsellinic acid), in contrast to no increase in H4K12 acetylation of the housekeeping tubA promoter. This augmented SM cluster acetylation requires LaeA for full effect and correlates with both increased transcript levels and metabolite production relative to wild type. H4K12 levels may thus represent a unique indicator of relative production potential, notably of SMs.

N-Glycan Modification in Aspergillus Species

ABSTRACT The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.

Nucleosome Positioning and Histone H3 Acetylation Are Independent Processes in the Aspergillus nidulans prnD-prnB Bidirectional Promoter

ABSTRACT In Aspergillus nidulans, proline can be used as a carbon and nitrogen source, and its metabolism requires the integration of three signals, including proline induction and nitrogen and carbon metabolite derepression. We have previously shown that the bidirectional promoter in the prnD-prnB intergenic region undergoes drastic chromatin rearrangements such that proline induction leads to the loss of positioned nucleosomes, whereas simultaneous carbon and nitrogen metabolite repression results in the partial repositioning of these nucleosomes. In the proline cluster, the inhibition of deacetylases by trichostatin A leads to partial derepression and is associated with a lack of nucleosome positioning. Here, we investigate the effect of histone acetylation in the proline cluster using strains deleted of essential components of putative A. nidulans histone acetyltransferase complexes, namely, gcnE and adaB, the orthologues of the Saccharomyces cerevisiae GCN5 and ADA2 genes, respectively. Surprisingly, GcnE and AdaB are not required for transcriptional activation and chromatin remodeling but are required for the repression of prnB and prnD and for the repositioning of nucleosomes in the divergent promoter region. Chromatin immunoprecipitation directed against histone H3 lysines K9 and K14 revealed that GcnE and AdaB participate in increasing the acetylation level of at least one nucleosome in the prnD-prnB intergenic region during activation, but these activities do not determine nucleosome positioning. Our results are consistent with a function of GcnE and AdaB in gene repression of the proline cluster, probably an indirect effect related to the function of CreA, the DNA-binding protein mediating carbon catabolite repression in A. nidulans.

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

Highlights ► Heterochromatin protein characterized in F. graminearum. ► Heterochromatic marks regulate secondary metabolism in this fungus. ► Aurofusarin and DON production under Hep1 regulation. ► Surprisingly, Hep1 positively regulates DON biosynthesis. ► Silencing of SM genes by heterochromatin could be conserved mechanism in ascomycetes.

RNA Silencing Gene Truncation in the Filamentous Fungus Aspergillus nidulans

ABSTRACT The genus Aspergillus is ideally suited for the investigation of RNA silencing evolution because it includes species that have experienced a variety of RNA silencing gene changes. Our work on this subject begins here with the model species Aspergillus nidulans. Filamentous ascomycete fungi generally each encode two of the core RNA silencing proteins, Dicer and Argonaute, but A. nidulans appears to have lost one of each to gene truncation events. Although a role in growth, development, or RNA silencing was not detected for the truncated genes, they do produce spliced and poly(A)-tailed transcripts, suggesting that they may have an undetermined biological function. Population analysis demonstrates that the truncated genes are fixed at the species level and that their full-length orthologs in a closely related species are also unstable. With these gene truncation events, A. nidulans encodes only a single intact Dicer and Argonaute. Their deletion results in morphologically and reproductively normal strains that are incapable of experimental RNA silencing. Thus, our results suggest that the remaining A. nidulans RNA silencing genes have a “nonhousekeeping” function, such as defense against viruses and transposons.

The Histone Acetyltransferase GcnE (GCN5) Plays a Central Role in the Regulation of Aspergillus Asexual Development

Acetylation of histones is a key regulatory mechanism of gene expression in eukaryotes. GcnE is an acetyltransferase of Aspergillus nidulans involved in the acetylation of histone H3 at lysine 9 and lysine 14. Previous works have demonstrated that deletion of gcnE results in defects in primary and secondary metabolism. Here we unveil the role of GcnE in development and show that a ∆gcnE mutant strain has minor growth defects but is impaired in normal conidiophore development. No signs of conidiation were found after 3 days of incubation, and immature and aberrant conidiophores were found after 1 week of incubation. Centroid linkage clustering and principal component (PC) analysis of transcriptomic data suggest that GcnE occupies a central position in Aspergillus developmental regulation and that it is essential for inducing conidiation genes. GcnE function was found to be required for the acetylation of histone H3K9/K14 at the promoter of the master regulator of conidiation, brlA, as well as at the promoters of the upstream developmental regulators of conidiation flbA, flbB, flbC, and flbD (fluffy genes). However, analysis of the gene expression of brlA and the fluffy genes revealed that the lack of conidiation originated in a complete absence of brlA expression in the ∆gcnE strain. Ectopic induction of brlA from a heterologous alcA promoter did not remediate the conidiation defects in the ∆gcnE strain, suggesting that additional GcnE-mediated mechanisms must operate. Therefore, we conclude that GcnE is the only nonessential histone modifier with a strong role in fungal development found so far.