Regulation of biosynthetic gene clusters

Besides structural genes like polyketides synthases, oxygenases, or reductases, also regulatory genes are embedded in secondary metabolite gene clusters. The encoded regulators, which transcriptionally control the structural genes, are divided into two classes: pathway specific transcription factors (TFs), controlling one specific gene cluster, and general TFs, controlling several different gene clusters or mediating environmental signals. In total, twelve TF superfamilies have been identified in fungi, three of which are exclusively present in fungi (Shelest, 2008). The largest fungal-specific superfamily is the zinc-cluster superfamily Zn(II)2Cys6. Its DNA-binding domain consists of six cysteine residues binding two zinc ions.

These TFs do not share a common function, but they are involved in a large number of different cellular processes, like sugar and amino acid metabolism, cell cycle, or stress response (MacPherson et al., 2006). Many gene clusters contain one or more TFs. A prominent example of a pathway-specific TF is the Zn(II)2Cys6-domain containing TF AflR, that positively regulates expression of the ST/AF gene cluster in Aspergilli (see chapter 1.2).

AflR binds to the palindromic sequence 5’-TCGN5CGA-3’ of the promoters of the AF/ST biosynthetic genes (Fernandes et al., 1998). A second motif 5’-TTAGGCCTAA-3’ was proposed to control autoregulation of aflR transcript (Chang et al., 1995). Deletion of aflR disrupts, while a modified expression of aflR changes the expression of the complete gene cluster (Ehrlich et al., 1998, Yu et al., 1996a).

Recent studies revealed that in some cases a cross-talk between assumed pathway-specific TFs occurs. In this way, some TFs control not only the gene cluster they are embedded in, but also different gene clusters located on even different chromosomes (Bergmann et al., 2010).

ScpR is a C2H2-type zinc finger TF, embedded in a cryptic NRPS gene cluster in A. nidulans, containing two NRPS genes inpA and inpB. Besides activation of these two genes, ScpR additionally activates expression of the asperfuranone gene cluster, containing the two PKS genes afoE and afoG, by binding to the promoter region of the asperfuranone-specific TF gene afoA.

Global regulatory factors do not activate one specific pathway, but several independent pathways by e.g. mediating environmental cues. The response to external signals like light, pH, temperature, or carbon and nitrogen sources is triggered by global TFs, containing mostly conserved Cys2His2 zinc finger domains, like CreA in A. nidulans, mediating carbon source changes (Dowzer et al., 1989) or PacC, mediating pH (Tilburn et al., 1995).

1.2.1.2 Epigenetic control

Chromatin is involved in all genetic processes in eukaryotic nuclei and changes in its structure go along with activated or silenced transcription of genes. Recent studies in fungi revealed, that this epigenetic control plays an important role in expression of biosynthetic gene clusters (Bok et al., 2009, Reyes-Dominguez et al., 2010, Shwab et al., 2007).

Chromatin consists of nuclear DNA, wrapped around an octamer of histone proteins. By post-translational modifications of histones, gene transcription is activated or silenced. These modifications include acetylation and methylation of lysines and arginines, phosphorylation of serines and threonines, and ubiquitination of lysines. Depending on its conformation induced by these modifications, chromatin is divided into active euchromatin or silenced heterochromatin. Hyperacetylated histones H3 and H4 correlate with transcriptional activity, whereas decreased acetylation is associated with transcriptional repression. However, methylations show different impacts on chromatin conformation. Methylated H3K4 facilitates euchromatin formation, whereas methylated H3K9, H3K27 and H4K20 are typical for heterochromatic states (Strauss et al., 2011), in which genes are silenced or repressed.

1.2.1.2.1 Methylation by S-adenosylmethionine (SAM)

Methylations of histones usually occur by transfer of a methyl group from S-adenosylmethionine (SAM, AdoMet). SAM was first discovered in 1953 by Catoni (Catoni, 1953) and since then extensively investigated, especially in mammals as it is proposed to have therapeutic benefits in human diseases (Chiang et al., 1996). Besides ATP, the ubiquitous enzyme substrate SAM is one of the most frequently used substrates and the major methyl group donor in all living organisms. In addition to histone methylation, it is involved in many methylation processes as protein, DNA, RNA, and phospholipid methylations, in which the methyl group is transferred by a methyltransferase to the corresponding substrate (Mato et al., 1997). About 15 methyltransferase superfamilies have been identified and their classifications are based on substrate specificity rather than sequence similarities (Loenen, 2006). Besides the function of SAM as methyl donor, it also acts as carboxy-aminopropyl donor in the synthesis of polyamines, like spermidine, the production of modified nucleotides in rRNA, or as transcriptional regulator by binding to riboswitches (Bjork et al., 1987, Bowman et al., 1973, Corbino et al., 2005, McDaniel et al., 2005, Winkler et al., 2005).

S-Adenosylmethionine synthetase (EC 2.5.1.6) is the only known enzyme that catalyzes the synthesis of SAM (18, Fig. 6) from methionine (17, Fig. 6) with ATP (Tabor et al., 1984).

The catalytic reaction occurs in two steps, in which the triphosphate is cleaved from ATP and further hydrolyzed to PPi and Pi before SAM is released (Mudd et al., 1958). The first crystal structure of a SAM synthetase, namely MetK of Escherichia coli, was determined in 1996 (Takusagawa et al., 1996a). MetK consists of four identical subunits forming two dimers among which the active sites lie. The triphosphate moiety interacts extensively with the amino acid residues in the active site of the enzyme in order to cleave it at both ends, while the adenine and ribose moiety shows weak interaction, what facilitates the release of the product (Takusagawa et al., 1996b).

Fig. 6: Biosynthesis of SAM. The SAM synthetase catalyzes the reaction from methionine (17) and ATP to the methyl group donor SAM (18).

1.2.1.2.2 The putative methyltransferase LaeA

LaeA (loss of aflR expression) was identified in 2004 as a regulator that complements an ST mutant strain, which was unable to express the pathway-specific regulator aflR (Bayram et al., 2011, Bok et al., 2004). Interestingly, it turned out that besides ST, LaeA was able to regulate the expression of many other secondary metabolites, like the antibiotic penicillin, the immunosuppressive gliotoxin, the cholesterol-lowering drug lovastatin, and several mycelial pigments. Due to its crucial function, LaeA has become famous as master regulator of secondary metabolism. LaeA is conserved in filamentous fungi, but not in S. cerevisiae, which possesses no secondary metabolism. LaeA is a nuclear protein with a classical nuclear localization signal (NLS) motif and a SAM-binding motif, showing sequence similarities to arginine and histone methyltransferases, indicating that LaeA acts by chromatin remodeling (Fig. 7). This hypothesis was corroborated by the finding that artificial introduction of additional genes into the ST gene cluster, which is under control of LaeA, resulted in LaeA dependent expression patterns (Bok et al., 2006a).

-OOC S

In A. nidulans it had been shown, that the silent ST gene cluster is marked by H3K9 trimethylation with high amounts of the heterochromatin protein HepA (Reyes-Dominguez et al., 2010). When the ST cluster is activated, trimethylated H3K9 and HepA levels decrease, whereas acetylated histone H3 levels increase. These chromatin modifications are restricted to genes located inside the cluster, while heterochromatic marks endure directly outside the cluster. It was suggested that LaeA counteracts H3K9 methylation inside the ST cluster, and by this, activates the specific TF AflR (Reyes-Dominguez et al., 2010). This epigenetic control explains why biosynthetic genes are clustered in fungi.

Fig. 7: Heterochromatic function of LaeA on the ST gene cluster. Modified from Keller et al., 2005. LaeA probably converts heterochromatin to euchromatin by interfering with deacetylases or methylases, and subsequently activates transcription of the ST gene cluster.