ECFG 9 Parallel Session 6 Regulation of Gene Expression

Astrid R Stricker, Robert L Mach

The filamentous ascomycete Hypocrea jecorina (Trichoderma reesei) offers one of the so far best characterized hydrolytic enzyme systems due to its broad application in pulp and paper, food and feed, as well as textile industry. In contrary to the wide variety of inducer molecules, which are only inducing parts of the hydrolytic enzyme system of this fungus, we have recently demonstrated that one major activating transcriptional factor (Xyr1) is an indispensible prerequisite for the hydrolase expression in H. jecorina. Here we present the mode of regulation of this key factor itself and the impact of its regulation on the expression of cellulolytic and xylanolytic enzyme-encoding genes. Whereas the expression of xyr1 is under strict Cre1-mediated carbon catabolite repression, its de novo synthesis is not required for a first hydrolase expression. On the other hand constitutive expression of xyr1 does not implicitly lead to hydrolase expression, but strictly requires the presence of at least one of the inducer molecules (e.g. xylose, xylobiose or sophorose). Summing up, these data strongly point to a posttranslational activation of Xyr1. We can provide evidence that Xyr1 is subject to phosphorylation, thereby gaining its DNA binding ability. However, additional narrow domain regulatory factors, such as Ace1, Ace2, and Xrp1 modify the expression of the hydrolytic genes. Respective models for the transcriptional regulation of the two major xylanase-encoding genes will be provided.

Peter Hortschansky¹, Martin Eisendle², Qusai Al-Abdallah¹, André D. Schmidt¹, Sebastian Bergmann¹, Marcel Thön¹, Olaf Kniemeyer¹, Beate Abt², Birgit Seeber², Ernst R. Werner³, Masashi Kato⁴, Axel A. Brakhage¹, Hubertus Haas²

1Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany, ²Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria, ³Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria, ⁴Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

The heterotrimeric CCAAT-binding complex (CBC) is evolutionary conserved in eukaryotic organisms including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the CBC consists of the subunits HapB, HapC and HapE. All HapC orthologs from eukaryotes contain three conserved cysteine residues in their core histone fold motif. Mutational and in vitro interaction analysis revealed that two of these cysteine residues are indispensable for stable HapC/HapE subcomplex formation and high affinity CBC DNA-binding. Furthermore, oxidised HapC is unable to participate in CBC assembly and located in the cytoplasm as a GFP-fusion, but can be reduced by the thioredoxin A system in vitro and in vivo. The latter was demonstrated by bimolecular fluorescence complementation (BiFC) analysis. Expression of trxA was upregulated in response to oxidative stress. Moreover, in a trxA deletion strain, the GFP-fusion of HapC is also mainly located in the cytoplasm. Therefore, redox regulation via thioredoxin very likely represents a general feature of the CBC in eukaryotes.

Recently, a putative fourth CBC subunit with an unknown function was identified in A. nidulans and designated HapX. We found that hapX expression is repressed by iron via the GATA-factor SreA and that various iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron starvation by the interaction of HapX with the CBC. These data suggest a model, in which HapX/CBC interaction is regulated at both transcriptional and post-translational levels. Iron starvation causes expression of hapX. Subsequent binding of HapX to the CBC results in transcriptional repression of iron-dependent pathways. During iron-replete conditions, hapX is repressed and, therefore, iron-dependent pathways are derepressed. Moreover, HapX/CBC interaction is inhibited by increased iron concentrations. This post-translational mechanism allows rapid adjustment to iron availability by disruption of the HapX/CBC complex. Mutual transcriptional control of hapX and sreA coordinates iron acquisition and iron-dependent pathways, thereby serving for both iron supply and prevention of iron toxicity. These data indicate that the CBC has a general role and that HapX function is confined to iron depleted conditions.

Filamentous fungi are able to use a wide variety of carbon sources. Many of these are metabolised to produce intermediates of the TCA cycle and therefore require gluconeogenesis to produce sugars. We are particularly interested in how the transcription of metabolic genes is rearranged in response to growth on acetate, fatty acids and aminoacids such as proline in Aspergillus nidulans. We have identified Zn(2)Cys(6) DNA binding proteins involved in these controls. FacB specifically regulates genes for acetate utilisation while FarA, FarB and ScfA regulate a very large number of genes required for fatty acid breakdown including proteins involved in peroxisomal biogenesis and proliferation. Genes for the glyoxalate cycle enzymes and other proteins required for both acetate and fatty acid utilisation are controlled independently by FacB and the fatty acid regulators. AcuK and AcuM are activators of genes encoding PEP carboxykinase and fructose-1,6 bisphosphatase which are required for growth on all gluconeogenic carbon sources. EMSA experiments have shown that these proteins bind as a heterodimer to sites in the 5' of relevant genes. Comparison of the 5' UTR of genes in Aspergillus spp. reveals conservation of these sites in genes for a surprisingly wide range of enzymes including those for components of the TCA cycle. Wider cross species comparisons including the hemiascomycetes Candida albicans and Yarrowia lipolytica indicates conservation of these control circuits.

Fungal mRNAs containing upstream open reading frames (uORFs) can be subject to post-transcriptional regulation. First, translation of the uORF can modulate translation of the main reading frame specified in the mRNA. Second, the uORF termination codon can be recognized as a "premature" termination codon by the machinery responsible for nonsense-mediated mRNA decay (NMD). Fungal mRNAs specifying the small subunit of arginine-specific carbamoyl phosphate synthetase contain a uORF specifying the evolutionarily conserved arginine attenuator peptide (AAP). The synthesis and amino acid sequence of the AAP are critical for cis-acting Arg-specific negative regulation of translation. In cell-free translation systems, AAP-mediated regulation depends on the AAP's ability to stall ribosomes at the uORF termination codon in response to Arg. Certain single missense mutations eliminate ribosome stalling in vitro and Arg-specific regulation in vivo and in vitro. The S. cerevisiae CPA1 and N. crassa AAPs are specified by uORFs whose start codons are in relatively poor translation initiation contexts, resulting in much leaky-scanning past the uORF. Furthermore, when Arg levels are low, there is no stalling of ribosomes on the uORF even when it is translated. However, when Arg is high, ribosomes that have synthesized the AAP stall, and this stalling blocks other scanning ribosomes from initiating translation at the downstream start codon. In addition to translational regulation, CPA1 expression is naturally regulated at the level of mRNA stability via a mechanism involving nonsense-mediated mRNA decay (NMD). Arg-regulated stalling at the CPA1 uORF stop codon triggers NMD and the data suggest a simple model that controlling the density of ribosomes at the uORF termination codon modulates NMD. Analyses of N. crassa arg-2 in isogenic NMD⁺ and nmd^- strains indicate that its levels are also controlled by NMD. We are developing methods to analyze N. crassa mRNA half-life to determine directly how the stability of arg-2 and other mRNAs is controlled, and to determine which trans-acting factors contribute to this control. These studies provide a basis for a general understanding of how ribosome occupancy of a uORF termination codon controls both translation and mRNA stability in eukaryotes.

Katharyn Sue, Brian Cheetham, Margaret Katz

XprG (PhoG) is a putative transcriptional activator in Aspergillus nidulans. xprG mutants have previously been shown to affect protease production under starvation conditions. XprG is also involved in the production of melanin and phosphate repressible acid phosphatase. In this study, we have used cDNA microarrays to identify other biological pathways in which XprG may be involved. We obtained expression profiles of xprG⁺ and xprGΔ strains, under two growth conditions; one containing a preferred carbon source (glucose) and the other containing no carbon source. A Two-way ANOVA comparison statistical test is being used to analyse any strain and condition effect on the expression profile, and also interactions between them. Results have identified new roles for XprG in mycotoxin synthesis and glucose transport, which have been validated biologically. XprG may also be involved in amino acid metabolism, conidiophore formation and regulation of the cell cycle.

HxkC is an atypical mitochondrial hexokinase which affects protease production. Mitochondrial hexokinases have been shown to regulate programmed cell death (PCD). There is genetic evidence that HxkC and XprG are involved in the same regulatory pathway, and that XprG is modulated by HxkC. Apoptotic markers, such as DNA laddering, have been found in hxkCΔ strains. This spontaneous cell death is suppressed in hxkCΔ double mutants containing a loss of function xprG2 mutation. We propose that XprG is a transcriptional activator, and is a major regulator of the cellular response to starvation.

Kazuo Shishido, Takehito Nakazawa, Shinya Kaneko, Yasumasa Miyazaki, Toru Jojima, Takashi Yamazaki, Shiho Katsukawa

Schizosaccharomyces pombe cdc5⁺ gene product (Sp.cdc5p)-related proteins (SPCDC5RPs) with c-Myb-type DNA-binding domains were isolated from various eukaryotic cells. The plant A. thaliana CDC5 and the human Cdc5 proteins were shown to bind to 7 bp and 12 bp-specific sequences, respectively. However no target gene was isolated. Later, it was found that Sp.cdc5p, S. cerevisiae Cef1 and human Cdc5L are essential for pre-mRNA splicing and a component of large splicing complexes. At present, Sp.cdc5p and SPCDC5RPs are regarded rather as splicing than as transcription factors. During the course of studying on the molecular mechanism of fruiting development of the basidiomycete Lentinula edodes, we isolated the homologue of Sp.cdc5⁺ gene, termed Le.cdc5. The Le.cdc5 product (Le.CDC5)(842 amino acid (aa) residues) possessed, in addition to c-Myb-type DNA-binding domains, a leucine zipper and two A-kinase-phosphorylation sites and was shown to bind a 7-bp sequence with the consensus sequence 5’GCAATGT3’. To demonstrate that the L.CDC5 protein functions as a transcription factor, we attempted to isolate its target gene(s). Genomic binding-site cloning experiment and inverse PCR experiment resulted in the isolation of a target gene, designated ctg1 (Le.CDC5 target gene). The ctg1 encodes a novel 159-aa protein with a leucine zipper-like sequence and contains a 7-bp Le.CDC5-binding sequence 5’GCAATCT3’ in its transcribed region downstream of the start codon. Chromatin immunoprecipitation analysis strongly suggested that intracellular Le.CDC5 binds to this 7-bp sequence present on L. edodes chromatin. The binding occurred most efficiently on chromatin from stipes of mature fruiting bodies. Based on the data of Northern-blot analysis, it might be possible to consider that one of the functions of ctg1 gene is an inhibition of stipe elongation. Two Le.CDC5-interaction partners were identified in L. edodes and named CIPA and CIPB. These proteins, however, did not interact with the Le.CDC5 phosphorylated by A kinase. The 127-aa CIPB protein was found to bind to a 6-bp sequence with the consensus sequence 5’CAACAC/T/G3’. The ctg1 contains eight 6-bp sequences: six in 5’-upstream region and two in transcribed region downstream of the start codon, all of which appear to bind to CIPB in vitro. We suggest that Le.CDC5 and CIPB cooperatively regulate ctg1 expression through an unusual manner.

Igor Morozov¹, Meriel G. Jones¹, David G. Spiller¹, Rene Novotny², Harald Berger², Joseph Strauss²

1The University of Liverpool, School of Biological Sciences, Biosciences Building, Crown Street, Liverpool L69 7ZB, United Kingdom, ²1Institut für Angewandte Genetik und Zellbiologie, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190, Vienna, Austria

The nitrogen metabolite signalling pathway in Aspergillus nidulans is a good model for gene regulation since it allows the response to a complex and changing environment to be monitored and manipulated in a precise and rapid manner. Transcriptional regulation of the pathway is coordinated by the transcription factor areA, whose activity is defined by the nitrogen state of the cell. AreA activity is in part modulated by differential degradation of its transcript in response to intracellular glutamine (Gln). Recently we reported that a large proportion of A. nidulans genes involved in nitrogen metabolism are subject to regulation at the level of transcript stability. Of the 13 genes tested, the ten that were subject to Gln signalled transcript degradation were also strongly regulated by AreA (areA, areB, niaD, niiA, crnA, gabA, nrtB, meaA, prnB and amdS).

Deadenylation of the poly(A) tail is the rate-limiting step in mRNA decay and this is a key step in the response to nitrogen regime change. Here we report that the PopB (Pop2) deadenylase mediates the nitrogen signal by accelerating the rate of the deadenylation in response to Gln. Moreover, in response to nitrogen availability, PopB modulates the formation, size and number of P-bodies, which are discrete, dynamic, cytoplasmic structures involved in the storage and degradation of the translationally repressed mRNAs. We have found that not only is the formation of the P-bodies regulated by the nitrogen metabolite signalling pathway but preliminary data suggests they become enriched with transcripts no longer required for nitrogen utilisation in response to Gln addition. This implies that the P-body is a specific site for signalled mRNA degradation and that P-body formation can be regulated by a signalling pathway. The implications of these novel results, regarding the role of PopB and function of P-bodies, will be discussed

Utilization of arginine as a source of nitrogen depends on the presence and inducibility of two arginine catabolic enzymes: arginase and ornithine aminotransferase (OAT) encoded by agaA and otaA genes, respectively. Both genes are induced by arginine and are under the control of nitrogen metabolite repression system which has been shown to be mediated by AREA general activator. The expression of agaA and otaA is repressed by ammonium. However, in areA600 complete loss-of-function mutant the expression of both genes is fully inducible. This implies that the induction of these genes does not directly or indirectly (by inducer exclusion) depends on AREA activator. Unexpectedly, there is no ammonium repression of agaA and otaA in areA600 mutant. There are two possible explanations of these results: AREA positively regulates a gene coding for a repressor of agaA and otaA, or AREA itself is the repressor of these genes.

Nitrogen repression of arginine catabolic genes also depends on the negatively acting factor AREB. Ammonium repression of agaA and otaA is lost in areB403/901 loss-of function mutant and is only partial in areB7 mutant (lack of the dimerization domain) what implies that a homo - or heterodimerization is probably necessary for the full activity of the repressor.

Analysis of different areA/areB double mutants suggests that these two GATA factors cooperate in repression of arginine catabolic genes under nitrogen repressing conditions.