The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element

The 5' regulatory region of the chh2 gene, encoding cellobiohydrolase II, of the filamentous fungus Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for cbh2 expression. The CAE consists of two separate, adjacent motifs, a CCAAT box on the template strand (ATTGG) and a GTAATA box on the coding strand, which co-operate in the induction of the gene by cellulose or sophorose. EMSA supershift experiments using an antibody against Aspergillus nidulans HAPC suggested that the complex which binds to the H. jecorina CCAAT box contains a HAPC homolog. To obtain direct evidence for this, we have cloned the hap2, hap3 and hap5 genes from H. jecorina. They encode proteins whose core regions display great similarity to Aspergillus HAPB, HAPC and HAPE and to known HAP homologs from other organisms. All three genes are transcribed in a carbon source-independent manner. A. nidulans deltahap strains were functionally complemented in vitro by the overexpressed H. jecorina HAP2, HAP3 and HAP5 proteins, and they thus represent subunits of the CCAAT-binding complex. Furthermore, all three proteins (HAP2, HAP3 and HAP5) were needed to bind to the CAE in the H. jecorina cbh2 gene promoter in vitro. We conclude that the CCAAT box on the template strand in CAE is bound by the H. jecorina equivalent of the HAP protein complex.     

The Snf1 kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the filamentous fungal counterpart Cre1

In Saccharomyces cerevisiae, the SNF1 gene product phosphorylates the carbon catabolite repressor protein Mig1 under conditions when glucose is limiting, thereby relieving the fungus from catabolite repression. We have investigated whether the corresponding counterpart of filamentous fungi-the Cre1 protein-is also phosphorylated by Snf1. To this end, snf1, an ortholog of SNF1, was isolated from the ascomycete Hypocrea jecorina. The gene encodes a protein with high similarity to Snf1 kinases from other eukaryotes in its N-terminal catalytic domain, but little similarity in the C-terminal half of the protein, albeit some short aa-areas were detected, however, which are conserved in filamentous fungi and in yeast. Expression of snf1 is independent of the carbon source. An overexpressed catalytic domain of H. jecorina Snf1 readily phosphorylated yeast Mig1, but not a Mig1 mutant form, in which all four identified Snf1 phosphorylation sites (Phi XRXXSXXX Phi) had been mutated. The enzyme did neither phosphorylate H. jecorina Cre1 nor histone H3, another substrate of Snf1 kinase in yeast. H. jecorina Snf1 also phosphorylated peptides comprising the strict Snf1 consensus, but notably did not phosphorylate peptides containing the regulatory serine residue in Cre1 (=Ser(241) in H. jecorina Cre1 and Ser(266) in Sclerotinia sclerotiorum CRE1). The use of cell-free extracts of H. jecorina as protein source for Snf1 showed phosphorylation of an unknown 36 kDa protein, which was present only in extracts from glucose-grown mycelia. We conclude that the Snf1 kinase from H. jecorina is not involved in the phosphorylation of Cre1.     

The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.     

Nucleosome transactions on the<Emphasis Type="Italic"> Hypocrea jecorina</Emphasis> (<Emphasis Type="Italic"> Trichoderma reesei</Emphasis>) cellulase promoter<Emphasis Type="Italic"> cbh2</Emphasis> associated with cellulase induction

The 5 regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes –1 and –2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome –1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5 regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome –1.Communicated by C. A. M. J. J. van den Hondel     

The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding

Eukaryotes have six minichromosome maintenance (MCM) proteins that are essential for DNA replication. The contribution of ATPase activity of MCM complexes to their function in replication is poorly understood. We have established a cell-free system competent for replication in which all MCM proteins are supplied by purified recombinant Xenopus MCM complexes. Recombinant MCM2-7 complex was able to assemble onto chromatin, load Cdc45 onto chromatin, and restore DNA replication in MCM-depleted extracts. Using mutational analysis in the Walker A motif of MCM6 and MCM7 of MCM2-7, we show that ATP binding and/or hydrolysis by MCM proteins is dispensable for chromatin loading and pre-replicative complex (pre-RC) assembly, but is required for origin unwinding during DNA replication. Moreover, this ATPase-deficient mutant complex did not support DNA replication in MCM-depleted extracts. Altogether, these results both demonstrate the ability of recombinant MCM proteins to perform all replication roles of MCM complexes, and further support the model that MCM2-7 is the replicative helicase. These data establish that mutations affecting the ATPase activity of the MCM complex uncouple its role in pre-RC assembly from DNA replication.     

Partial complementation of pyruvate dehydrogenase deficiency by independently expressed lipoyl and catalytic domains of the dihydrolipoamide acetyltransferase component

Two compatible plasmids encoding a hybrid lipoyl domain and a defective pyruvate dehydrogenase (PDH) complex which lacks lipoyl domains, were co-expressed in a strain of Escherichia coli deleted for the PDH complex genes. In vivo complementation between the mutant complexes and the independent lipoyl domains was observed using growth tests in liquid and solid media. However, no PDH complex activity could be detected in the corresponding cell-free extracts. This suggests that untethered lipoyl domains can interact productively with the three types of active site in the multienzyme complex, but this association is disrupted in cell-free extracts.