Photomorphogenesis in the hypogeous fungus Tuber borchii: isolation and characterization of Tbwc-1, the homologue of the blue-light photoreceptor of Neurospora crassa.

Truffles form a group of plant-symbiotic Ascomycetes whose hypogeous life cycle is poorly understood. Here we present initial evidence for the influence of light on Tuber borchii mycelial growth and the identification and cloning of a gene, Tbwc-1, homologous to a blue-light photoreceptor of Neurospora crassa. Blue-light irradiation of T. borchii colonies inhibits their apical growth. It also alters apical growth in N. crassa. In Neurospora, the response is controlled by a nuclear photoreceptor, NcWC-1 (White Collar-1), which consists of a sensor domain (LOV) and a transcriptional factor moiety. We isolated a gene (Tbwc-1) whose deduced amino acid sequence shows a high similarity and colinearity of domains with NcWC-1, except for the polyglutamine regions. As previously found in Neurospora, Tbwc-1 mRNA is under light control and its steady state level increases upon irradiation. In silico analysis of the TbWC-1 sensor domain (LOV) supports the hypothesis that TbWC-1 is a photoreceptor, while the absence of the two polyglutamine regions involved in transcriptional activation in Neurospora suggests that this function in Tuber could be lost.

The bromodomain: a chromatin browser?

Reversible modification of histone tails is a regulatory step in chromatin remodeling. The N-terminal tails of histones are signaling platforms that carry amino acid residues for post-translational modification and contribute to chromosomal higher order structure. These modifications are performed by a number of chromatin modulators such as histone (h) acetyltransferase, h-deacetylase, h-methyltransferase and h-kinase. Large numbers of these enzymes as well as other chromatin-associated proteins share the bromodomain, a signature protein motif. Structural studies reveal not only wide structural conservation of bromodomains but also envision a possible role of this domain in the recognition of specific modified residues in the histone tails. The widespread presence of bromodomains in leukemogenic and cancer genes has provided a fundamental tool for studies of the role of epigenetic and chromatin remodeling in malignant diseases.

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p.

The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

Characterization of the Neurospora crassa mus-25 mutant: the gene encodes a protein which is homologous to the Saccharomyces cerevisiae Rad54 protein.

Characterization of the Neurospora crassa mus-25 mutant suggests that it is defective in recombination repair and belongs to the uvs-6 epistasis group. It shows a high sensitivity to the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to UV radiation. It is barren (i.e. does not produce ascospores) in homozygous crosses. The frequency of MMS-induced mutations at the ad-3 loci is approximately three times higher than in the wild type. The ratio of homologous to nonhomologous integration of the pMTR::HYG plasmid is much lower than in wild type. The mus-25 mutant is epistatic to the mei-3 mutant for MMS sensitivity. mei-3, which is a homololog of the Saccharomyces cerevisiae gene RAD51, is a member of the uvs-6 epistasis group which contains several genes that are homologous to recombination repair genes in other organisms. The mus-25 gene was cloned by identifying a genomic DNA fragment which complements the MMS sensitivity of the mutant. The amino acid sequence deduced from the cloned DNA showed a high degree of homology to the Rad54 protein, which is involved in recombinational repair in S. cerevisiae. Comparison of the nucleotide sequences of the genomic and cDNAs of the mus-25 gene revealed an ORF of 2505 bp with a single 118-bp intron beginning immediately after the second nucleotide of the AUG start codon. The molecular weight of the deduced gene product was 93.5 kDa. The transcript level was raised within 60 min after UV irradiation or MMS treatment, as also observed for the expression of the other N. crassa recombinational repair genes, suggesting the existence of a common mechanism which induces expression of the recombinational repair genes in response to DNA damage.

Role of a white collar-1-white collar-2 complex in blue-light signal transduction.

Mutations in either white collar-1 (wc-1) or white collar-2 (wc-2) lead to a loss of most blue-light-induced phenomena in Neurospora crassa. Sequence analysis and in vitro experiments show that WC-1 and WC-2 are transcription factors regulating the expression of light-induced genes. The WC proteins form homo- and heterodimers in vitro; this interaction could represent a fundamental step in the control of their activity. We demonstrate in vivo that the WC proteins are assembled in a white collar complex (WCC) and that WC-1 undergoes a change in mobility due to light-induced phosphorylation events. The phosphorylation level increases progressively upon light exposure, producing a hyperphosphorylated form that is degraded and apparently replaced in the complex by a newly synthesized WC-1. WC-2 is unmodified and also does not change quantitatively in the time frame examined. Light-dependent phosphorylation of WC-1 also occurs in a wc-2 mutant, suggesting that a functional WC-2 is dispensable for this light-specific event. These results suggest that light-induced phosphorylation and degradation of WC-1 could play a role in the transient expression of blue-light-regulated genes. Our findings suggest a mechanism by which WC-1 and WC-2 mediate light responses in Neurospora.

The bromodomain of Gcn5p interacts in vitro with specific residues in the N terminus of histone H4.

Whereas the histone acetyltransferase activity of yeast Gcn5p has been widely studied, its structural interactions with the histones and the role of the carboxy-terminal bromodomain are still unclear. Using a glutathione S-transferase pull down assay we show that Gcn5p binds the amino-terminal tails of histones H3 and H4, but not H2A and H2B. The deletion of bromodomain abolishes this interaction and bromodomain alone is able to interact with the H3 and H4 N termini. The amino acid residues of the H4 N terminus involved in the binding with Gcn5p have been studied by site-directed mutagenesis. The substitution of amino acid residues R19 or R23 of the H4 N terminus with a glutamine (Q) abolishes the interaction with Gcn5p and the bromodomain. These residues differ from those known to be acetylated or to be involved in binding the SIR proteins. This evidence and the known dispensability of the bromodomain for Gcn5p acetyltransferase activity suggest a new structural role for the highly evolutionary conserved bromodomain.

Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospora crassa white collar proteins.

The genes coding for white collar-1 and white collar-2 (wc-1 and wc-2) have been isolated previously, and their products characterized as Zn-finger transcription factors involved in the control of blue light-induced genes. Here, we show that the PAS dimerization domains present in both proteins enable the WC-1 and WC-2 proteins to dimerize in vitro. Homodimers and heterodimers are formed between the white collar (WC) proteins. A computer analysis of WC-1 reveals a second domain, called LOV, also identified in NPH1, a putative blue light photoreceptor in plants and conserved in redox-sensitive proteins and in the phytochromes. The WC-1 LOV domain does not dimerize with canonical PAS domains, but it is able to self-dimerize. The isolation of three blind wc-1 strains, each with a single amino acid substitution only in the LOV domain, reveals that this region is essential for blue light responses in Neurospora. The demonstration that the WC-1 proteins in these LOV mutants are still able to self-dimerize suggests that this domain plays an additional role, essential in blue light signal transduction.

Regulation of expression of GLT1, the gene encoding glutamate synthase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression. A collection of increasingly larger 5' deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation. The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5' deletion derivatives. Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.

GCN5, a yeast transcriptional coactivator, induces chromatin reconfiguration of HIS3 promoter in vivo.

Gcn5p, the nuclear histone acetyltransferase (HAT A), is a component of the multiprotein adaptor complex, ADA. Its role as a transcriptional coactivator is required for full induction of most of the genes regulated by GCN4. In this study we present experimental evidence demonstrating that, during gene activation, the nuclease sensitive region of HIS3 promoter, harbouring the poly (dA:dT) and the GCN4 binding site, is invaded by nucleosomes in a gcn5 disrupted strain. These data demonstrate, for the first time, that Gcn5p affects directly the chromatin organization of a chromosomal gene during its transcriptional activation.

White collar proteins: PASsing the light signal in Neurospora crassa.

The filamentous fungus Neurospora crassa is an excellent paradigm for the study of blue light signal transduction. The isolation and characterization of the genes for two central regulators of the blue light response, white collar-1 and white collar-2, have begun to shed light on the mechanism of blue light signal transduction in fungi. These proteins are not only proposed to encode blue-light-activated transcription factors but also to be elements of the blue light signal transduction pathway.

Blue light regulation in Neurospora crassa.

The fungus Neurospora crassa has been shown to be a paradigm for photobiological, biochemical, and genetic studies of blue light perception and signal transduction. Several different developmental and morphological processes of Neurospora are regulated by blue light and can be divided into early and late blue light responses. The characterization of two central regulator proteins of blue light signal transduction in Neurospora crassa, WC1 and WC2, and the isolation of light-regulated genes, indicate transcriptional control as a central step in blue light signalling.

Sequence of the GLT1 gene from Saccharomyces cerevisiae reveals the domain structure of yeast glutamate synthase.

Glutamate synthase (GOGAT) and glutamine synthetase play a crucial role in ammonium assimilation and glutamate biosynthesis in the yeast Saccharomyces cerevisiae. The GOGAT enzyme has been purified and the GOGAT structural gene (GLT1) has been cloned, showing that this enzyme is a homotrimeric protein with a monomeric size of 199 kDa. We report the GLT1 nucleotide sequence and the amino acid sequence of its deduced protein product. Our results show that there is a high conservation with the corresponding genes of Escherichia coli, Medicago sativa (alfalfa) and Zea mais (maize). Binding domains for glutamine, cofactors (FMN and NADH) and the cysteine clusters (which comprise the iron-sulfur centres) were tentatively identified on the basis of sequence comparison with GOGAT sequences from E. coli, alfalfa and maize.

White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein.

The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.

The cpc-2 gene of Neurospora crassa encodes a protein entirely composed of WD-repeat segments that is involved in general amino acid control and female fertility.

Phenotypic and molecular studies of the mutation U142 indicate that the cpc-2+ gene is required to activate general amino acid control under conditions of amino acid limitation in the vegetative growth phase, and for formation of protoperithecia in preparation for the sexual phase of the life cycle of Neurospora crassa. The cpc-2 gene was cloned by complementation of the cpc-2 mutation in a his-2ts bradytrophic background. Genomic and cDNA sequence analysis indicated a 1636 bp long open reading frame interrupted by four introns. The deduced 316 amino acid polypeptide reveals 70% positional identity over its full length with G-protein beta-subunit-related polypeptides found in humans, rat (RACK1), chicken, tobacco and Chlamydomonas. With the exception of RACK1 the function of these proteins is obscure. All are entirely made up of seven WD-repeats. Expression studies of cpc-2 revealed one abundant transcript in the wild type; in the mutant its level is drastically reduced. In mutant cells transformed with the complementing sequence, the transcript level, enzyme regulation and female fertility are restored. In the wild type the cpc-2 transcript is down-regulated under conditions of amino acid limitation. With cpc-2 a new element involved in general amino acid control has been identified, indicating a function for a WD-repeat protein that belongs to a class that is conserved throughout the evolution of eukaryotes.

Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide.

Purification of the glutamate synthase (GOGAT) enzyme from Saccharomyces cerevisiae showed that it is an oligomeric enzyme composed of three identical 199-kDa subunits. The GOGAT structural gene was isolated by screening a yeast genomic library with a yeast PCR probe. This probe was obtained by amplification with degenerate oligonucleotides designed from conserved regions of known GOGAT genes. The derived amino-terminal sequence of the GOGAT gene was confirmed by direct amino-terminal sequence analysis of the purified protein of 199 kDa. Northern (RNA) analysis allowed the identification of an mRNA of about 7 or 8 kb. An internal fragment of the GOGAT gene was used to obtain null GOGAT mutants completely devoid of GOGAT activity. The results show that S. cerevisiae has a single NADH-GOGAT enzyme, consisting of three 199-kDa monomers, that differs from the one found in prokaryotic microorganisms but is similar to those found in other eukaryotic organisms such as alfalfa.

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs.

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs.

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.

The Neurospora crassa carotenoid biosynthetic gene (albino 3) reveals highly conserved regions among prenyltransferases.

In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al-3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophosphate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNA clones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in dark-grown and light-induced mycelia. The analysis revealed that the al-3 gene is not expressed in the dark and that its transcription is induced by blue light (Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276). The al-3 gene encodes a polypeptide of 428 amino acids. Comparison of the deduced amino acid sequence of al-3 with the sequences of prenyltransferases of other species, from bacteria to humans, showed three highly conserved homologous regions. These homologous regions may be involved in the formation of the catalytic site of the prenyltransferases.