Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis.

The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today's truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a 'symbiosis toolbox'. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at approximately 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for approximately 58% of the genome. In contrast, this genome only contains approximately 7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis-'the symbiosis toolbox'-evolved along different ways in ascomycetes and basidiomycetes.

Epigenetic and Posttranslational Modifications in Light Signal Transduction and the Circadian Clock in Neurospora crassa.

Blue light, a key abiotic signal, regulates a wide variety of physiological processes in many organisms. One of these phenomena is the circadian rhythm presents in organisms sensitive to the phase-setting effects of blue light and under control of the daily alternation of light and dark. Circadian clocks consist of autoregulatory alternating negative and positive feedback loops intimately connected with the cellular metabolism and biochemical processes. Neurospora crassa provides an excellent model for studying the molecular mechanisms involved in these phenomena. The White Collar Complex (WCC), a blue-light receptor and transcription factor of the circadian oscillator, and Frequency (FRQ), the circadian clock pacemaker, are at the core of the Neurospora circadian system. The eukaryotic circadian clock relies on transcriptional/translational feedback loops: some proteins rhythmically repress their own synthesis by inhibiting the activity of their transcriptional factors, generating self-sustained oscillations over a period of about 24 h. One of the basic mechanisms that perpetuate self-sustained oscillations is post translation modification (PTM). The acronym PTM generically indicates the addition of acetyl, methyl, sumoyl, or phosphoric groups to various types of proteins. The protein can be regulatory or enzymatic or a component of the chromatin. PTMs influence protein stability, interaction, localization, activity, and chromatin packaging. Chromatin modification and PTMs have been implicated in regulating circadian clock function in Neurospora. Research into the epigenetic control of transcription factors such as WCC has yielded new insights into the temporal modulation of light-dependent gene transcription. Here we report on epigenetic and protein PTMs in the regulation of the Neurospora crassa circadian clock. We also present a model that illustrates the molecular mechanisms at the basis of the blue light control of the circadian clock.

Synthesis of a novel series of thiazole-based histone acetyltransferase inhibitors.

Acetylation, which targets a broad range of histone and non-histone proteins, is a reversible mechanism and plays a critical role in eukaryotic genes activation/deactivation. Acetyltransferases are very well conserved through evolution. This allows the use of a simple model organism, such as budding yeast, for the study of their related processes and to discover specific inhibitors. Following a simple yeast-based chemogenetic approach, we have identified a novel HAT (histone acetyltransferase) inhibitor active both in vitro and in vivo. This new synthetic compound, 1-(4-(4-chlorophenyl)thiazol-2-yl)-2-(propan-2-ylidene)hydrazine, named BF1, showed substrate selectivity for histone H3 acetylation and inhibitory activity in vitro on recombinant HAT Gcn5 and p300. Finally, we tested BF1 on human cells, HeLa as control and two aggressive cancer cell lines: a neuroblastoma from neuronal tissue and glioblastoma from brain tumour. Both global acetylation of histone H3 and specific acetylation at lysine 18 (H3AcK18) were lowered by BF1 treatment. Collectively, our results show the efficacy of this novel HAT inhibitor and propose the utilization of BF1 as a new, promising tool for future pharmacological studies.

Chemogenomic profiling of the cellular effects associated with histone H3 acetylation impairment by a quinoline-derived compound.

We report the results of a chemogenomic profiling aimed to explore the mode of action of a quinolic analogue of the p300 histone acetyltransferase (HAT) inhibitor anacardic acid, named MC1626. This compound reduced histone H3 acetylation in a dose-dependent manner and the HATs Gcn5 and Rtt109, which specifically target H3 lysines, were the only ones that caused chemical-genetic synthetic sickness with MC1626 when mutated. Deletion of specific Gcn5 (e.g., Ada1) and Rtt109 (e.g., Asf1) multiprotein complex components also enhanced MC1626 sensitivity. In addition to N-terminal H3 lysines, MC1626 inhibits H3-K56 acetylation, a histone modification that, in yeast, is exclusively supported by Rtt109 and indirectly influences DNA integrity. Several DNA repair mutants were found to be sensitive to MC1626. Functional links between histone acetylation impairment by MC1626 and mitochondrion as well as cytoskeleton functionality were also revealed, thus extending the range of non-nuclear processes that are influenced by histone acetylation.

The Neurospora crassa White Collar-1 dependent blue light response requires acetylation of histone H3 lysine 14 by NGF-1.

Blue light-induced transcription in Neurospora crassa is regulated by the White Collar-1 (WC-1) photoreceptor. We report that residue K14 of histone H3 associated with the light-inducible albino-3 (al-3) promoter becomes transiently acetylated after photoinduction. This acetylation depends on WC-1. The relevance of this chromatin modification was directly evaluated in vivo by construction of a Neurospora strain with a mutated histone H3 gene (hH3(K14Q)). This strain phenocopies a wc-1 blind mutant and shows a strong reduction of light-induced transcriptional activation of both al-3 and vivid (vvd), another light-inducible gene. We mutated Neurospora GCN Five (ngf-1), which encodes a homologue of the yeast HAT Gcn5p, to generate a strain impaired in H3 K14 acetylation and found that it was defective in photoinduction. Together, our findings reveal a direct link between histone modification and light signaling in Neurospora and contribute to the developing understanding of the molecular mechanisms operating in light-inducible gene activation.

Photoreceptors in the dark: A functional white collar-like complex and other putative light-sensing components encoded by the genome of the subterranean fungus Tuber melanosporum.

Light is perceived and transduced by fungi, where it modulates processes as diverse as growth and morphogenesis, sexual development and secondary metabolism. A special case in point is that of fungi with a subterranean, light-shielded habitat such as Tuber spp. Using as reference the genome sequence of the black truffle Tuber melanosporum, we used bioinformatic prediction tools and expression data to gain insight on the photoreceptor systems of this hypogeous ectomycorrhizal fungus. These include a chromophore-less opsin, a putative red-light-sensing phytochrome not expressed at detectable levels in any of the examined lifecycle stages, and a nearly canonical two-component (WC-1/WC-2) photoreceptor system similar to the Neurospora white collar complex (WCC). Multiple evidence, including expression at relatively high levels in all lifecycle stages except for fruiting-bodies and the results of heterologous functional complementation experiments conducted in Neurospora, suggests that the Tuber WCC is likely functional and capable of responding to blue-light. The other putative T. melanosporum photoreceptor components, especially the chromophore-less opsin and the likely non-functional phytochrome, may instead represent signatures of adaptation to a hypogeous (light-shielded) lifestyle.

SAGA DUB-Ubp8 Deubiquitylates Centromeric Histone Variant Cse4.

Aneuploidy, the unbalanced segregation of chromosomes during cell division, is recurrent in many tumors and the cause of birth defects and genetic diseases. Centromeric chromatin represents the chromosome attachment site to the mitotic spindle, marked by specialized nucleosomes containing a specific histone variant, CEN-H3/Cse4, in yeast. Mislocalization of Cse4 outside the centromere is deleterious and may cause aberrant chromosome behavior and mitotic loss. For this reason, ubiquitylation by the E3-ubiquitin ligase Psh1 and subsequent proteolysis tightly regulates its restricted localization. Among multiproteic machineries, the SAGA complex is not merely engaged in acetylation but also directly involved in deubiquitylation. In this study, we investigated the role of SAGA-DUB's Ubp8-driven deubiquitylation of the centromeric histone variant Cse4 in budding yeast. We found that Ubp8 works in concert with the E3-ubiquitin ligase Psh1, and that its loss causes defective deubiquitylation and the accumulation of a short ubiquitin oligomer on Cse4. We also show that lack of Ubp8 and defective deubiquitylation increase mitotic instability, cause faster Cse4 proteolysis and induce mislocalization of the centromeric histone outside the centromere. Our data provide evidence for a fundamental role of DUB-Ubp8 in deubiquitylation and the stability of the centromeric histone in budding yeast.

Nanocapsules: coating for living cells.

One of the most promising tools for future applications in science and medicine is the use of nanotechnologies. Especially self-assembly systems, e.g., polyelectrolyte (PE) capsules prepared by means of the layer-by-layer technique with tailored properties, fulfill the requirements for nano-organized systems in a satisfactory manner. The nano-organized shells are suitable as coating for living cells or artificial tissue to prevent immune response. With these shells, material can be delivered to predefined organs. In this paper, some preliminary results are presented, giving a broad overview over the possibilities to use nano-organized capsules. Based on the observations that the cells while duplicating break the capsule a mutant yeast strain (Saccharomyces cerevisiae), which express GFP-tubulin under galactose promotion, was investigated by means of confocal laser scanning microscopy. The measurements reveal an increased surface charge in the region of buds developed prior encapsulation. In order to test the used PE pair for cytotoxicity, germinating conidia of the fungi Neurospora crassa were coated. The investigation with fluorescence microscopy shows a variation in the surface charge for the growing region and the conidium poles. The capsules exhibit interesting properties as valuable tool in science and a promising candidate for application in the field of medicine.

Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.

Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.

Physical association of the WC-1 photoreceptor and the histone acetyltransferase NGF-1 is required for blue light signal transduction in Neurospora crassa.

In Neurospora crassa and other filamentous fungi, light-dependent-specific phenomena are regulated by transcription factors WC-1 and WC-2. In addition to its transcriptional activity, WC-1 is able to directly sense light stimuli through a LOV sensor domain. Its location in the nucleus and heterodimerization with WC-2, together with the presence of a zinc-finger DNA-binding domain and an environmental sensor domain, all resemble the functional evolutionary architecture adopted by vertebrate nuclear receptors (NRs). Here we describe a scenario in which WC-1 represents a functional orthologue of NRs and acts through association with the chromatin-modifying coactivator NGF-1, which encodes a homologue of the yeast Gcn5p acetyltransferase. To support this view, we show a direct association between WC-1 and NGF-1 that depends on a WC-1 region containing a conserved functional LXXLL motif, a signature previously described as being an exclusive feature of NR/coactivator interaction. Our data suggest that a WC-1/NGF-1 complex is preassembled in the dark on light-inducible promoters and that, after exposure to light stimulation, NGF-1-associated HAT activity leads to histone H3 acetylation and transcriptional activation. Finally, we provide evidence for a NGF-1-independent acetylated form of WC-1. Overall our data indicate that Neurospora and higher eukaryotes share a common mechanism for the signal transduction of environmental stimuli.

A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone.

Acetylation is a key modulator of genome accessibility through decondensation of the chromatin structure. The balance between acetylation and opposite deacetylation is, in fact, a prerequisite for several cell functions and differentiation. To find modulators of the histone acetyltransferase Gcn5p, we performed a phenotypic screening on a set of newly synthesized molecules derived from thiazole in budding yeast Saccharomyces cerevisiae. We selected compounds that induce growth inhibition in yeast strains deleted in genes encoding known histone acetyltransferases. A novel molecule CPTH2, cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone, was selected based on its inhibitory effect on the growth of a gcn5Delta strain. We demonstrated a specific chemical-genetic interaction between CPTH2 and HAT Gcn5p, indicating that CPTH2 inhibits the Gcn5p dependent functional network. CPTH2 inhibited an in vitro HAT reaction, which is reverted by increasing concentration of histone H3. In vivo, it decreased acetylation of bulk histone H3 at the specific H3-AcK14 site. On the whole, our results demonstrate that CPTH2 is a novel HAT inhibitor modulating Gcn5p network in vitro and in vivo.

Gcn5p plays an important role in centromere kinetochore function in budding yeast.

We report that the histone acetyltransferase Gcn5p is involved in cell cycle progression, whereas its absence induces several mitotic defects, including inefficient nuclear division, chromosome loss, delayed G(2) progression, and spindle elongation. The fidelity of chromosome segregation is finely regulated by the close interplay between the centromere and the kinetochore, a protein complex hierarchically assembled in the centromeric DNA region, while disruption of GCN5 in mutants of inner components results in sick phenotype. These synthetic interactions involving the ADA complex lay the genetic basis for the critical role of Gcn5p in kinetochore assembly and function. We found that Gcn5p is, in fact, physically linked to the centromere, where it affects the structure of the variant centromeric nucleosome. Our findings offer a key insight into a Gcn5p-dependent epigenetic regulation at centromere/kinetochore in mitosis.

The role of loop ZA and Pro371 in the function of yeast Gcn5p bromodomain revealed through molecular dynamics and experiment.

Biological experiments were combined with molecular dynamics simulations to understand the importance of amino acidic residues present in the bromodomain of the yeast histone acetyltransferase Gcn5p. It was found that residue Pro371 plays an important role in the molecular recognition of the acetylated histone H4 tail by Gcn5p bromodomain. Crystallographic analysis of the complex showed that this residue does not directly interact with the histone substrate. It has been demonstrated that a double mutation Pro371Thr and Met372Ala in the Gcn5p bromodomain impairs chromatin remodeling activity. It is demonstrated here that, in this double mutant and in the fully deleted bromodomain strain, there is lower growth under amino acid deprivation conditions. By in vitro surface plasmon resonance (Biacore) experiments it is shown that the binding affinity of the double mutation to acetyl lysine 16 histone H4 peptide decreases. Molecular dynamics simulations were used to explain this loss in acetyl lysine-Gcn5p bromodomain affinity, in the double mutant. By comparing nanosecond molecular dynamics trajectories of the native as well as the single and doubly mutated bromodomain, it is concluded that the presence of Pro371 is important to the functionality of the Gcn5p bromodomain. In the simulation a point mutation involving this highly conserved residue induced an increase in the flexibility of the ZA loop, which in turn modulated the exposure of the binding pocket to the acetyl lysine. The combined double mutations (Pro371Thr-Met372Ala) not only markedly perturb the motion of the ZA loop but also destabilize the entire structure of the bromodomain.

Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics.

Mycorrhizal ascomycetes are ecologically and commercially important fungi that have proved impervious to genetic transformation so far. We report here on the successful transient transformation of Tuber borchii, an ectomycorrhizal ascomycete that colonizes a variety of trees and produces highly prized hypogeous fruitbodies known as "truffles". A hypervirulent Agrobacterium tumefaciens strain bearing the binary plasmid pBGgHg was used for transformation. The genes for hygromycin resistance and the enhanced green fluorescent protein (EGFP), both under the control of vector-borne promoters, were employed as selection markers. Patches of dark and fluorescent hyphae were observed upon fluorescence microscopic examination of hygromycin-resistant mycelia. The presence of EGFP was confirmed by both confocal microscopy and PCR analysis. The lack in the transformed mycelia of the DNA coding for kanamicin resistance (a trait encoded by a vector-borne gene located outside of the T-DNA region) indicates that Agrobacterium-mediated gene transfer correctly occurred in T. borchii.