Patrizia Filetici

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p

The bromodomain is an ∼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 Å resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15–29 of histone H4 acetylated at the ζ‐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.

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.

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 199kDa.

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

ABSTRACT 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 G2 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.

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.

CPTH6, a thiazole-derivative, induces histone hypoacetylation and apoptosis in human leukemia cells

Purpose: We previously identified novel thiazole derivatives able to reduce histone acetylation and histone acetyltransferase (HAT) activity in yeast. Among these compounds, 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) has been selected and used throughout this study. Experimental Design: The effect of CPTH6 on histone acetylation, cell viability and differentiation, cell-cycle distribution, and apoptosis in a panel of acute myeloid leukemia and solid tumor cell lines has been evaluated. Results: Here, we showed that CPTH6 leads to an inhibition of Gcn5 and pCAF HAT activity. Moreover, it inhibits H3/H4 histones and α-tubulin acetylation of a panel of leukemia cell lines. Concentration- and time-dependent inhibition of cell viability, paralleled by accumulation of cells in the G0/G1 phase and depletion from the S/G2M phases, was observed. The role of mitochondrial pathway on CPTH6-induced apoptosis was shown, being a decrease of mitochondrial membrane potential and the release of cytochrome c, from mitochondria to cytosol, induced by CPTH6. Also the involvement of Bcl-2 and Bcl-xL on CPTH6-induced apoptosis was found after overexpression of the two proteins in leukemia cells. Solid tumor cell lines from several origins were shown to be differently sensitive to CPTH6 treatment in terms of cell viability, and a correlation between the inhibitory efficacy on H3/H4 histones acetylation and cytotoxicity was found. Differentiating effect on leukemia and neuroblastoma cell lines was also induced by CPTH6. Conclusions: These results make CPTH6 a suitable tool for discovery of molecular targets of HAT and, potentially, for the development of new anticancer therapies, which warrants further investigations. Clin Cancer Res; 18(2); 475–86. ©2011 AACR.

Quinoline derivative MC1626, a putative GCN5 histone acetyltransferase (HAT) inhibitor, exhibits hat-independent activity against Toxoplasma gondii

ABSTRACT We report that quinoline derivative MC1626, first described as an inhibitor of the histone acetyltransferase (HAT) GCN5, is active against the protozoan parasite Toxoplasma gondii in vitro. However, MC1626 does not inhibit Toxoplasma GCN5 HATs or reduce HAT-mediated activity; rather, this quinoline may target the plastid organelle called the apicoplast.

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.

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

Neurospora and higher eukaryotes share a common mechanism for the signal transduction of environmental stimuli. A scenario is given in which the Neurospora WC-1 photoreceptor represents a function orthologous to that of vertebrate nuclear receptors, acting through the association with the HAT NGF-1 via a vertebrate-like LXXLL motif.

The UGA3-GLT1 intergenic region constitutes a promoter whose bidirectional nature is determined by chromatin organization in Saccharomyces cerevisiae

Transcription of an important number of divergent genes of Saccharomyces cerevisiae is controlled by intergenic regions, which constitute factual bidirectional promoters. However, few of such promoters have been characterized in detail. The analysis of the UGA3‐GLT1 intergenic region has provided an interesting model to study the joint action of two global transcriptional activators that had been considered to act independently. Our results show that Gln3p and Gcn4p exert their effect upon cis‐acting elements, which are shared in a bidirectional promoter. Accordingly, when yeast is grown on a low‐quality nitrogen source, or under amino acid deprivation, the expression of both UGA3 and GLT1 is induced through the action of both these global transcriptional modulators that bind to a region of the bidirectional promoter. In addition, we demonstrate that chromatin organization plays a major role in the bidirectional properties of the UGA3‐GLT1 promoter, through the action of an upstream Abf1p‐binding consensus sequence and a polydAdTtract. Mutations in these cis‐elements differentially affect transcription of UGA3 and GLT1, and thus alter the overall relative expression. This is the first example of an intergenic region constituting a promoter whose bidirectional character is determined by chromatin organization.