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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.

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.

La paniculitis mesentérica es una rara entidad de caracte - rísticas benignas que afecta al tejido adiposo del mesenterio intestinal y que puede progresar de distintas formas, desde la resolución espontánea hasta la fibrosis. La etiología aún es incierta, siendo causas probables el trauma, infecciones o cirugía. Presentamos el caso de un paciente de sexo masculino de 64 años de edad que comenzó de forma súbita con dolor abdominal y leucocitosis. Se realizó diagnóstico de paniculi - tis mesentérica mediante una tomografía computada y el pa - ciente evolucionó con resolución espontánea a los dos meses.

El páncreas heterotópico es la presencia de tejido pancreático fuera de la localización anatómica del páncreas. Es una entidad rara que puede presentarse en cualquier parte del tracto gastrointestinal, siendo el estómago y el intestino delgado los sitios más comunes. Usualmente es asintomático, pero puede hacerse clínicamente evidente cuando se complica con cambios patológicos como inflamación, sangrado, obstrucción y transformación maligna. Presentamos un paciente de 49 años de edad de sexo masculino que consultó por presentar dolor epigástrico recurrente. La endoscopía digestiva alta reveló un tumor submucoso en el antro. El estudio histopatológico posterior a la cirugía demostró tejido pancreático heterotópico. El páncreas ectópico debe considerarse en el diagnóstico diferencial de los tumores gástricos submucosos.

La neumatosis intestinal es una entidad muy infrecuente asociada a varias patologías, como el infarto intestino-mesentérico, la enterocolitis necrotizante y la enfermedad pulmonar obstructiva crónica. Se caracteriza por la presencia de gas en la subserosa o submucosa a través del tracto gastrointestinal. Presentamos el caso de un paciente de sexo masculino de 63 años de edad que consultó por dolor en abdomen superior, vómitos y fiebre elevada (39º) nueve días después de una gastrectomía total por cáncer. La radiografía directa de abdomen constató distensión intestinal y la tomografía computada (TC) demostró distensión intestinal, edema mesentérico, neumatosis intestinal a través del intestino delgado y neumatosis portal, preferentemente en el lóbulo hepático izquierdo. Se realizó una laparotomía de urgencia que reveló únicamente distensión intestinal por adherencias, sin evidenciar necrosis intestinal. El paciente evolucionó desfavorablemente, falleciendo posteriormente. Reportamos un nuevo caso y revisamos la literatura de la neumatosis intestinal asociada con neumatosis portal.The pneumatosis intestinalis is a very infrequent condition associated with a number of diseases, such as mesenteric infarction, necrotizing enterocolitis, and obstructive pulmonary disease characterized by the presence of subserosal or submucosal gas cyst throughout the gastrointestinal tract. A 63- year- old man complained of upper abdominal pain, vomiting and high fever (39º C) on the nine day after total gastrectomy for cancer. Abdominal X-ray revealed intestinal distension. The abdominal Computed Tomography (CT) showed intestinal dilatation, mesenteric oedema, diffuse pneumatosis throughout the small intestine and gas in the portal venous system predominantly in the left hepatic lobe. It was performed emergency activity that revealed intestinal distension secondary to adhesion without intestinal necrosis. The patient had a downhill course and died thereafter. We report a new case and reviewed the literature of pneumatosis intestinalis associated with hepatic portal venous gas.

We conducted a clinical and electromyographical study in patients with Chagas' disease in the indeterminate or chronic stages of the illness. Altogether 841 patients were examined. Only 511 were admitted within the protocol; the remainder patients were rejected because they showed other causes able to damage the nervous system. Fifty two (10.17%) out of the 511 patients showed signs and symptoms of peripheral nervous system involvement in the form of sensory impairment and diminished tendon jerks suggesting the presence of neuropathy. Forty five of them were submitted to a conventional electromyographical examination. Fifteen of mem showed normal results, while the remainder 30 disclosed a reduced interference pattern, being most of the remaining motor unit potentials fragmented or poliphasic, reduced sensory and motor conduction velocities and diminished amplitude of the sensory action potential. The findings suggest that some chagasic patients in the indeterminate or chronic stages of the disease may develop a clinical mild sensory-motor peripheral neuropathy.El estúdio presente fue diseftado con ei objeto de pesquizar Ia existência de manifestaciones clinicas en pacientes afectados por enfermedad de Chagas, en estádio indeterminado o crônico, que tuviesen, ai menos, 2 reacciones serologicas positivas. En total fueron examinados 841 enfermos. De ellos solo 511 fueron admitidos en ei protocolo; los restantes fueron rechazados por mostrar Ia presencia de otras causas que hubiesen podido danar su sistema nervioso. Dentro de los 511 pacientes admitidos, 52 (10.17%) evidenciaron alteraciones objetivas y subjetivas de Ia sensibilidad y disminucion de los reflejos osteotendinosos. Estos signos y sintomas, que sugieren la presencia de neuropatia, podian combinarse de diferente manera. Como complemento dei examen clinico, se efectuo estúdio electromiografico convencional en 45 de estos pacientes. En 15 los hallazgos fueron normales, en tanto que en los restantes 30 pudo demostrarse reduccion de la capacidad de reclutamiento voluntário de unidad motora, presencia de potenciales de unidad motora fragmentados o polifasicos, reduccion de la velocidad de conduccion motora y sensitiva y disminucion de la amplitud dei potencial sensitivo. Los resultados obtenidos sefialan que una proporcion de los pacientes en el estádio indeterminado o crônico de la infeccion pueden desarrollar una moderada neuropatia sensitivo-motora con expresion clinica.

Abstract Background We describe a patient who underwent transurethral resection of the prostate for urinary obstructive symptoms and had histological findings of adenocarcinoma of the prostate with prostatic localization of chronic lymphocitic leukemia (CLL).The contemporary presence of CLL, adenocarcinoma of the prostate and residual prostatic gland after transurethral resection has never been reported before and the authors illustrate how they managed this unusual patient. Case presentation A 79-years-old white man, presented with acute urinary retention, had a peripheral blood count with an elevated lymphocytosis (21.250/mL) with a differential of 65.3% lymphocytes and the prostate-specific antigen (PSA) value was 3.38 ng/mL with a percent free PSA of 8.28%. The transrectal ultrasound (TRUS) indicated an isoechonic and homogenic enlarged prostate of 42 cm3 and the abdomen ultrasound found a modest splenomegaly and no peripheral lymphadenophaty. The patient underwent transurethral resection of the prostate and had a pathological finding of adenocarcinoma in the prostate with a Gleason Score 4 (2+2) of less than 5% of the material (clinical stage T1a), associated with a diffused infiltration of chronic lymphocitic leukemia elements. Conclusions The incidental finding of a prostatic localization of a low-grade non-Hodgkin's lymphoma does not modify eventually further treatments for neither prostate cancer nor lymphoma. The presence of a low-grade and low-stage lymphoma, confirmed by a hematological evaluation, and the simultaneous evidence of an adenocarcinoma after transurethral resection of the prostate for acute urinary retention do not require any immediate treatment due to its long-term survival rate and the follow-up remains based on periodical PSA evaluation and complete blood count.

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. [ABSTRACT FROM PUBLISHER]

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADPGDH) 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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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., (Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Canzonetta et al.)

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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., (Copyright 2005 John Wiley & Sons, Ltd.)

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.

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., (Copyright 1998 Academic Press.)

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.