Genome-wide recruitment profiling of transcription factor Crz1 in response to high pH stress

Background: Exposure of the budding Saccharomyces cerevisiae to an alkaline environment produces a robust transcriptional response involving hundreds of genes. Part of this response is triggered by an almost immediate burst of calcium that activates the Ser/Thr protein phosphatase calcineurin. Activated calcineurin dephosphorylates the transcription factor (TF) Crz1, which moves to the nucleus and binds to calcineurin/Crz1 responsive gene promoters. In this work we present a genome-wide study of the binding of Crz1 to gene promoters in response to high pH stress. Results: Environmental alkalinization promoted a time-dependent recruitment of Crz1 to 152 intergenic regions, the vast majority between 1 and 5 min upon stress onset. Positional evaluation of the genomic coordinates combined with existing transcriptional studies allowed identifying 140 genes likely responsive to Crz1 regulation. Gene Ontology analysis confirmed the relevant impact of calcineurin/Crz1 on a set of genes involved in glucose utilization, and uncovered novel targets, such as genes responsible for trehalose metabolism. We also identified over a dozen of genes encoding TFs that are likely under the control of Crz1, suggesting a possible mechanism for amplification of the signal at the transcription level. Further analysis of the binding sites allowed refining the consensus sequence for Crz1 binding to gene promoters and the effect of chromatin accessibility in the timing of Crz1 recruitment to promoters. Conclusions: The present work defines at the genomic-wide level the kinetics of binding of Crz1 to gene promoters in response to alkaline stress, confirms diverse previously known Crz1 targets and identifies many putative novel ones. Because of the relevance of calcineurin/Crz1 in signaling diverse stress conditions, our data will contribute to understand the transcriptional response in other circumstances that also involve calcium signaling, such as exposition to sexual pheromones or saline stress

Histone H1 Post-Translational Modifications : Update and Future Perspectives

Histone H1 is the most variable histone and its role at the epigenetic level is less characterized than that of core histones. In vertebrates, H1 is a multigene family, which can encode up to 11 subtypes. The H1 subtype composition is different among cell types during the cell cycle and differentiation. Mass spectrometry-based proteomics has added a new layer of complexity with the identification of a large number of post-translational modifications (PTMs) in H1. In this review, we summarize histone H1 PTMs from lower eukaryotes to humans, with a particular focus on mammalian PTMs. Special emphasis is made on PTMs, whose molecular function has been described. Post-translational modifications in H1 have been associated with the regulation of chromatin structure during the cell cycle as well as transcriptional activation, DNA damage response, and cellular differentiation. Additionally, PTMs in histone H1 that have been linked to diseases such as cancer, autoimmune disorders, and viral infection are examined. Future perspectives and challenges in the profiling of histone H1 PTMs are also discussed

Complex evolutionary history of the mammalian histone H1.1-H1.5 gene family

H1 is involved in chromatin higher-order structure and gene regulation. H1 has a tripartite structure. The central domain is stably folded in solution, while the N- and C-terminal domains are intrinsically disordered. The terminal domains are encoded by DNA of low sequence complexity, and are thus prone to short insertions/deletions (indels). We have examined the evolution of the H1.1-H1.5 gene family from 27 mammalian species. Multiple sequence alignment has revealed a strong preferential conservation of the number and position of basic residues among paralogs, suggesting that overall H1 basicity is under a strong purifying selection. The presence of a conserved pattern of indels, ancestral to the splitting of mammalian orders, in the N- and C-terminal domains of the paralogs, suggests that slippage may have favored the rapid divergence of the subtypes and that purifying selection has maintained this pattern because it is associated with function. Evolutionary analyses have found evidences of positive selection events in H1.1, both before and after the radiation of mammalian orders. Positive selection ancestral to mammalian radiation involved changes at specific sites that may have contributed to the low relative affinity of H1.1 for chromatin. More recent episodes of positive selection were detected at codon positions encoding amino acids of the C-terminal domain of H1.1, which may modulate the folding of the CTD. The detection of putative recombination points in H1.1-H1.5 subtypes suggests that this process may has been involved in the acquisition of the tripartite H1 structure

Linker histone partial phosphorylation : effects on secondary structure and chromatin condensation

Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation

Disulfide driven folding for a conditionally disordered protein

Altres ajuts: ICREA, ICREA-Academia 2015 to S.V.Conditionally disordered proteins are either ordered or disordered depending on the environmental context. The substrates of the mitochondrial intermembrane space (IMS) oxidoreductase Mia40 are synthesized on cytosolic ribosomes and diffuse as intrinsically disordered proteins to the IMS, where they fold into their functional conformations; behaving thus as conditionally disordered proteins. It is not clear how the sequences of these polypeptides encode at the same time for their ability to adopt a folded structure and to remain unfolded. Here we characterize the disorder-to-order transition of a Mia40 substrate, the human small copper chaperone Cox17. Using an integrated real-time approach, including chromatography, fluorescence, CD, FTIR, SAXS, NMR, and MS analysis, we demonstrate that in this mitochondrial protein, the conformational switch between disordered and folded states is controlled by the formation of a single disulfide bond, both in the presence and in the absence of Mia40. We provide molecular details on how the folding of a conditionally disordered protein is tightly regulated in time and space, in such a way that the same sequence is competent for protein translocation and activity

Estudios estructurales y caracterización de la unión al DNA del dominio C-terminal de la histona H1 : efecto de la fosforilación /

Descripció del recurs: el 21-08-2008Consultable des del TDXTítol obtingut de la portada digitalitzadaLa histona H1 es la responsable de la condensación de la cromatina en la fibra de 30 nm. Se ha descrito que la H1 tiene preferencia por el DNA tipo SAR. Hemos mostrado que los subtipos H1a-e, H1º y H1t individualmente muestran preferencia por las SAR. La H1 está compuesta por 3 dominios: N-terminal, globular y C-terminal. El análisis independiente de los dominios mostró que el C-terminal determina la preferencia por las SAR de la H1. Las protaminas, relacionadas evolutivamente con la histona H1 y muy ricas en arginina también tienen preferencia por las SAR. La unión preferencial a las SAR del C-terminal y la protamina se pierde en presencia de distamicina, lo que indica que la interacción involucra el surco menor del DNA. La preferencia por las SAR está determinada por los bloques AT homopoliméricos. El dominio C-terminal de las histonas H1º (C-H1º) y H1t (C-H1t) se encuentra desestructurado en disolución acuosa, pero en presencia de DNA y 140 mM de NaCl se estructura completamente. La estructura secundaria obtenida está caracterizada por la presencia de hélice α, estructura β, giros y lazos abiertos. El TFE también induce estructura secundaria en los dominios C-terminales con características similares a las encontradas en los complejos con DNA. La estructura de los dominios C-terminales unidos al DNA es extremadamente estable. La desnaturalización de los dominios C-terminales unidos a DNA es cooperativa. El acoplamiento entre la unión al DNA y la inducción de estructura secundaria en el C-terminal permite incluir este dominio en el grupo de las proteínas intrínsecamente desordenadas que funcionan mediante el reconocimiento molecular. Las elevadas concentraciones de solutos macromoleculares en el interior de la célula hacen que una fracción importante del volumen intracelular no esté disponible. Los dominios C-H1º y C-H1t se estructuran en presencia de Ficoll 70 (30%) y el PEG 6000 (30%) por efectos del volumen excluido. La estructura secundaria inducida es similar a la encontrada en el C-terminal unido a DNA. Los resultados de SAXS indican que la compactación del C-terminal en presencia de aglomerantes macromoleculares es propia de un estado globular. La compactación va acompañada de la aparición de un núcleo hidrófobico. Sin embargo, el dominio C-terminal no está estructurado cooperativamente en presencia de agentes aglomerantes. Estas características indican que el C-terminal en presencia de agentes aglomerantes se encuentra en un estado de glóbulo fundido. La formación del glóbulo fundido en la célula aceleraría el paso al estado nativo o unido al DNA y facilitaría la difusión y el intercambio de la H1 en la cromatina. La histona H1 es fosforilada por las quinasas dependientes de ciclinas (CDKs). Esta fosforilación es dependiente del ciclo celular con el máximo de fosfatos en la metafase de la mitosis. La mayoría de las dianas de las CDKs se encuentran en el dominio C-terminal. La fosforilación de los tres motivos TPXK del C-H1º induce un cambio estructural caracterizado por la disminución de la proporción de hélice α y un aumento de la estructura β. La magnitud del cambio depende de la relación proteína/DNA. A relaciones cercanas a la saturación la conformación de la proteína es del tipo todo-β. La fosforilación de uno o dos de los tres sitios TPXK presentes en el C-H1º tiene efectos estructurales específicos. La fosforilación en T118 es la que afecta más profundamente la estructura con una disminución importante de la hélice α, acompañada de la aparición de un porcentaje significativo de ovillo estadístico. La neutralización de la carga positiva de las lisinas por los grupos fosfato del DNA puede ser una de las causas principales de la estructuración del dominio C-terminal en los complejos con el DNA. A pH alcalino se induce estructuración en el C-H1º no fosforilado y trifosforilado, la cual refleja los cambios dependientes de fosforilación encontrados en los complejos con DNA.H1 linker histones are thought to be primarily responsible for the condensation of the 30 nm chromatin fibre. Histone H1 preferentially binds to scaffold-associated regions (SARs). Here we show that the mammalian somatic subtypes H1a,b,c,d,e and H1_ and themale germline-specific subtype H1t, all preferentially bind to SARs. Experiments with the isolated domains show that whilst the C-terminal domain maintains strong and preferential binding, the N-terminal and globular domains show weak binding and poor specificity for the SAR. The preferential binding of SAR by the H1 molecule thus appears to be determined by its highly basic C-terminal domain. Salmine, a typical fish protamine, which could have its evolutionary origin in histone H1, also shows preferential binding to the SAR. The interaction of distamycin, a minor groove binder with high affinity for homopolymeric oligo(dA).oligo(dT) tracts, abolishes preferential binding of the C-terminal domain of histone H1 and protamine to the SAR, suggesting the involvement of the DNA minor groove in the interaction. The carboxyl-terminal domain of linker histone H1 subtypes H1º (C-H1º) and H1t (C-H1t) has little structure in aqueous solution but becomes extensively folded upon interaction with DNA. The secondary structure elements present in the bound carboxylterminal domain include α-helix, β-structure, turns, and open loops. The addition of TFE also induces secondary structure in the C-terminal domain that is very similar to the structure of the DNA bound. Examination of the changes in the amide I components in the 20-80 °C temperature interval showed that the secondary structure of the DNA-bound C-H1t is for the most part extremely stable. The H1 carboxyl-terminal domain appears to belong to the so-called disordered proteins, undergoing coupled binding and folding. In the cellular environment macromolecules and small molecule solutes are present at high concentrations so that a significant fraction of the intracellular space is not available to other macromolecules.The C-terminal domains C-H1º and C-H1t are significantly structured in the presence of Ficoll 70 (30%) and PEG (30%) The proportions of secondary structure motifs were comparable to those of the DNA-bound domain. The small-angle X-ray scattering showed that in crowding agents the C-terminus had the compaction of a globular state. Progressive dissipation of the secondary structure and a lineal increase in partial heat capacity (Cp) with temperature together with increased binding of ANS indicated that the C-terminus is not cooperatively folded in crowded conditions. These results indicate that the C-terminus in crowding agents is in a molten globule state. Folding of the C-terminus in crowded conditions may increase the rate of the transition toward the DNA bound state and facilitate H1 diffusion inside cell nuclei. Histone H1 is phosphorylated in a cell cycle-dependent manner by cyclin-dependent kinases (CDKs). The highest number of phosphorylated sites is found in mitosis. The majority of the phosphorylation sites for CDKs are located on the C-terminal domain. Complete phosphorylation of C-H1º is associated to a major structural change. This structural rearrengement implies the loss of almost all the α-helix and a large increase in β-structure. The extent of the conformational change appears to be dependent on triphosphorylation and the protein/DNA ratio. The final state of the structural change consists in an all-β protein at ratios near saturation. Phosphorylation of one or two site have distintic structural effects. Phosphorylation of T118 affects the secondary structure the most, with a decrease of the α-helix and the appearence of random coil. Charge neutralization provided by DNA phosphate groups is an important factor in the folding of the C-terminal domain of histone H1 when bound to DNA. Alkaline pH induces secondary structure in C-H1º unphosphorylated and triphosphorylated that reflects the structural changes associated to phosphorylation

Història evolutiva de la família d'histones H1.1-H1.5 de mamífers

El Grup de Recerca d'Expressió Gènica i Regulació Cel·lular Eucariòtica del Departament de Bioquímica i Biologia Molecular de la Facultat de Biociències (UAB), ha elaborat un nou model d'evolució molecular per a una part de la familia multigènica de la histona H1 (H1.1-H1.5). Aquest model sustenta la diferenciació funcional dels subtipus. Està basat en la presència d'una forta selecció negativa global i en altres processos com la recombinació i la selecció positiva d'aminoacids especifícs.El Grupo de Investigación de Expresión Génica y Regulación Celular Eucariótica del Departamento de Bioquímica y Biología Molecular de la Facultad de Biociencias (UAB), ha elaborado un nuevo modelo de evolución molecular para una parte de la familia multigénica de la histona H1 (H1.1-H1.5). Este modelo sustenta la diferenciación funcional de los subtipos de H1. Se basa en la presencia de una fuerte selección negativa global y en otros procesos como la recombinación y la selección positiva de aminoácidos específicos.The Gene Expression and Eukaryotic Cell Regulation Research Group of the Biochemistry and Molecular Biology Department of Biosciences Faculty (UAB), have proposed a new model for the molecular evolution of a part of the H1 multigenic family (H1.1-H1.5). This model is in favor of the functional differentiation of the subtypes. It is based on the presence of a strong negative selection and includes other processes such as recombination and positive selection of specific amino acids

Història evolutiva de la família d'histones H1.1-H1.5 de mamífers

El Grup de Recerca d'Expressió Gènica i Regulació Cel·lular Eucariòtica del Departament de Bioquímica i Biologia Molecular de la Facultat de Biociències (UAB), ha elaborat un nou model d'evolució molecular per a una part de la familia multigènica de la histona H1 (H1.1-H1.5). Aquest model sustenta la diferenciació funcional dels subtipus. Està basat en la presència d'una forta selecció negativa global i en altres processos com la recombinació i la selecció positiva d'aminoacids especifícs.El Grupo de Investigación de Expresión Génica y Regulación Celular Eucariótica del Departamento de Bioquímica y Biología Molecular de la Facultad de Biociencias (UAB), ha elaborado un nuevo modelo de evolución molecular para una parte de la familia multigénica de la histona H1 (H1.1-H1.5). Este modelo sustenta la diferenciación funcional de los subtipos de H1. Se basa en la presencia de una fuerte selección negativa global y en otros procesos como la recombinación y la selección positiva de aminoácidos específicos.The Gene Expression and Eukaryotic Cell Regulation Research Group of the Biochemistry and Molecular Biology Department of Biosciences Faculty (UAB), have proposed a new model for the molecular evolution of a part of the H1 multigenic family (H1.1-H1.5). This model is in favor of the functional differentiation of the subtypes. It is based on the presence of a strong negative selection and includes other processes such as recombination and positive selection of specific amino acids

Phosphorylation of the carboxy-terminal domain of histone H1 : effects on secondary structure and DNA condensation

Linker histone H1 plays an important role in chromatin folding. Phosphorylation by cyclin-dependent kinases is the main post-translational modification of histone H1. We studied the effects of phosphorylation on the secondary structure of the DNA-bound H1 carboxy-terminal domain (CTD), which contains most of the phosphorylation sites of the molecule. The effects of phosphorylation on the secondary structure of the DNA-bound CTD were site-specific and depended on the number of phosphate groups. Full phosphorylation significantly increased the proportion of β-structure and decreased that of α-helix. Partial phosphorylation increased the amount of undefined structure and decreased that of α-helix without a significant increase in β-structure. Phosphorylation had a moderate effect on the affinity of the CTD for the DNA, which was proportional to the number of phosphate groups. Partial phosphorylation drastically reduced the aggregation of DNA fragments by the CTD, but full phosphorylation restored to a large extent the aggregation capacity of the unphosphorylated domain. These results support the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of H1 partial phosphorylation in interphase chromatin relaxation. More generally, our results suggest that the effects of phosphorylation are mediated by specific structural changes and are not simply a consequence of the net charge

Proteasome-dependent degradation of histone subtypes is mediated by its C-terminal domain

Altres ajuts: acords transformatius de la UABHistone H1 is involved in chromatin compaction and dynamics. In human cells, the H1 complement is formed by different amounts of somatic H1 subtypes, H1.0-H1.5 and H1X. The amount of each variant depends on the cell type, the cell cycle phase, and the time of development and can be altered in disease. However, the mechanisms regulating H1 protein levels have not been described. We have analyzed the contribution of the proteasome to the degradation of H1 subtypes in human cells using two different inhibitors: MG132 and bortezomib. H1 subtypes accumulate upon treatment with both drugs, indicating that the proteasome is involved in the regulation of H1 protein levels. Proteasome inhibition caused a global increase in cytoplasmatic H1, with slight changes in the composition of H1 bound to chromatin and chromatin accessibility and no alterations in the nucleosome repeat length. The analysis of the proteasome degradation pathway showed that H1 degradation is ubiquitin-independent. The whole protein and its C-terminal domain can be degraded directly by the 20S proteasome in vitro. Partial depletion of PA28γ revealed that this regulatory subunit contributes to H1 degradation within the cell. Our study shows that histone H1 protein levels are under tight regulation to prevent its accumulation in the nucleus. We revealed a new regulatory mechanism for histone H1 degradation, where the C-terminal disordered domain is responsible for its targeting and degradation by the 20S proteasome, a process enhanced by the regulatory subunit PA28γ