Nucleosomal signatures impose nucleosome positioning in coding and noncoding sequences in the genome.

In the yeast genome, a large proportion of nucleosomes occupy well-defined and stable positions. While the contribution of chromatin remodelers and DNA binding proteins to maintain this organization is well established, the relevance of the DNA sequence to nucleosome positioning in the genome remains controversial. Through quantitative analysis of nucleosome positioning, we show that sequence changes distort the nucleosomal pattern at the level of individual nucleosomes in three species of Schizosaccharomyces and in Saccharomyces cerevisiae This effect is equally detected in transcribed and nontranscribed regions, suggesting the existence of sequence elements that contribute to positioning. To identify such elements, we incorporated information from nucleosomal signatures into artificial synthetic DNA molecules and found that they generated regular nucleosomal arrays indistinguishable from those of endogenous sequences. Strikingly, this information is species-specific and can be combined with coding information through the use of synonymous codons such that genes from one species can be engineered to adopt the nucleosomal organization of another. These findings open the possibility of designing coding and noncoding DNA molecules capable of directing their own nucleosomal organization.

Comparative analysis of methods for genome-wide nucleosome cartography.

Nucleosomes contribute to compacting the genome into the nucleus and regulate the physical access of regulatory proteins to DNA either directly or through the epigenetic modifications of the histone tails. Precise mapping of nucleosome positioning across the genome is, therefore, essential to understanding the genome regulation. In recent years, several experimental protocols have been developed for this purpose that include the enzymatic digestion, chemical cleavage or immunoprecipitation of chromatin followed by next-generation sequencing of the resulting DNA fragments. Here, we compare the performance and resolution of these methods from the initial biochemical steps through the alignment of the millions of short-sequence reads to a reference genome to the final computational analysis to generate genome-wide maps of nucleosome occupancy. Because of the lack of a unified protocol to process data sets obtained through the different approaches, we have developed a new computational tool (NUCwave), which facilitates their analysis, comparison and assessment and will enable researchers to choose the most suitable method for any particular purpose. NUCwave is freely available at http://nucleosome.usal.es/nucwave along with a step-by-step protocol for its use.

Nucleosomal organization of replication origins and meiotic recombination hotspots in fission yeast.

In Schizosaccharomyces pombe, DNA replication origins (ORIs) and meiotic recombination hotspots lack consensus sequences and show a bias towards mapping to large intergenic regions (IGRs). To explore whether this preference depended on underlying chromatin features, we have generated genome-wide nucleosome profiles during mitosis and meiosis. We have found that meiotic double-strand break sites (DSBs) colocalize with nucleosome-depleted regions (NDRs) and that large IGRs include clusters of NDRs that overlap with almost half of all DSBs. By contrast, ORIs do not colocalize with NDRs and they are regulated independently of DSBs. Physical relocation of NDRs at ectopic loci or modification of their genomic distribution during meiosis was paralleled by the generation of new DSB sites. Over 80% of all meiotic DSBs colocalize with NDRs that are also present during mitosis, indicating that the recombination pattern is largely dependent on constitutive properties of the genome and, to a lesser extent, on the transcriptional profile during meiosis. The organization of ORIs and of DSBs regions in S. pombe reveals similarities and differences relative to Saccharomyces cerevisiae.

PTPN13 regulates cellular signalling and ß-catenin function during megakaryocytic differentiation.

PTPN13 is a high-molecular weight intracellular phosphatase with several isoforms that exhibits a highly modular structure. Although in recent years different roles have been described for PTPN13, we are still far from understanding its function in cell biology. Here we show that PTPN13 expression is activated during megakaryocytic differentiation at the protein and mRNA level. Our results show that the upregulation of PTPN13 inhibits megakaryocytic differentiation, while PTPN13 silencing triggers differentiation. The ability of PTPN13 to alter megakaryocytic differentiation can be explained by its capacity to regulate ERK and STAT signalling. Interestingly, the silencing of ß-catenin produced the same effect as PTPN13 downregulation. We demonstrate that both proteins coimmunoprecipitate and colocalise. Moreover, we provide evidence showing that PTPN13 can regulate ß-catenin phosphorylation, stability and transcriptional activity. Therefore, the ability of PTPN13 to control megakaryocytic differentiation must be intimately linked to the regulation of ß-catenin function. Moreover, our results show for the first time that PTPN13 is stabilised upon Wnt signalling, which makes PTPN13 an important player in canonical Wnt signalling. Our results show that PTPN13 behaves as an important regulator of megakaryocytic differentiation in cell lines and also in murine haematopoietic progenitors. This importance can be explained by the ability of PTPN13 to regulate cellular signalling, and especially through the regulation of ß-catenin stability and function. Our results hold true for different megakaryocytic cell lines and also for haematopoietic progenitors, suggesting that these two proteins may play a relevant role during in vivo megakaryopoiesis.

BicOverlapper 2.0: visual analysis for gene expression.

MOTIVATION: Systems biology demands the use of several point of views to get a more comprehensive understanding of biological problems. This usually leads to take into account different data regarding the problem at hand, but it also has to do with using different perspectives of the same data. This multifaceted aspect of systems biology often requires the use of several tools, and it is often hard to get a seamless integration of all of them, which would help the analyst to have an interactive discourse with the data. RESULTS: Focusing on expression profiling, BicOverlapper 2.0 visualizes the most relevant aspects of the analysis, including expression data, profiling analysis results and functional annotation. It also integrates several state-of-the-art numerical methods, such as differential expression analysis, gene set enrichment or biclustering. AVAILABILITY AND IMPLEMENTATION: BicOverlapper 2.0 is available at: http://vis.usal.es/bicoverlapper2

Clustered regulatory elements at nucleosome-depleted regions punctuate a constant nucleosomal landscape in Schizosaccharomyces pombe.

BACKGROUND: Nucleosomes facilitate the packaging of the eukaryotic genome and modulate the access of regulators to DNA. A detailed description of the nucleosomal organization under different transcriptional programmes is essential to understand their contribution to genomic regulation. RESULTS: To visualize the dynamics of individual nucleosomes under different transcriptional programmes we have generated high-resolution nucleosomal maps in Schizosaccharomyces pombe. We show that 98.5% of the genome remains almost invariable during mitosis and meiosis while remodelling is limited to approximately 1100 nucleosomes in the promoters of a subset of meiotic genes. These inducible nucleosome-depleted regions (NDR) and also those constitutively present in the genome overlap precisely with clusters of binding sites for transcription factors (TF) specific for meiosis and for different functional classes of genes, respectively. Deletion of two TFs affects only a small fraction of all the NDRs to which they bind in vivo, indicating that TFs collectively contribute to NDR maintenance. CONCLUSIONS: Our results show that the nucleosomal profile in S. pombe is largely maintained under different physiological conditions and patterns of gene expression. This relatively constant landscape favours the concentration of regulators in constitutive and inducible NDRs. The combinatorial analysis of binding motifs in this discrete fraction of the genome will facilitate the definition of the transcriptional regulatory networks.

Gcn5 facilitates Pol II progression, rather than recruitment to nucleosome-depleted stress promoters, in Schizosaccharomyces pombe.

In the fission yeast, the MAP kinase Sty1 and the transcription factor Atf1 regulate up to 400 genes in response to environmental signals, and both proteins have been shown to bind to their promoters in a stress-dependent manner. In a genetic search, we have isolated the histone H3 acetyltransferase Gcn5, a component of the SAGA complex, as being essential for oxidative stress survival and activation of those genes. Upon stress, Gcn5 is recruited to promoters and coding sequences of stress genes in a Sty1- and Atf1-dependent manner, causing both an enhanced acetylation of histone H3 and nucleosome eviction. Unexpectedly, recruitment of RNA polymerase II (Pol II) is not impaired in Δgcn5 cells. We show here that stress genes display a 400-bp long nucleosome depleted region upstream of the transcription start site even prior to activation. Stress treatment does not alter promoter nucleosome architecture, but induces eviction of the downstream nucleosomes at stress genes, which is not observed in Δgcn5 cells. We conclude that, while Pol II is recruited to nucleosome-free stress promoters in a transcription factor dependent manner, Gcn5 mediates eviction of nucleosomes positioned downstream of promoters, allowing efficient Pol II progression along the genes.

Analysis of DNA strand-specific differential expression with high density tiling microarrays.

BACKGROUND: DNA microarray technology allows the analysis of genome structure and dynamics at genome-wide scale. Expression microarrays (EMA) contain probes for annotated open reading frames (ORF) and are widely used for the analysis of differential gene expression. By contrast, tiling microarrays (TMA) have a much higher probe density and provide unbiased genome-wide coverage. The purpose of this study was to develop a protocol to exploit the high resolution of TMAs for quantitative measurement of DNA strand-specific differential expression of annotated and non-annotated transcripts. RESULTS: We extensively filtered probes present in Affymetrix Genechip Yeast Genome 2.0 expression and GeneChip S. pombe 1.0FR tiling microarrays to generate custom Chip Description Files (CDF) in order to compare their efficiency. We experimentally tested the potential of our approach by measuring the differential expression of 4904 genes in the yeast Schizosaccharomyces pombe growing under conditions of oxidative stress. The results showed a Pearson correlation coefficient of 0.943 between both platforms, indicating that TMAs are as reliable as EMAs for quantitative expression analysis. A significant advantage of TMAs over EMAs is the possibility of detecting non-annotated transcripts generated only under specific physiological conditions. To take full advantage of this property, we have used a target-labelling protocol that preserves the original polarity of the transcripts and, therefore, allows the strand-specific differential expression of non-annotated transcripts to be determined. By using a segmentation algorithm prior to generating the corresponding custom CDFs, we identified and quantitatively measured the expression of 510 transcripts longer than 180 nucleotides and not overlapping previously annotated ORFs that were differentially expressed at least 2-fold under oxidative stress. CONCLUSIONS: We show that the information derived from TMA hybridization can be processed simultaneously for high-resolution qualitative and quantitative analysis of the differential expression of well-characterized genes and of previously non-annotated and antisense transcripts. The consistency of the performance of TMA, their genome-wide coverage and adaptability to updated genome annotations, and the possibility of measuring strand-specific differential expression makes them a tool of choice for the analysis of gene expression in any organism for which TMA platforms are available.

BicOverlapper: a tool for bicluster visualization.

UNLABELLED: BicOverlapper is a tool to visualize biclusters from gene-expression matrices in a way that helps to compare biclustering methods, to unravel trends and to highlight relevant genes and conditions. A visual approach can complement biological and statistical analysis and reduce the time spent by specialists interpreting the results of biclustering algorithms. The technique is based on a force-directed graph where biclusters are represented as flexible overlapped groups of genes and conditions. AVAILABILITY: The BicOverlapper software and supplementary material are available at http://vis.usal.es/bicoverlapper

A visual analytics approach for understanding biclustering results from microarray data.

BACKGROUND: Microarray analysis is an important area of bioinformatics. In the last few years, biclustering has become one of the most popular methods for classifying data from microarrays. Although biclustering can be used in any kind of classification problem, nowadays it is mostly used for microarray data classification. A large number of biclustering algorithms have been developed over the years, however little effort has been devoted to the representation of the results. RESULTS: We present an interactive framework that helps to infer differences or similarities between biclustering results, to unravel trends and to highlight robust groupings of genes and conditions. These linked representations of biclusters can complement biological analysis and reduce the time spent by specialists on interpreting the results. Within the framework, besides other standard representations, a visualization technique is presented which is based on a force-directed graph where biclusters are represented as flexible overlapped groups of genes and conditions. This microarray analysis framework (BicOverlapper), is available at http://vis.usal.es/bicoverlapper CONCLUSION: The main visualization technique, tested with different biclustering results on a real dataset, allows researchers to extract interesting features of the biclustering results, especially the highlighting of overlapping zones that usually represent robust groups of genes and/or conditions. The visual analytics methodology will permit biology experts to study biclustering results without inspecting an overwhelming number of biclusters individually.

A species-specific nucleosomal signature defines a periodic distribution of amino acids in proteins.

Nucleosomes are the basic structural units of chromatin. Most of the yeast genome is organized in a pattern of positioned nucleosomes that is stably maintained under a wide range of physiological conditions. In this work, we have searched for sequence determinants associated with positioned nucleosomes in four species of fission and budding yeasts. We show that mononucleosomal DNA follows a highly structured base composition pattern, which differs among species despite the high degree of histone conservation. These nucleosomal signatures are present in transcribed and non-transcribed regions across the genome. In the case of open reading frames, they correctly predict the relative distribution of codons on mononucleosomal DNA, and they also determine a periodicity in the average distribution of amino acids along the proteins. These results establish a direct and species-specific connection between the position of each codon around the histone octamer and protein composition.

Promoter nucleosome dynamics regulated by signalling through the CTD code.

The phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a key role in delineating transcribed regions within chromatin by recruiting histone methylases and deacetylases. Using genome-wide nucleosome mapping, we show that CTD S2 phosphorylation controls nucleosome dynamics in the promoter of a subset of 324 genes, including the regulators of cell differentiation ste11 and metabolic adaptation inv1. Mechanistic studies on these genes indicate that during gene activation a local increase of phospho-S2 CTD nearby the promoter impairs the phospho-S5 CTD-dependent recruitment of Set1 and the subsequent recruitment of specific HDACs, which leads to nucleosome depletion and efficient transcription. The early increase of phospho-S2 results from the phosphorylation of the CTD S2 kinase Lsk1 by MAP kinase in response to cellular signalling. The artificial tethering of the Lsk1 kinase at the ste11 promoter is sufficient to activate transcription. Therefore, signalling through the CTD code regulates promoter nucleosomes dynamics.

New insights into the RNA-based mechanism of action of the anticancer drug 5'-fluorouracil in eukaryotic cells.

5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR analysis allowed us to detect splicing defects of a significant number of intron-containing mRNA, in addition to identify some rRNA and tRNA processing defects after 5FU treatment. Interestingly, our studies also revealed that 5FU specifically induced the expression of certain genes implicated in the processing of mRNA, tRNA and rRNA precursors, and in the post-transcriptional modification of uracil residues in RNA. The transcription of several tRNA genes was also significantly induced after drug exposure. These transcriptional changes might represent a cellular response mechanism to counteract 5FU damage since deletion strains for some of these up-regulated genes were hypersensitive to 5FU. Moreover, most of these RNA processing genes have human orthologs that participate in conserved pathways, suggesting that they could be novel targets to improve the efficacy of 5FU-based treatments.