Exalign: a new method for comparative analysis of exon–intron gene structures

The evolution of genes is usually studied and reconstructed at the sequence level, that is, by comparing and aligning their genomic, transcript or protein sequences. However, including the exon–intron structure of genes in the analysis can provide further and useful information, for example to draw reliable phylogenetic relationships left unsolved by traditional sequence-based evolutionary studies, or to shed further light on patterns of intron gain and loss. In spite of this, no tool especially devised for this task is currently available. In this work we present Exalign, an algorithm designed to retrieve, compare and search for the exon-intron structure of existing gene annotations, that has been implemented in a software tool freely accessible through a web interface as well as available for download. We present different applications of our method, from the reconstruction of the evolutionary history of homologous gene families to the detection of as of today unknown cases of intron loss in human and rodents, and, remarkably, two never reported intron gain events in human and mouse. The web interface for accessing Exalign is available at http://www.pesolelab.it/exalign/ or http://www.beacon.unimi.it/exalign

MoD Tools: regulatory motif discovery in nucleotide sequences from co-regulated or homologous genes

Understanding the complex mechanisms regulating gene expression at the transcriptional and post-transcriptional levels is one of the greatest challenges of the post-genomic era. The MoD (MOtif Discovery) Tools web server comprises a set of tools for the discovery of novel conserved sequence and structure motifs in nucleotide sequences, motifs that in turn are good candidates for regulatory activity. The server includes the following programs: Weeder, for the discovery of conserved transcription factor binding sites (TFBSs) in nucleotide sequences from co-regulated genes; WeederH, for the discovery of conserved TFBSs and distal regulatory modules in sequences from homologous genes; RNAProfile, for the discovery of conserved secondary structure motifs in unaligned RNA sequences whose secondary structure is not known. In this way, a given gene can be compared with other co-regulated genes or with its homologs, or its mRNA can be analyzed for conserved motifs regulating its post-transcriptional fate. The web server thus provides researchers with different strategies and methods to investigate the regulation of gene expression, at both the transcriptional and post-transcriptional levels. Available at and

Gain-of-function p53 mutants have widespread genomic locations partially overlapping with p63

p53 and p63 are transcription factors -TFs- playing master roles in the DNA-damage response and in the development and maintenance of pluristratified epithelia, respectively. p53 mutations are common in epithelial tumors and HaCaT keratinocytes harbor two p53 alleles -H179Y and R282Q- with gain-of-function (GOF) activity. Indeed, functional inactivation of mutp53 affects the growth rate of HaCaT. We investigated the strategy of mutp53, by performing ChIP-Seq experiments of mutp53 and p63 and analyzed the transcriptome after mutp53 inactivation. Mutp53 bind to 7135 locations in vivo, with a robust overlap with p63. De novo motifs discovery recovered a p53/p63RE with high information content in sites bound by p63 and mutp53/p63, but not by mutp53 alone: these sites are rather enriched in elements of other TFs. The HaCaT p63 locations are only partially overlapping with those of normal keratinocytes; importantly, and enriched in mutp53 sites which delineate a functionally different group of target genes. Our data favour a model whereby mutp53 GOF mutants act both by tethering growth-controlling TFs and highjacking p63 to new locations

Transcriptional Network of p63 in Human Keratinocytes

p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin

Pscan: finding over-represented transcription factor binding site motifs in sequences from co-regulated or co-expressed genes

The first step in gene expression, transcription, is modulated by the interaction of transcription factors with their corresponding binding sites on the DNA sequence. Pscan is a software tool that scans a set of sequences (e.g. promoters) from co-regulated or co-expressed genes with motifs describing the binding specificity of known transcription factors and assesses which motifs are significantly over- or under-represented, providing thus hints on which transcription factors could be common regulators of the genes studied, together with the location of their candidate binding sites in the sequences. Pscan does not resort to comparisons with orthologous sequences and experimental results show that it compares favorably to other tools for the same task in terms of false positive predictions and computation time. The website is free and open to all users and there is no login requirement. Address: http://www.beaconlab.it/pscan

YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation

The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its related protein TAZ, mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and TAZ regulate a large number of target genes, acting as co-activators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis we identified two molecules which could have a role in the altered genome-wide methylation profile: the long non-coding RNA Ephemeron, whose rapid upregulation is crucial for ESCs transition into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency

Hmga2 protein loss alters nuclear envelope and 3D chromatin structure

The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function

Assessment of orthologous splicing isoforms in human and mouse orthologous genes

<p>Abstract</p> <p>Background</p> <p>Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level.</p> <p>Results</p> <p>As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns.</p> <p>Conclusions</p> <p>We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still maintaining the conventional definition of gene orthology, a new concept of "splicing orthology" can be defined at transcript level.</p

WeederH: an algorithm for finding conserved regulatory motifs and regions in homologous sequences

BACKGROUND: This work addresses the problem of detecting conserved transcription factor binding sites and in general regulatory regions through the analysis of sequences from homologous genes, an approach that is becoming more and more widely used given the ever increasing amount of genomic data available. RESULTS: We present an algorithm that identifies conserved transcription factor binding sites in a given sequence by comparing it to one or more homologs, adapting a framework we previously introduced for the discovery of sites in sequences from co-regulated genes. Differently from the most commonly used methods, the approach we present does not need or compute an alignment of the sequences investigated, nor resorts to descriptors of the binding specificity of known transcription factors. The main novel idea we introduce is a relative measure of conservation, assuming that true functional elements should present a higher level of conservation with respect to the rest of the sequence surrounding them. We present tests where we applied the algorithm to the identification of conserved annotated sites in homologous promoters, as well as in distal regions like enhancers. CONCLUSION: Results of the tests show how the algorithm can provide fast and reliable predictions of conserved transcription factor binding sites regulating the transcription of a gene, with better performances than other available methods for the same task. We also show examples on how the algorithm can be successfully employed when promoter annotations of the genes investigated are missing, or when regulatory sites and regions are located far away from the genes

Tools and data services registry: a community effort to document bioinformatics resources

Life sciences are yielding huge data sets that underpin scientific discoveries fundamental to improvement in human health, agriculture and the environment. In support of these discoveries, a plethora of databases and tools are deployed, in technically complex and diverse implementations, across a spectrum of scientific disciplines. The corpus of documentation of these resources is fragmented across the Web, with much redundancy, and has lacked a common standard of information. The outcome is that scientists must often struggle to find, understand, compare and use the best resources for the task at hand. Here we present a community-driven curation effort, supported by ELIXIR—the European infrastructure for biological information—that aspires to a comprehensive and consistent registry of information about bioinformatics resources. The sustainable upkeep of this Tools and Data Services Registry is assured by a curation effort driven by and tailored to local needs, and shared amongst a network of engaged partners. As of November 2015, the registry includes 1785 resources, with depositions from 126 individual registrations including 52 institutional providers and 74 individuals. With community support, the registry can become a standard for dissemination of information about bioinformatics resources: we welcome everyone to join us in this common endeavour. The registry is freely available at https://bio.tools