Federico Zambelli

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.

Motif discovery and transcription factor binding sites before and after the next-generation sequencing era

Motif discovery has been one of the most widely studied problems in bioinformatics ever since genomic and protein sequences have been available. In particular, its application to the de novo prediction of putative over-represented transcription factor binding sites in nucleotide sequences has been, and still is, one of the most challenging flavors of the problem. Recently, novel experimental techniques like chromatin immunoprecipitation (ChIP) have been introduced, permitting the genome-wide identification of protein–DNA interactions. ChIP, applied to transcription factors and coupled with genome tiling arrays (ChIP on Chip) or next-generation sequencing technologies (ChIP-Seq) has opened new avenues in research, as well as posed new challenges to bioinformaticians developing algorithms and methods for motif discovery.

A perspective of promoter architecture from the CCAAt box

The CCAAT box is an important promoter element regulated by NF-Y, a conserved trimer with histone-like features. We describe a new Position Specific Frequency Matrix (PSFM): we derived from 328 NF-Y promoters from the literature the p-CCAAT, and refined it by analysing ChIP on chip data (g-CCAAT). Interestingly, g-CCAAT- has distinct features, such as variations within the CCAAT pentanucleotide. We validated the NF-Y-dependency of several promoters with functional assays. We examined the presence of these PSFMs in all human promoters and detail a number of parameters of CCAAT boxes: position, orientation, distance from TSS, presence of TATA, CpG islands and enrichments of nearby TF elements. The CCAAT genes fall into different GO categories, with cell-cycle and chromatin/transcription specifically enriched. Additional findings surfaced: (i) the CCAAT-TATA combination, often mentioned in textbooks, is an exception, rather than the rule. CCAAT promoters are less precise in terms of TSS. (ii) There is a good correlation between CCAAT and CpG islands; (iii) selective TFs sites are enriched in CCAAT promoters, with precise stereoalignements of some of them. In conclusion, the new features of the CCAAT box and the link with the neighbouring elements will help in the functional classification of promoters.

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 .

Identification of pathways directly regulated by SHORT VEGETATIVE PHASE during vegetative and reproductive development in Arabidopsis

BackgroundMADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism.ResultsTo compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively.ConclusionsOur analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them.

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.

PscanChIP: finding over-represented transcription factor-binding site motifs and their correlations in sequences from ChIP-Seq experiments

Chromatin immunoprecipitation followed by sequencing with next-generation technologies (ChIP-Seq) has become the de facto standard for building genome-wide maps of regions bound by a given transcription factor (TF). The regions identified, however, have to be further analyzed to determine the actual DNA-binding sites for the TF, as well as sites for other TFs belonging to the same TF complex or in general co-operating or interacting with it in transcription regulation. PscanChIP is a web server that, starting from a collection of genomic regions derived from a ChIP-Seq experiment, scans them using motif descriptors like JASPAR or TRANSFAC position-specific frequency matrices, or descriptors uploaded by users, and it evaluates both motif enrichment and positional bias within the regions according to different measures and criteria. PscanChIP can successfully identify not only the actual binding sites for the TF investigated by a ChIP-Seq experiment but also secondary motifs corresponding to other TFs that tend to bind the same regions, and, if present, precise positional correlations among their respective sites. The web interface is free for use, and there is no login requirement. It is available at http://www.beaconlab.it/pscan_chip_dev.

ASPicDB: a database of annotated transcript and protein variants generated by alternative splicing

Alternative splicing is emerging as a major mechanism for the expansion of the transcriptome and proteome diversity, particularly in human and other vertebrates. However, the proportion of alternative transcripts and proteins actually endowed with functional activity is currently highly debated. We present here a new release of ASPicDB which now provides a unique annotation resource of human protein variants generated by alternative splicing. A total of 256 939 protein variants from 17 191 multi-exon genes have been extensively annotated through state of the art machine learning tools providing information of the protein type (globular and transmembrane), localization, presence of PFAM domains, signal peptides, GPI-anchor propeptides, transmembrane and coiled-coil segments. Furthermore, full-length variants can be now specifically selected based on the annotation of CAGE-tags and polyA signal and/or polyA sites, marking transcription initiation and termination sites, respectively. The retrieval can be carried out at gene, transcript, exon, protein or splice site level allowing the selection of data sets fulfilling one or more features settled by the user. The retrieval interface also enables the selection of protein variants showing specific differences in the annotated features. ASPicDB is available at http://www.caspur.it/ASPicDB/.

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

BackgroundThis 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.ResultsWe 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.ConclusionResults 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.

SpliceAid-F: a database of human splicing factors and their RNA-binding sites

A comprehensive knowledge of all the factors involved in splicing, both proteins and RNAs, and of their interaction network is crucial for reaching a better understanding of this process and its functions. A large part of relevant information is buried in the literature or collected in various different databases. By hand-curated screenings of literature and databases, we retrieved experimentally validated data on 71 human RNA-binding splicing regulatory proteins and organized them into a database called ‘SpliceAid-F’ (http://www.caspur.it/SpliceAidF/). For each splicing factor (SF), the database reports its functional domains, its protein and chemical interactors and its expression data. Furthermore, we collected experimentally validated RNA–SF interactions, including relevant information on the RNA-binding sites, such as the genes where these sites lie, their genomic coordinates, the splicing effects, the experimental procedures used, as well as the corresponding bibliographic references. We also collected information from experiments showing no RNA–SF binding, at least in the assayed conditions. In total, SpliceAid-F contains 4227 interactions, 2590 RNA-binding sites and 1141 ‘no-binding’ sites, including information on cellular contexts and conditions where binding was tested. The data collected in SpliceAid-F can provide significant information to explain an observed splicing pattern as well as the effect of mutations in functional regulatory elements.