Anna Maria D'Erchia

p53FamTaG: a database resource of human p53, p63 and p73 direct target genes combining in silico prediction and microarray data

BackgroundThe p53 gene family consists of the three genes p53, p63 and p73, which have polyhedral non-overlapping functions in pivotal cellular processes such as DNA synthesis and repair, growth arrest, apoptosis, genome stability, angiogenesis, development and differentiation. These genes encode sequence-specific nuclear transcription factors that recognise the same responsive element (RE) in their target genes. Their inactivation or aberrant expression may determine tumour progression or developmental disease. The discovery of several protein isoforms with antagonistic roles, which are produced by the expression of different promoters and alternative splicing, widened the complexity of the scenario of the transcriptional network of the p53 family members. Therefore, the identification of the genes transactivated by p53 family members is crucial to understand the specific role for each gene in cell cycle regulation. We have combined a genome-wide computational search of p53 family REs and microarray analysis to identify new direct target genes. The huge amount of biological data produced has generated a critical need for bioinformatic tools able to manage and integrate such data and facilitate their retrieval and analysis.DescriptionWe have developed the p53FamTaG database (p53 FAMily TArget Genes), a modular relational database, which contains p53 family direct target genes selected in the human genome searching for the presence of the REs and the expression profile of these target genes obtained by microarray experiments. p53FamTaG database also contains annotations of publicly available databases and links to other experimental data.The genome-wide computational search of the REs was performed using PatSearch, a pattern-matching program implemented in the DNAfan tool. These data were integrated with the microarray results we produced from the overexpression of different isoforms of p53, p63 and p73 stably transfected in isogenic cell lines, allowing the comparative study of the transcriptional activity of all the proteins in the same cellular background.p53FamTaG database is available free at http://www2.ba.itb.cnr.it/p53FamTaG/Conclusionp53FamTaG represents a unique integrated resource of human direct p53 family target genes that is extensively annotated and provides the users with an efficient query/retrieval system which displays the results of our microarray experiments and allows the export of RE sequences. The database was developed for supporting and integrating high-throughput in silico and experimental analyses and represents an important reference source of knowledge for research groups involved in the field of oncogenesis, apoptosis and cell cycle regulation.

ASPicDB: A database resource for alternative splicing analysis

MOTIVATION Alternative splicing has recently emerged as a key mechanism responsible for the expansion of transcriptome and proteome complexity in human and other organisms. Although several online resources devoted to alternative splicing analysis are available they may suffer from limitations related both to the computational methodologies adopted and to the extent of the annotations they provide that prevent the full exploitation of the available data. Furthermore, current resources provide limited query and download facilities. RESULTS ASPicDB is a database designed to provide access to reliable annotations of the alternative splicing pattern of human genes and to the functional annotation of predicted splicing isoforms. Splice-site detection and full-length transcript modeling have been carried out by a genome-wide application of the ASPic algorithm, based on the multiple alignments of gene-related transcripts (typically a Unigene cluster) to the genomic sequence, a strategy that greatly improves prediction accuracy compared to methods based on independent and progressive alignments. Enhanced query and download facilities for annotations and sequences allow users to select and extract specific sets of data related to genes, transcripts and introns fulfilling a combination of user-defined criteria. Several tabular and graphical views of the results are presented, providing a comprehensive assessment of the functional implication of alternative splicing in the gene set under investigation. ASPicDB, which is regularly updated on a monthly basis, also includes information on tissue-specific splicing patterns of normal and cancer cells, based on available EST sequences and their library source annotation. AVAILABILITY www.caspur.it/ASPicDB

The Fatty Acid Synthase Gene is a Conserved p53 Family Target from Worm to Human

The discovery that the p53 family consists of three members (p53, p63 and p73) in vertebrates and of a single homolog in invertebrates has raised the challenge of understanding the functions of the ancestor and how they have evolved and differentiated within the duplicated genes in vertebrates. Here, we report that the fatty acid synthase (FAS) gene, encoding for a key enzyme involved in the biogenesis of membrane lipids in rapidly proliferating cells, is a conserved target of the p53 family throughout the evolution. We show that CEP-1, the C. elegans p53 homolog, is able to bind the two p53 family responsive elements (REs) identified in the worm fasn-1 gene. Moreover, we demonstrate that fasn-1 expression is modulated by CEP-1 in vivo, by comparing wild-type and CEP-1 knockout worms. In human, luciferase and chromatin immunoprecipitation assays demonstrate that TAp73α and ΔNp63α, but not p53, TAp73β and TAp63α bind the two p53 REs of the human FASN gene. We show that the ectopic expression of TAp73β and ΔNp63α leads to an increase of FASN mRNA levels, while their silencing produces a decrease of FASN expression. Furthermore, we present data showing a correlation between ΔNp63α and FASN expression in cellular proliferation. Of relevant importance is that fasn-1 is the first CEP-1 direct target gene identified so far in C. elegans and our results suggest a new CEP-1 role in cellular proliferation and development, besides the one already described in apoptosis of germ cells. These data confirm the hypothesis that the ancestral functions of the single invertebrate gene may have been spread out among the three vertebrate members, each of them have acquired specific role in cell cycle regulation.

REDIportal: a comprehensive database of A-to-I RNA editing events in humans

RNA editing by A-to-I deamination is the prominent co-/post-transcriptional modification in humans. It is carried out by ADAR enzymes and contributes to both transcriptomic and proteomic expansion. RNA editing has pivotal cellular effects and its deregulation has been linked to a variety of human disorders including neurological and neurodegenerative diseases and cancer. Despite its biological relevance, many physiological and functional aspects of RNA editing are yet elusive. Here, we present REDIportal, available online at http://srv00.recas.ba.infn.it/atlas/, the largest and comprehensive collection of RNA editing in humans including more than 4.5 millions of A-to-I events detected in 55 body sites from thousands of RNAseq experiments. REDIportal embeds RADAR database and represents the first editing resource designed to answer functional questions, enabling the inspection and browsing of editing levels in a variety of human samples, tissues and body sites. In contrast with previous RNA editing databases, REDIportal comprises its own browser (JBrowse) that allows users to explore A-to-I changes in their genomic context, empathizing repetitive elements in which RNA editing is prominent.

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application

BackgroundThe study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.).Moreover, the huge volume of data generated by NGS platforms introduces unprecedented computational and technological challenges to efficiently analyze and store sequence data and results.MethodsIn order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq).ResultsThrough a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs.

Tissue-specific mtDNA abundance from exome data and its correlation with mitochondrial transcription, mass and respiratory activity.

Eukaryotic cells contain a population of mitochondria, variable in number and shape, which in turn contain multiple copies of a tiny compact genome (mtDNA) whose expression and function is strictly coordinated with the nuclear one. mtDNA copy number varies between different cell or tissues types, both in response to overall metabolic and bioenergetics demands and as a consequence or cause of specific pathological conditions. Here we present a novel and reliable methodology to assess the effective mtDNA copy number per diploid genome by investigating off-target reads obtained by whole-exome sequencing (WES) experiments. We also investigate whether and how mtDNA copy number correlates with mitochondrial mass, respiratory activity and expression levels. Analyzing six different tissues from three age- and sex-matched human individuals, we found a highly significant linear correlation between mtDNA copy number estimated by qPCR and the frequency of mtDNA off target WES reads. Furthermore, mtDNA copy number showed highly significant correlation with mitochondrial gene expression levels as measured by RNA-Seq as well as with mitochondrial mass and respiratory activity. Our methodology makes thus feasible, at a large scale, the investigation of mtDNA copy number in diverse cell-types, tissues and pathological conditions or in response to specific treatments.

p73 and p63 Sustain Cellular Growth by Transcriptional Activation of Cell Cycle Progression Genes

Despite extensive studies on the role of tumor suppressor p53 protein and its homologues, p73 and p63, following their overexpression or cellular stress, very little is known about the regulation of the three proteins in cells during physiologic cell cycle progression. We report a role for p73 and p63 in supporting cellular proliferation through the transcriptional activation of the genes involved in G(1)-S and G(2)-M progression. We found that in MCF-7 cells, p73 and p63, but not p53, are modulated during the cell cycle with a peak in S phase, and their silencing determines a significant suppression of proliferation compared with the control. Chromatin immunoprecipitation analysis shows that in cycling cells, p73 and p63 are bound to the p53-responsive elements (RE) present in the regulatory region of cell cycle progression genes. On the contrary, when the cells are arrested in G(0)-G(1), p73 detaches from the REs and it is replaced by p53, which represses the expression of these genes. When the cells move in S phase, p73 is recruited again and p53 is displaced or is weakly bound to the REs. These data open new possibilities for understanding the involvement of p73 and p63 in cancer. The elevated concentrations of p73 and p63 found in many cancers could cause the aberrant activation of cell growth progression genes and therefore contribute to cancer initiation or progression under certain conditions.

Molecular clock and gene function.

Molecular phylogenies based on the molecular clock require the comparison of orthologous genes. Orthologous and paralogous genes usually have very different evolutionary fates. In general, orthologs keep the same functions in species, whereas, particularly over a long time span, paralogs diverge functionally and may become pseudogenes or get lost. In eukaryotic genomes, because of the degree of redundancy of genetic information, homologous genes are grouped in gene families, the evolution of which may differ greatly between the various organisms. This implies that each gene in a species does not always have an ortholog in another species and thus, due to multiple duplication events following a speciation, many orthologous clades of paralogs are generated. We are often dealing with a one-to-many or many-to-many relationship between genes. In this paper, we analyze the evolution of two gene families, the p53 gene family and the porin gene family. The evolution of the p53 family shows a one-to-many gene relationship going from invertebrates to vertebrates. In invertebrates only a single gene has been found, while in vertebrates three members of the family, namely p53, p63, and p73, are present. The evolution of porin (VDAC) genes (VDAC1, VDAC2, and VDAC3) is an example of a many-to-many gene relationship going from yeast to mammals. However, the porin gene redundancy found in invertebrates and possibly in some fishes may indicate a tendency to duplicate the genetic material, rather than a real need for function innovation.

Connecting p63 to cellular proliferation: the example of the adenosine deaminase target gene.

An unresolved issue regards the role of p73 and p63, the two homologs of the p53 oncosuppressor gene, in normal cells and in tumour development. Specific target genes for each protein need to be identified and characterized in order to understand the specific role of each protein in tumour initiation and progression as well as in oncosuppression and development. We tested whether p63 is implicated in transcriptional events related to sustaining cell proliferation by transactivation of antiapoptotic and cell survival target genes such as Adenosine Deaminase (ADA), an important gene involved in cell proliferation. We demonstrate that ADA is a direct target gene of p63 isoforms. In human keratinocytes, the rate of proliferation and the high level of ADA transcript diminished upon elimination of p63 by small interfering RNA. Reporter assays and chromatin immunoprecipitation experiments indicate a physical interaction of p63 with the two putative p53 binding sites we identified in the ADA gene. Moreover, in response to p53 stabilization and ?Np63 downregulation in normal keratinocytes after U.V. treatment, we found a change in the transcriptional pattern of the p53 family target genes, consistent with the different roles played by p53 and p63 in tumour suppression and cellular proliferation. In fact p53 upregulation determined an increase in p21, which in turn mediated the cell cycle arrest, while the downregulation of ?Np63 determined a marked decrease in ADA transcript. The experiments reported here support the hypothesis that TAp63 and ?Np63 might contribute to tumour genesis not exclusively by antagonizing p53, but by conferring a proliferative potential on cancer cells through the transactivation of target genes indispensable for cell division, such as the Adenosine Deaminase gene.

TRIM8 modulates p53 activity to dictate cell cycle arrest

p53 is a central hub in controlling cell proliferation. To maintain genome integrity in response to cellular stress, p53 directly regulates the transcription of genes involved in cell cycle arrest, DNA repair, apoptosis and/or senescence. An array of post-translational modifications and protein-protein interactions modulates its stability and activities in order to avoid malignant transformation. However, to date it is still not clear how cells decide their own fate in response to different types of stress. We described here that the human TRIM8 protein, a member of the TRIM family, is a new modulator of the p53-mediated tumor suppression mechanism. We showed that under stress conditions, such as UV exposure, p53 induced the expression of TRIM8, which in turn stabilized p53 leading to cell cycle arrest and reduction of cell proliferation through enhancement of CDKN1A (p21) and GADD45 expression. TRIM8 silencing reduced the capacity of p53 to activate genes involved in cell cycle arrest and DNA repair, in response to cellular stress. Concurrently, TRIM8 overexpression induced the degradation of the MDM2 protein, the principal regulator of p53 stability. Co-immunoprecipitation experiments showed that TRIM8 physically interacted with p53, impairing its interaction with MDM2. Altogether, our results reveal a previously unknown regulatory pathway controlling p53 activity and suggest TRIM8 as a novel therapeutic target to enhance p53 tumor suppressor activity.