Alessandra Traini

Estrogen Receptor α Controls a Gene Network in Luminal-Like Breast Cancer Cells Comprising Multiple Transcription Factors and MicroRNAs

Luminal-like breast tumor cells express estrogen receptor alpha (ERalpha), a member of the nuclear receptor family of ligand-activated transcription factors that controls their proliferation, survival, and functional status. To identify the molecular determinants of this hormone-responsive tumor phenotype, a comprehensive genome-wide analysis was performed in estrogen stimulated MCF-7 and ZR-75.1 cells by integrating time-course mRNA expression profiling with global mapping of genomic ERalpha binding sites by chromatin immunoprecipitation coupled to massively parallel sequencing, microRNA expression profiling, and in silico analysis of transcription units and receptor binding regions identified. All 1270 genes that were found to respond to 17beta-estradiol in both cell lines cluster in 33 highly concordant groups, each of which showed defined kinetics of RNA changes. This hormone-responsive gene set includes several direct targets of ERalpha and is organized in a gene regulation cascade, stemming from ligand-activated receptor and reaching a large number of downstream targets via AP-2gamma, B-cell activating transcription factor, E2F1 and 2, E74-like factor 3, GTF2IRD1, hairy and enhancer of split homologue-1, MYB, SMAD3, RARalpha, and RXRalpha transcription factors. MicroRNAs are also integral components of this gene regulation network because miR-107, miR-424, miR-570, miR-618, and miR-760 are regulated by 17beta-estradiol along with other microRNAs that can target a significant number of transcripts belonging to one or more estrogen-responsive gene clusters.

Use of MSAP Markers to Analyse the Effects of Salt Stress on DNA Methylation in Rapeseed (Brassica napus var. oleifera)

Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP) approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone) and salinity-sensitive (Toccata) rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4) and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site-specific methylation differences in the rapeseed genome, as detected by MSAP analysis.

Patchwork sequencing of tomato San Marzano and Vesuviano varieties highlights genome-wide variations

BackgroundInvestigation of tomato genetic resources is a crucial issue for better straight evolution and genetic studies as well as tomato breeding strategies. Traditional Vesuviano and San Marzano varieties grown in Campania region (Southern Italy) are famous for their remarkable fruit quality. Owing to their economic and social importance is crucial to understand the genetic basis of their unique traits.ResultsHere, we present the draft genome sequences of tomato Vesuviano and San Marzano genome. A 40x genome coverage was obtained from a hybrid Illumina paired-end reads assembling that combines de novo assembly with iterative mapping to the reference S. lycopersicum genome (SL2.40). Insertions, deletions and SNP variants were carefully measured. When assessed on the basis of the reference annotation, 30% of protein-coding genes are predicted to have variants in both varieties. Copy genes number and gene location were assessed by mRNA transcripts mapping, showing a closer relationship of San Marzano with reference genome. Distinctive variations in key genes and transcription/regulation factors related to fruit quality have been revealed for both cultivars.ConclusionsThe effort performed highlighted varieties relationships and important variants in fruit key processes useful to dissect the path from sequence variant to phenotype.

Genome Microscale Heterogeneity among Wild Potatoes Revealed by Diversity Arrays Technology Marker Sequences.

Tuber-bearing potato species possess several genes that can be exploited to improve the genetic background of the cultivated potato Solanum tuberosum. Among them, S. bulbocastanum and S. commersonii are well known for their strong resistance to environmental stresses. However, scant information is available for these species in terms of genome organization, gene function, and regulatory networks. Consequently, genomic tools to assist breeding are meager, and efficient exploitation of these species has been limited so far. In this paper, we employed the reference genome sequences from cultivated potato and tomato and a collection of sequences of 1,423 potato Diversity Arrays Technology (DArT) markers that show polymorphic representation across the genomes of S. bulbocastanum and/or S. commersonii genotypes. Our results highlighted microscale genome sequence heterogeneity that may play a significant role in functional and structural divergence between related species. Our analytical approach provides knowledge of genome structural and sequence variability that could not be detected by transcriptome and proteome approaches.

Evolutionary meta-analysis of solanaceous resistance gene and solanum resistance gene analog sequences and a practical framework for cross-species comparisons

Cross-species comparative genomics approaches have been employed to map and clone many important disease resistance (R) genes from Solanum species-especially wild relatives of potato and tomato. These efforts will increase with the recent release of potato genome sequence and the impending release of tomato genome sequence. Most R genes belong to the prominent nucleotide binding site-leucine rich repeat (NBS-LRR) class and conserved NBS-LRR protein motifs enable survey of the R gene space of a plant genome by generation of resistance gene analogs (RGA), polymerase chain reaction fragments derived from R genes. We generated a collection of 97 RGA from the disease-resistant wild potato S. bulbocastanum, complementing smaller collections from other Solanum species. To further comparative genomics approaches, we combined all known Solanum RGA and cloned solanaceous NBS-LRR gene sequences, nearly 800 sequences in total, into a single meta-analysis. We defined R gene diversity bins that reflect both evolutionary relationships and DNA cross-hybridization results. The resulting framework is amendable and expandable, providing the research community with a common vocabulary for present and future study of R gene lineages. Through a series of sequence and hybridization experiments, we demonstrate that all tested R gene lineages are of ancient origin, are shared between Solanum species, and can be successfully accessed via comparative genomics approaches.

Gene models from ESTs (GeneModelEST): an application on the Solanum lycopersicum genome.

BackgroundThe structure annotation of a genome is based either on ab initio methodologies or on similaritiy searches versus molecules that have been already annotated. Ab initio gene predictions in a genome are based on a priori knowledge of species-specific features of genes. The training of ab initio gene finders is based on the definition of a data-set of gene models. To accomplish this task the common approach is to align species-specific full length cDNA and EST sequences along the genomic sequences in order to define exon/intron structure of mRNA coding genes.ResultsGeneModelEST is the software here proposed for defining a data-set of candidate gene models using exclusively evidence derived from cDNA/EST sequences.GeneModelEST requires the genome coordinates of the spliced-alignments of ESTs and of contigs (tentative consensus sequences) generated by an EST clustering/assembling procedure to be formatted in a General Feature Format (GFF) standard file. Moreover, the alignments of the contigs versus a protein database are required as an NCBI BLAST formatted report file.The GeneModelEST analysis aims to i) evaluate each exon as defined from contig spliced alignments onto the genome sequence; ii) classify the contigs according to quality levels in order to select candidate gene models; iii) assign to the candidate gene models preliminary functional annotations.We discuss the application of the proposed methodology to build a data-set of gene models of Solanum lycopersicum, whose genome sequencing is an ongoing effort by the International Tomato Genome Sequencing Consortium.ConclusionThe contig classification procedure used by GeneModelEST supports the detection of candidate gene models, the identification of potential alternative transcripts and it is useful to filter out ambiguous information. An automated procedure, such as the one proposed here, is fundamental to support large scale analysis in order to provide species-specific gene models, that could be useful as a training data-set for ab initio gene finders and/or as a reference gene list for a human curated annotation.

Euchromatic and heterochromatic compositional properties emerging from the analysis of Solanum lycopersicum BAC sequences

The consortium responsible for the sequencing of the tomato (Solanum lycopersicum) genome initially focused on the sequencing of the euchromatic regions using a BAC-by-BAC strategy. We analyzed the compositional features of the whole collection of BAC sequences publically available. This analysis highlights specific peculiarities of heterochromatic and euchromatic BACs, in particular: the whole BAC collection has i) a large variability in repeat and gene content, ii) a positive and significant correlation of LTR retrotransposons of the Gypsy class with the repeat content and iii) the preferential location of the SINEs (short interspersed nuclear elements) in BAC sequences showing a low repeat content. Our results point out a typical design of the tomato chromosomes and pave the way for further investigations on the relationship between DNA primary structure and chromatin organization in Solanaceae genomes.