Genome-wide profiling of Populus small RNAs

<p>Abstract</p> <p>Background</p> <p>Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced <it>Populus trichocarpa </it>using a concatenation strategy in combination with 454 sequencing.</p> <p>Results</p> <p>The most abundant size class of sRNAs were 24 nt. Long Terminal Repeats were particularly associated with 24 nt sRNAs. Additionally, some repetitive elements were associated with 22 nt sRNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the proposed sex-determining locus and a major cluster of <it>NBS-LRR </it>genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and <it>NBS-LRR </it>disease resistance genes, classes of genes that have been significantly expanded in <it>Populus</it>. Additional loci enriched for sRNA production were identified and characterised. We identified 15 novel predicted microRNAs (miRNAs), including miRNA*sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs).</p> <p>Conclusions</p> <p>The short RNA population of <it>P. trichocarpa </it>is at least as complex as that of <it>Arabidopsis thaliana</it>. We provide a first genome-wide view of short RNA production for <it>P. trichocarpa </it>and identify new, non-conserved miRNAs.</p

An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression.

Breast cancer is the most diagnosed malignancy and the second leading cause of cancer mortality in females. Previous association studies have identified variants on 2q35 associated with the risk of breast cancer. To identify functional susceptibility loci for breast cancer, we interrogated the 2q35 gene desert for chromatin architecture and functional variation correlated with gene expression. We report a novel intergenic breast cancer risk locus containing an enhancer copy number variation (enCNV; deletion) located approximately 400Kb upstream to IGFBP5, which overlaps an intergenic ERα-bound enhancer that loops to the IGFBP5 promoter. The enCNV is correlated with modified ERα binding and monoallelic-repression of IGFBP5 following estrogen treatment. We investigated the association of enCNV genotype with breast cancer in 1,182 cases and 1,362 controls, and replicate our findings in an independent set of 62,533 cases and 60,966 controls from 41 case control studies and 11 GWAS. We report a dose-dependent inverse association of 2q35 enCNV genotype (percopy OR=0.68 95%CI 0.55-0.83, P=0.0002; replication OR=0.77 95%CI 0.73-0.82, P=2.1x10(-19)) and identify 13 additional linked variants (r(2)>0.8) in the 20Kb linkage block containing the enCNV (P=3.2x10(-15) - 5.6x10(-17)). These associations were independent of previously reported 2q35 variants, rs13387042/rs4442975 and rs16857609, and were stronger for ER-positive than ER-negative disease. Together, these results suggest that 2q35 breast cancer risk loci may be mediating their effect through IGFBP5

The role of autophagy in the cross-talk between epithelial-mesenchymal transitioned tumor cells and cancer stem-like cells

Epithelial-mesenchymal transition (EMT) and cancer stem-like cells (CSC) are becoming highly relevant targets in anticancer drug discovery. A large body of evidence suggests that epithelial-mesenchymal transitioned tumor cells (EMT tumor cells) and CSCs have similar functions. There is also an overlap regarding the stimuli that can induce the generation of EMT tumor cells and CSCs. Moreover, direct evidence has been brought that EMT can give rise to CSCs. It is unclear however, whether EMT tumor cells should be considered CSCs or if they have to undergo further changes. In this article we summarize available evidence suggesting that, indeed, additional programs must be engaged and we propose that macroautophagy (hereafter, autophagy) represents a key trait distinguishing CSCs from EMT tumor cells. Thus, CSCs have often been reported to be in an autophagic state and blockade of autophagy inhibits CSCs. On the other hand, there is ample evidence showing that EMT and autophagy are distinct events. CSCs, however, represent, by themselves, a heterogeneous population. Thus, CSCs have been distinguished in predominantly noncycling and cycling CSCs, the latter representing CSCs that self-renew and replenish the pool of differentiated tumor cells. We now suggest that the non-cycling CSC subpopulation is in an autophagic state. We propose also two models to explain the relationship between EMT tumor cells and these two major CSC subpopulations: a branching model in which EMT tumor cells can give rise to cycling or non-cycling CSCs, respectively, and a hierarchical model in which EMT tumor cells are first induced to become autophagic CSCs and, subsequently, cycling CSCs. Finally, we address the therapeutic consequences of these insights

Phylogenetic analysis of metastatic progression in breast cancer using somatic mutations and copy number aberrations.

Several studies using genome-wide molecular techniques have reported various degrees of genetic heterogeneity between primary tumours and their distant metastases. However, it has been difficult to discern patterns of dissemination owing to the limited number of patients and available metastases. Here, we use phylogenetic techniques on data generated using whole-exome sequencing and copy number profiling of primary and multiple-matched metastatic tumours from ten autopsied patients to infer the evolutionary history of breast cancer progression. We observed two modes of disease progression. In some patients, all distant metastases cluster on a branch separate from their primary lesion. Clonal frequency analyses of somatic mutations show that the metastases have a monoclonal origin and descend from a common 'metastatic precursor'. Alternatively, multiple metastatic lesions are seeded from different clones present within the primary tumour. We further show that a metastasis can be horizontally cross-seeded. These findings provide insights into breast cancer dissemination

Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsiz

Evidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation.

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the 'iCOGS' genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84-0.87; P=1.7 × 10(-43)) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology

Scalable Transcriptome Preparation for Massive Parallel Sequencing

Background: The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity. Methodology: In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation. Conclusion/Significance: The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods

Genome-wide profiling of Populus small RNAs

Background: Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing. Results: The most abundant size class of sRNAs were 24 nt. Long Terminal Repeats were particularly associated with 24 nt sRNAs. Additionally, some repetitive elements were associated with 22 nt sRNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the proposed sex-determining locus and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified and characterised. We identified 15 novel predicted microRNAs (miRNAs), including miRNA*sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs). Conclusions: The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis thaliana. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs.Copyrighted by BioMed Central

Translational Database Selection and Multiplexed Sequence Capture for Up Front Filtering of Reliable Breast Cancer Biomarker Candidates

Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples

Deep Annotation of Populus trichocarpa microRNAs from Diverse Tissue Sets

Populus trichocarpa is an important woody model organism whose entire genome has been sequenced. This resource has facilitated the annotation of microRNAs (miRNAs), which are short non-coding RNAs with critical regulatory functions. However, despite their developmental importance, P. trichocarpa miRNAs have yet to be annotated from numerous important tissues. Here we significantly expand the breadth of tissue sampling and sequencing depth for miRNA annotation in P. trichocarpa using high-throughput smallRNA (sRNA) sequencing. miRNA annotation was performed using three individual next-generation sRNA sequencing runs from separate leaves, xylem, and mechanically treated xylem, as well as a fourth run using a pooled sample containing vegetative apices, male flowers, female flowers, female apical buds, and male apical and lateral buds. A total of 276 miRNAs were identified from these datasets, including 155 previously unannotated miRNAs, most of which are P. trichocarpa specific. Importantly, we identified several xylem-enriched miRNAs predicted to target genes known to be important in secondary growth, including the critical reaction wood enzyme xyloglucan endo-transglycosylase/hydrolase and vascular-related transcription factors. This study provides a thorough genome-wide annotation of miRNAs in P. trichocarpa through deep sRNA sequencing from diverse tissue sets. Our data significantly expands the P. trichocarpa miRNA repertoire, which will facilitate a broad range of research in this major model system