We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the United States Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed, for these and qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.

RNA-seq facilitates unbiased genome-wide gene-expression profiling. However, its concordance with the well-established microarray platform must be rigorously assessed for confident uses in clinical and regulatory application. Here we use a comprehensive study design to generate Illumina RNA-seq and Affymetrix microarray data from the same set of liver samples of rats under varying degrees of perturbation by 27 chemicals representing multiple modes of action (MOA). The cross-platform concordance in terms of differentially expressed genes (DEGs) or enriched pathways is highly correlated with treatment effect size, gene-expression abundance and the biological complexity of the MOA. RNA-seq outperforms microarray (90% versus 76%) in DEG verification by quantitative PCR and the main gain is its improved accuracy for low expressed genes. Nonetheless, predictive classifiers derived from both platforms performed similarly. Therefore, the endpoint studied and its biological complexity, transcript abundance, and intended application are important factors in transcriptomic research and for decision-making.

RNA-directed DNA methylation, one of several RNA interference-mediated pathways in the nucleus, has been documented in plants and in human cells. Despite progress in identifying the DNA methyltransferases, histone-modifying enzymes and RNA interference proteins needed for RNA-directed DNA methylation, the mechanism remains incompletely understood. We screened for mutants defective in RNA-directed DNA methylation and silencing of a transgene promoter in Arabidopsis thaliana and identified three drd complementation groups. DRD1 is a SNF2-like protein required for RNA-directed de novo methylation. We report here that DRD2 and DRD3 correspond to the second-largest subunit and largest subunit, respectively, of a fourth class of DNA-dependent RNA polymerase (polymerase IV) that is unique to plants. DRD3 is a functionally diversified homolog of NRPD1a or SDE4, identified in a separate screen for mutants defective in post-transcriptional gene silencing. The identical DNA methylation patterns observed in all three drd mutants suggest that DRD proteins cooperate to create a substrate for RNA-directed de novo methylation.

SummaryArabidopsis thaliana is a host for the sugar beet cyst nematode Heterodera schachtii. Juvenile nematodes invade the roots and induce the development of a syncytium, which functions as a feeding site for the nematode. Here, we report on the transcriptome of syncytia induced in the roots of Arabidopsis. Microaspiration was employed to harvest pure syncytium material, which was then used to prepare RNA for hybridization to Affymetrix GeneChips. Initial data analysis showed that the gene expression in syncytia at 5 and 15 days post-infection did not differ greatly, and so both time points were compared together with control roots. Out of a total of 21 138 genes, 18.4% (3893) had a higher expression level and 15.8% (3338) had a lower expression level in syncytia, as compared with control roots, using a multipletesting corrected false discovery rate of below 5%. A gene ontology (GO) analysis of up-and downregulated genes showed that categories related to high metabolic activity were preferentially upregulated. A principal component analysis was applied to compare the transcriptome of syncytia with the transcriptome of different Arabidopsis organs (obtained by the AtGenExpress project), and with specific root tissues. This analysis revealed that syncytia are transcriptionally clearly different from roots (and all other organs), as well as from other root tissues.

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.

RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits. Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation.

B7-H3 belongs to the B7 superfamily, a group of molecules that costimulate or downmodulate T-cell responses. Although it was shown that B7-H3 could inhibit T-cell responses, several studies -most of them performed in murine systems -found B7-H3 to act in a costimulatory manner. In this study, we have specifically addressed a potential functional dualism of human B7-H3 by assessing the effect of this molecule under varying experimental conditions as well as on different T-cell subsets. We show that B7-H3 does not costimulate human T cells. In the presence of strong activating signals, B7-H3 potently and consistently down-modulated human T-cell responses. This inhibitory effect was evident when analysing proliferation and cytokine production and affected naïve as well as pre-activated T cells. Furthermore, we demonstrate that B7-H3-T-cell interaction is characterised by an early suppression of IL-2 and that T-cell inhibition can be reverted by exogenous IL-2. Since the triggering receptor expressed on myeloid cells like transcript 2 (TREML2/TLT-2) has been recently described as costimulatory receptor of murine B7-H3 we have extensively analysed interaction of human B7-H3 with TREML2/TLT-2. In these experiments we found no evidence for such an interaction. Furthermore, our data do not point to a role for murine TREML2 as a receptor for murine B7-H3. Key words: Costimulatory molecules . Immune regulation . T cells Supporting Information available online IntroductionFor fine-tuning the immune response, several costimulatory and coinhibitory signals are needed, in addition to signal 1 provided via the peptide-MHC/TCR-complex interaction. CD80 (B7-1) and CD86 (B7-2) serve as primary costimulatory ligands. Recently, additional members of the B7 family -the so-called B7 homologshave been identified [1]. The functional role of several of these B7 homologs is still controversially discussed. One of these molecules is B7-H3, which was originally described as a potent costimulatory molecule and inducer of IFN-g in human T cells [2]. In contrast, Ling et al. found human B7-H3 to strongly down-regulate T-cell proliferation and cytokine production [3]. It was suggested that the presence of two B7-H3 receptors with different functions could explain these divergent results [3]. Recent data that showed opposing effects of B7-H3 on resting and cytokine-activated T cells as well as contradicting results on the function of murine B7-H3 would also be in support for such a constellation [4][5][6][7]. Such receptor molecules could either be differentially regulated on T cells or be expressed on different T-cell subsets. Depending on the experimental system used the effects of the costimulatory or the inhibitory receptor could prevail and explain the discrepancies in different studies.Here, we have specifically addressed a potential functional dualism of B7-H3 by studying B7-H3 effects under varying experimental conditions as well as on different subsets of human T cells. Our results point to a potent and consistent inhibitory role of h...

The global amino acid compositions as deduced from the complete genomic sequences of six thermophilic archaea, two thermophilic bacteria, 17 mesophilic bacteria and two eukaryotic species were analysed by hierarchical clustering and principal components analysis. Both methods showed an influence of several factors on amino acid composition. Although GC content has a dominant effect, thermophilic species can be identified by their global amino acid compositions alone. This study presents a careful statistical analysis of factors that affect amino acid composition and also yielded specific features of the average amino acid composition of thermophilic species. Moreover, we introduce the first example of a 'compositional tree' of species that takes into account not only homologous proteins, but also proteins unique to particular species. We expect this simple yet novel approach to be a useful additional tool for the study of phylogeny at the genome level.