Identification of active regulatory regions from DNA methylation data

We have recently shown that transcription factor binding leads to defined reduction in DNA methylation, allowing for the identification of active regulatory regions from high-resolution methylomes. Here, we present MethylSeekR, a computational tool to accurately identify such footprints from bisulfite-sequencing data. Applying our method to a large number of published human methylomes, we demonstrate its broad applicability and generalize our previous findings from a neuronal differentiation system to many cell types and tissues. MethylSeekR is available as an R package at www.bioconductor.or

Overestimation of alternative splicing caused by variable probe characteristics in exon arrays

In higher eukaryotes, alternative splicing is a common mechanism for increasing transcriptome diversity. Affymetrix exon arrays were designed as a tool for monitoring the relative expression levels of hundreds of thousands of known and predicted exons with a view to detecting alternative splicing events. In this article, we have analyzed exon array data from many different human and mouse tissues and have uncovered a systematic relationship between transcript-fold change and alternative splicing as reported by the splicing index. Evidence from dilution experiments and deep sequencing suggest that this effect is of technical rather than biological origin and that it is driven by sequence features of the probes. This effect is substantial and results in a 12-fold overestimation of alternative splicing events in genes that are differentially expressed. By cross-species exon array comparison, we could further show that the systematic bias persists even across species boundaries. Failure to consider this effect in data analysis would result in the reproducible false detection of apparently conserved alternative splicing events. Finally, we have developed a software in R called COSIE (Corrected Splicing Indices for Exon arrays) that for any given set of new exon array experiments corrects for the observed bias and improves the detection of alternative splicing (available at www.fmi.ch/groups/gbioinfo

The mammalian TRIM-NHL protein TRIM71/LIN-41 is a repressor of mRNA function

TRIM-NHL proteins are conserved regulators of development and differentiation but their molecular function has remained largely elusive. Here, we report an as yet unrecognized activity for the mammalian TRIM-NHL protein TRIM71 as a repressor of mRNAs. We show that TRIM71 is associated with mRNAs and that it promotes translational repression and mRNA decay. We have identified Rbl1 and Rbl2, two transcription factors whose down-regulation is important for stem cell function, as TRIM71 targets in mouse embryonic stem cells. Furthermore, one of the defining features of TRIM-NHL proteins, the NHL domain, is necessary and sufficient to target TRIM71 to RNA, while the RING domain that confers ubiquitin ligase activity is dispensable for repression. Our results reveal strong similarities between TRIM71 and Drosophila BRAT, the best-studied TRIM-NHL protein and a well-documented translational repressor, suggesting that BRAT and TRIM71 are part of a family of mRNA repressors regulating proliferation and differentiatio

Engineering of a conditional allele reveals multiple roles of XRN2 in Caenorhabditis elegans development and substrate specificity in microRNA turnover

Although XRN2 proteins are highly conserved eukaryotic 5′→3′ exonucleases, little is known about their function in animals. Here, we characterize Caenorhabditis elegans XRN2, which we find to be a broadly and constitutively expressed nuclear protein. An xrn-2 null mutation or loss of XRN2 catalytic activity causes a molting defect and early larval arrest. However, by generating a conditionally mutant xrn-2ts strain de novo through an approach that may be also applicable to other genes of interest, we reveal further functions in fertility, during embryogenesis and during additional larval stages. Consistent with the known role of XRN2 in controlling microRNA (miRNA) levels, we can demonstrate that loss of XRN2 activity stabilizes some rapidly decaying miRNAs. Surprisingly, however, other miRNAs continue to decay rapidly in xrn-2ts animals. Thus, XRN2 has unanticipated miRNA specificity in vivo, and its diverse developmental functions may relate to distinct substrates. Finally, our global analysis of miRNA stability during larval stage 1 reveals that miRNA passenger strands (miR*s) are substantially less stable than guide strands (miRs), supporting the notion that the former are mostly byproducts of biogenesis rather than a less abundant functional specie

QuasR: quantification and annotation of short reads in R

Summary: QuasR is a package for the integrated analysis of high-throughput sequencing data in R, covering all steps from read preprocessing, alignment and quality control to quantification. QuasR supports different experiment types (including RNA-seq, ChIP-seq and Bis-seq) and analysis variants (e.g. paired-end, stranded, spliced and allele-specific), and is integrated in Bioconductor so that its output can be directly processed for statistical analysis and visualization. Availability and implementation: QuasR is implemented in R and C/C++. Source code and binaries for major platforms (Linux, OS X and MS Windows) are available from Bioconductor (www.bioconductor.org/packages/release/bioc/html/QuasR.html). The package includes a ‘vignette' with step-by-step examples for typical work flows. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Computational discovery of animal small RNA genes and targets

Though recently discovered, small RNAs appear to play a wealth of regulatory roles, being involved in degradation of target mRNAs, translation silencing of target genes, chromatin remodeling and transposon silencing. Presented here are the computational tools that I developed to annotate and characterize small RNA genes and to identify their targets. One of these tools is oligomap, a novel software for fast and exhaustive identi�cation of nearly-perfect matches of small RNAs in sequence databases. Oligomap is part of an automated annotation pipeline used in our laboratory to annotate small RNA sequences. The application of these tools to samples of small RNAs obtained from mouse and human germ cells together with subsequent computational analyses lead to the discovery of a new class of small RNAs which are now called piRNAs. The computational analysis revealed that piRNAs have a strong uridine preference at their 5' end, that unlike miRNAs, piRNAs are not excised from fold-back precursors but rather from long primary transcripts, and that the genome organization of their genes is conserved between human and mouse even though piRNAs on the sequence level are poorly conserved. In vertebrates, the most studied class of small regulatory RNAs are the miRNAs which bind to mRNAs and block translation. A computational framework is introduced to identify miRNA targets in mammals, ies, worms and �sh. The method uses extensive cross species conservation information to predict miRNA binding sites that are under evolutionary pressure. A downstream analysis of predicted miRNA targets revealed novel properties of miRNA target sites, one of which is a positional bias of miRNA target sites in long mammalian 3' untranslated regions. Intersection of our predictions with biochemical pathway annotation data suggested novel functions for some of the miRNAs. To gain further insights into the mechanism of miRNA targeting, I studied microarray data obtained in siRNA experiments. SiRNAs have been shown to produce o�- targets that resemble miRNA targets. This analysis suggests the presence of additional determinants of miRNA target site functionality (beyond complementarity between the miRNA 5' end and the target) in the close vicinity (about 150 nucleotides) of the miRNA-complementary site. Finally, as part of a study aiming to reduce siRNA o�-target e�ects by introducing chemical modi�cations in the siRNA, I performed microarray data analysis of siRNA transfection experiments. Presented are the methods used to quantify o�- target activity of siRNAs carrying di�erent types of chemical modi�cations. The analysis revealed that o�-targets caused by the passenger strand of the siRNA can be reduced by 5'-O-methylation

Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes

The cold shock domain is one of the most highly conserved motifs between bacteria and higher eukaryotes. Y-box-binding proteins represent a subfamily of cold shock domain proteins with pleiotropic functions, ranging from transcription in the nucleus to translation in the cytoplasm. These proteins have been investigated in all major model organisms except Caenorhabditis elegans. In this study, we set out to fill this gap and present a functional characterization of CEYs, the C. elegans Y-box-binding proteins. We find that, similar to other organisms, CEYs are essential for proper gametogenesis. However, we also report a novel function of these proteins in the formation of large polysomes in the soma. In the absence of the somatic CEYs, polysomes are dramatically reduced with a simultaneous increase in monosomes and disomes, which, unexpectedly, has no obvious impact on animal biology. Because transcripts that are enriched in polysomes in wild-type animals tend to be less abundant in the absence of CEYs, our findings suggest that large polysomes might depend on transcript stabilization mediated by CEY protein

Ribonuclease-mediated control of body fat

Obesity is a global health issue, arousing interest in molecular mechanisms controlling fat. Transcriptional regulation of fat has received much attention, and key transcription factors involved in lipid metabolism, such as SBP-1/SREBP, LPD-2/C/EBP, and MDT-15, are conserved from nematodes to mammals. However, there is a growing awareness that lipid metabolism can also be controlled by post-transcriptional mechanisms. Here, we show that the Caenorhabditis elegans RNase, REGE-1, related to MCPIP1/Zc3h12a/Regnase-1, a key regulator of mammalian innate immunity, promotes accumulation of body fat. Using exon-intron split analysis, we find that REGE-1 promotes fat by degrading the mRNA encoding ETS-4, a fat-loss-promoting transcription factor. Because ETS-4, in turn, induces rege-1 transcription, REGE-1 and ETS-4 appear to form an auto-regulatory module. We propose that this type of fat regulation may be of key importance when, if faced with an environmental change, an animal must rapidly but precisely remodel its metabolism.</p

Dietary soy and meat proteins induce distinct physiological and gene expression changes in rats

This study reports on a comprehensive comparison of the effects of soy and meat proteins given at the recommended level on physiological markers of metabolic syndrome and the hepatic transcriptome. Male rats were fed semi-synthetic diets for 1 wk that differed only regarding protein source, with casein serving as reference. Body weight gain and adipose tissue mass were significantly reduced by soy but not meat proteins. The insulin resistance index was improved by soy, and to a lesser extent by meat proteins. Liver triacylglycerol contents were reduced by both protein sources, which coincided with increased plasma triacylglycerol concentrations. Both soy and meat proteins changed plasma amino acid patterns. The expression of 1571 and 1369 genes were altered by soy and meat proteins respectively. Functional classification revealed that lipid, energy and amino acid metabolic pathways, as well as insulin signaling pathways were regulated differently by soy and meat proteins. Several transcriptional regulators, including NFE2L2, ATF4, Srebf1 and Rictor were identified as potential key upstream regulators. These results suggest that soy and meat proteins induce distinct physiological and gene expression responses in rats and provide novel evidence and suggestions for the health effects of different protein sources in human diets

Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation

Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced micro- RNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these prodifferentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.Comment: 45 pages 5 figure