Carsten O. Daub

An atlas of active enhancers across human cell types and tissues

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.

The regulated retrotransposon transcriptome of mammalian cells

Although repetitive elements pervade mammalian genomes, their overall contribution to transcriptional activity is poorly defined. Here, as part of the FANTOM4 project, we report that 6–30% of cap-selected mouse and human RNA transcripts initiate within repetitive elements. Analysis of approximately 250,000 retrotransposon-derived transcription start sites shows that the associated transcripts are generally tissue specific, coincide with gene-dense regions and form pronounced clusters when aligned to full-length retrotransposon sequences. Retrotransposons located immediately 5′ of protein-coding loci frequently function as alternative promoters and/or express noncoding RNAs. More than a quarter of RefSeqs possess a retrotransposon in their 3′ UTR, with strong evidence for the reduced expression of these transcripts relative to retrotransposon-free transcripts. Finally, a genome-wide screen identifies 23,000 candidate regulatory regions derived from retrotransposons, in addition to more than 2,000 examples of bidirectional transcription. We conclude that retrotransposon transcription has a key influence upon the transcriptional output of the mammalian genome.

Tiny RNAs associated with transcription start sites in animals

It has been reported that relatively short RNAs of heterogeneous sizes are derived from sequences near the promoters of eukaryotic genes. As part of the FANTOM4 project, we have identified tiny RNAs with a modal length of 18 nt that map within −60 to +120 nt of transcription start sites (TSSs) in human, chicken and Drosophila. These transcription initiation RNAs (tiRNAs) are derived from sequences on the same strand as the TSS and are preferentially associated with G+C-rich promoters. The 5′ ends of tiRNAs show peak density 10–30 nt downstream of TSSs, indicating that they are processed. tiRNAs are generally, although not exclusively, associated with highly expressed transcripts and sites of RNA polymerase II binding. We suggest that tiRNAs may be a general feature of transcription in metazoa and possibly all eukaryotes.

Hidden layers of human small RNAs

BackgroundSmall RNA attracts increasing interest based on the discovery of RNA silencing and the rapid progress of our understanding of these phenomena. Although recent studies suggest the possible existence of yet undiscovered types of small RNAs in higher organisms, many studies to profile small RNA have focused on miRNA and/or siRNA rather than on the exploration of additional classes of RNAs.ResultsHere, we explored human small RNAs by unbiased sequencing of RNAs with sizes of 19–40 nt. We provide substantial evidences for the existence of independent classes of small RNAs. Our data shows that well-characterized non-coding RNA, such as tRNA, snoRNA, and snRNA are cleaved at sites specific to the class of ncRNA. In particular, tRNA cleavage is regulated depending on tRNA type and tissue expression. We also found small RNAs mapped to genomic regions that are transcribed in both directions by bidirectional promoters, indicating that the small RNAs are a product of dsRNA formation and their subsequent cleavage. Their partial similarity with ribosomal RNAs (rRNAs) suggests unrevealed functions of ribosomal DNA or interstitial rRNA. Further examination revealed six novel miRNAs.ConclusionOur results underscore the complexity of the small RNA world and the biogenesis of small RNAs.

Estimating mutual information using B-spline functions – an improved similarity measure for analysing gene expression data

BackgroundThe information theoretic concept of mutual information provides a general framework to evaluate dependencies between variables. In the context of the clustering of genes with similar patterns of expression it has been suggested as a general quantity of similarity to extend commonly used linear measures. Since mutual information is defined in terms of discrete variables, its application to continuous data requires the use of binning procedures, which can lead to significant numerical errors for datasets of small or moderate size.ResultsIn this work, we propose a method for the numerical estimation of mutual information from continuous data. We investigate the characteristic properties arising from the application of our algorithm and show that our approach outperforms commonly used algorithms: The significance, as a measure of the power of distinction from random correlation, is significantly increased. This concept is subsequently illustrated on two large-scale gene expression datasets and the results are compared to those obtained using other similarity measures.A C++ source code of our algorithm is available for non-commercial use from kloska@scienion.de upon request.ConclusionThe utilisation of mutual information as similarity measure enables the detection of non-linear correlations in gene expression datasets. Frequently applied linear correlation measures, which are often used on an ad-hoc basis without further justification, are thereby extended.

Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting and reveals Argonaute association with RNA fragments of diverse origin.

While several studies have focused on the relationship between individual miRNA loci or classes of small RNA with human Argonaute (AGO) proteins, a comprehensive, global analysis of the RNA content associating with different AGO proteins has yet to be performed. We have compared the content of deep sequenced RNA extracted from immunoprecipitation experiments with the AGO1, AGO2, and AGO3 proteins. Consistent with previous observations, sequence tags derived from miRNA loci globally associate in approximately equivalent amounts with AGO1, AGO2, and AGO3. Exceptions include miR-182, miR-222, and miR-223*, which could be coupled to processes targeting the loci for interaction with specific AGO proteins. A closer inspection of the data, however, supports the presence of an unusual sorting mechanism wherein a subset of miRNA loci give rise to distinct isomirs which preferentially associate with distinct AGO proteins in a significantly differential manner. We also identify the complete set of short RNA derived from non-miRNA sources including tRNA, snRNA, snoRNA, vRNA, and mRNA associating with the AGO proteins, many of which are predicted to play roles in post-transcriptional gene silencing. We also observe enrichment of tags mapping to promoter regions of genes, suggesting that a fraction of the recently-identified promoter-associated small RNAs in humans could function through interaction with AGO proteins. Finally, we observe antisense miRNA transcripts are frequently present in low copy numbers across a range of diverse miRNA loci and these transcripts appear to associate with AGO proteins.

Gateways to the FANTOM5 promoter level mammalian expression atlas

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.

TagDust--a program to eliminate artifacts from next generation sequencing data.

Motivation: Next-generation parallel sequencing technologies produce large quantities of short sequence reads. Due to experimental procedures various types of artifacts are commonly sequenced alongside the targeted RNA or DNA sequences. Identification of such artifacts is important during the development of novel sequencing assays and for the downstream analysis of the sequenced libraries. Results: Here we present TagDust, a program identifying artifactual sequences in large sequencing runs. Given a user-defined cutoff for the false discovery rate, TagDust identifies all reads explainable by combinations and partial matches to known sequences used during library preparation. We demonstrate the quality of our method on sequencing runs performed on Illuminas Genome Analyzer platform. Availability: Executables and documentation are available from http://genome.gsc.riken.jp/osc/english/software/. Contact: timolassmann@gmail.com

MiR-107 and MiR-185 Can Induce Cell Cycle Arrest in Human Non Small Cell Lung Cancer Cell Lines

Background MicroRNAs (miRNAs) are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5′ seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentially play critical roles in carcinogenesis. Methodology/Principal Findings Introduction of synthetic miR-107 or miR-185 suppressed growth of the human non-small cell lung cancer cell lines. Flow cytometry analysis revealed these miRNAs induce a G1 cell cycle arrest in H1299 cells and the suppression of cell cycle progression is stronger than that by Let-7 miRNA. By the gene expression analyses with oligonucleotide microarrays, we find hundreds of genes are affected by transfection of these miRNAs. Using miRNA-target prediction analyses and the array data, we listed up a set of likely targets of miR-107 and miR-185 for G1 cell cycle arrest and validate a subset of them using real-time RT-PCR and immunoblotting for CDK6. Conclusions/Significance We identified new cell cycle regulating miRNAs, miR-107 and miR-185, localized in frequently altered chromosomal regions in human lung cancers. Especially for miR-107, a large number of down-regulated genes are annotated with the gene ontology term ‘cell cycle’. Our results suggest that these miRNAs may contribute to regulate cell cycle in human malignant tumors.

Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)–4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5′ ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as “divergently regulated”). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular “inputs” such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional “outputs” such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.