We present an update to our Galaxy-based web server for processing and visualizing deeply sequenced data. Its core tool set, deepTools, allows users to perform complete bioinformatic workflows ranging from quality controls and normalizations of aligned reads to integrative analyses, including clustering and visualization approaches. Since we first described our deepTools Galaxy server in 2014, we have implemented new solutions for many requests from the community and our users. Here, we introduce significant enhancements and new tools to further improve data visualization and interpretation. deepTools continue to be open to all users and freely available as a web service at deeptools.ie-freiburg.mpg.de. The new deepTools2 suite can be easily deployed within any Galaxy framework via the toolshed repository, and we also provide source code for command line usage under Linux and Mac OS X. A public and documented API for access to deepTools functionality is also available.

We present a Galaxy based web server for processing and visualizing deeply sequenced data. The web server's core functionality consists of a suite of newly developed tools, called deepTools, that enable users with little bioinformatic background to explore the results of their sequencing experiments in a standardized setting. Users can upload pre-processed files with continuous data in standard formats and generate heatmaps and summary plots in a straight-forward, yet highly customizable manner. In addition, we offer several tools for the analysis of files containing aligned reads and enable efficient and reproducible generation of normalized coverage files. As a modular and open-source platform, deepTools can easily be expanded and customized to future demands and developments. The deepTools webserver is freely available at http://deeptools.ie-freiburg.mpg.de and is accompanied by extensive documentation and tutorials aimed at conveying the principles of deep-sequencing data analysis. The web server can be used without registration. deepTools can be installed locally either stand-alone or as part of Galaxy.

Despite an abundance of new studies about topologically associating domains (TADs), the role of genetic information in TAD formation is still not fully understood. Here we use our software, HiCExplorer (hicexplorer.readthedocs.io) to annotate >2800 high-resolution (570 bp) TAD boundaries in Drosophila melanogaster. We identify eight DNA motifs enriched at boundaries, including a motif bound by the M1BP protein, and two new boundary motifs. In contrast to mammals, the CTCF motif is only enriched on a small fraction of boundaries flanking inactive chromatin while most active boundaries contain the motifs bound by the M1BP or Beaf-32 proteins. We demonstrate that boundaries can be accurately predicted using only the motif sequences at open chromatin sites. We propose that DNA sequence guides the genome architecture by allocation of boundary proteins in the genome. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogenome.ie-freiburg.mpg.de.

Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.

Heterochromatin is required to restrict aberrant expression of retrotransposons, but it remains poorly defined due to the underlying repeat-rich sequences. We dissected Suv39h-dependent histone H3 lysine 9 trimethylation (H3K9me3) by genome-wide ChIP sequencing in mouse embryonic stem cells (ESCs). Refined bioinformatic analyses of repeat subfamilies indicated selective accumulation of Suv39h-dependent H3K9me3 at interspersed repetitive elements that cover ∼5% of the ESC epigenome. The majority of the ∼8,150 intact long interspersed nuclear elements (LINEs) and endogenous retroviruses (ERVs), but only a minor fraction of the >1.8 million degenerate and truncated LINEs/ERVs, are enriched for Suv39h-dependent H3K9me3. Transcriptional repression of intact LINEs and ERVs is differentially regulated by Suv39h and other chromatin modifiers in ESCs but governed by DNA methylation in committed cells. These data provide a function for Suv39h-dependent H3K9me3 chromatin to specifically repress intact LINE elements in the ESC epigenome.

Summary DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are poorly understood. A role for three-dimensional chromatin organization has been proposed. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells Rif1 coats late replicating domains and, together with Lamin B1 identifies the majority of the late replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during G1, thereby revealing a novel function of Rif1 as organizer of nuclear architecture. Loss of Rif1 affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during S-phase Rif1 ensures temporal coordination of replication of interacting domains. In summary our study identifies Rif1 as the first molecular link between nuclear architecture organization and replication-timing establishment in mammals.

The C source code for our TRAP program is freely available for non-commercial use at http://www.molgen.mpg.de/~manke/papers/TFaffinities/

Periodontitis is a widespread, complex inflammatory disease of the mouth, which results in a loss of gingival tissue and alveolar bone, with aggressive periodontitis (AgP) as its most severe form. To identify genetic risk factors for periodontitis, we conducted a genome-wide association study in German AgP patients. We found AgP to be strongly associated with the intronic SNP rs1537415, which is located in the glycosyltransferase gene GLT6D1. We replicated the association in a panel of Dutch generalized and localized AgP patients. In the combined analysis including 1758 subjects, rs1537415 reached a genome-wide significance level of P= 5.51 x 10(-9), OR = 1.59 (95% CI 1.36-1.86). The associated rare G allele of rs1537415 showed an enrichment of 10% in periodontitis cases (48.4% in comparison with 38.8% in controls). Fine-mapping and a haplotype analysis indicated that rs1537415 showed the strongest association signal. Sequencing identified no further associated variant. Tissue-specific expression analysis of GLT6D1 indicated high transcript levels in the leukocytes, the gingiva and testis. Analysis of potential transcription factor binding sites at this locus predicted a significant reduction of GATA-3 binding affinity, and an electrophoretic mobility assay indicated a T cell specific reduction of protein binding for the G allele. Overexpression of GATA-3 in HEK293 cells resulted in allele-specific binding of GATA-3, indicating the identity of GATA-3 as the binding protein. The identified association of GLT6D1 with AgP implicates this locus as an important susceptibility factor, and GATA-3 as a potential signaling component in the pathophysiology of periodontitis.