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Meszaros B.,Hungarian Academy of Sciences | Kumar M.,Structural and Computational Biology Unit | Gibson T.J.,Structural and Computational Biology Unit | Uyar B.,Berlin Institute for Medical Systems Biology | Dosztanyi Z.,Eötvös Loránd University
Science Signaling | Year: 2017

Degrons are the elements that are used by E3 ubiquitin ligases to target proteins for degradation. Most degrons are short linear motifs embedded within the sequences of modular proteins. As regulatory sites for protein abundance, they are important for many different cellular processes, such as progression through the cell cycle and monitoring cellular hypoxia. Degrons enable the elimination of proteins that are no longer required, preventing their possible dysfunction. Although the human genome encodes ∼600 E3 ubiquitin ligases, only a fraction of these enzymes have well-defined target degrons. Thus, for most cellular proteins, the destruction mechanisms are poorly understood. This is important for many diseases, especially for cancer, a disease that involves the enhanced expression of oncogenes and the persistence of encoded oncoproteins coupled with reduced abundance of tumor suppressors. Loss0of-function mutations occur in the degrons of several oncoproteins, such as the transcription factors MYC and NRF2, and in various mitogenic receptors, such as NOTCH1 and several receptor tyrosine kinases. Mutations eliminating the function of the β-catenin degron are found in many cancers and are considered one of the most abundant mutations driving carcinogenesis. In this Review, we describe the current knowledge of degrons in cancer and suggest that increased research on the "dark degrome" (unknown degron-E3 relationships) would enhance progress in cancer research. © 2017 The Authors, some rights reserved.

Leucci E.,Copenhagen University | Zriwil A.,Copenhagen University | Gregersen L.H.,Copenhagen University | Gregersen L.H.,Berlin Institute for Medical Systems Biology | And 7 more authors.
Oncogene | Year: 2012

MicroRNAs are important regulators of gene expression in normal development and disease. miR-9 is overexpressed in several cancer forms, including brain tumours, hepatocellular carcinomas, breast cancer and Hodgkin lymphoma (HL). Here we demonstrated a relevance for miR-9 in HL pathogenesis and identified two new targets Dicer1 and HuR. HL is characterized by a massive infiltration of immune cells and fibroblasts in the tumour, whereas malignant cells represent only 1% of the tumour mass. These infiltrates provide important survival and growth signals to the tumour cells, and several lines of evidence indicate that they are essential for the persistence of HL. We show that inhibition of miR-9 leads to derepression of DICER and HuR, which in turn results in a decrease in cytokine production by HL cells followed by an impaired ability to attract normal inflammatory cells. Finally, inhibition of miR-9 by a systemically delivered antimiR-9 in a xenograft model of HL increases the protein levels of HuR and DICER1 and results in decreased tumour outgrowth, confirming that miR-9 actively participates in HL pathogenesis and points to miR-9 as a potential therapeutic target. © 2012 Macmillan Publishers Limited All rights reserved.

Rodelsperger C.,Charité - Medical University of Berlin | Rodelsperger C.,Max Planck Institute for Molecular Genetics | Dieterich C.,Berlin Institute for Medical Systems Biology
PLoS ONE | Year: 2010

Whole genome gene order evolution in higher eukaryotes was initially considered as a random process. Gene order conservation or conserved synteny was seen as a feature of common descent and did not imply the existence of functional constraints. This view had to be revised in the light of results from sequencing dozens of vertebrate genomes. It became apparent that other factors exist that constrain gene order in some genomic regions over long evolutionary time periods. Outside of these regions, genomes diverge more rapidly in terms of gene content and order. We have developed CYNTENATOR, a progressive gene order alignment software, to identify genomic regions of conserved synteny over a large set of diverging species. CYNTENATOR does not depend on nucleotide-level alignments and a priori homology assignment. Our software implements an improved scoring function that utilizes the underlying phylogeny. In this manuscript, we report on our progressive gene order alignment approach, a and give a comparison to previous software and an analysis of 17 vertebrate genomes for conservation in gene order. CYNTENATOR has a runtime complexity of O(n 3) and a space complexity of O(n2) with n being the gene number in a genome. CYNTENATOR performs as good as state-of-the-art software on simulated pairwise gene order comparisons, but is the only algorithm that works in practice for aligning dozens of vertebrate-sized gene orders. Lineage-specific characterization of gene order across 17 vertebrate genomes revealed mechanisms for maintaining conserved synteny such as enhancers and coregulation by bidirectional promoters. Genes outside conserved synteny blocks show enrichments for genes involved in responses to external stimuli, stimuli such as immunity and olfactory response in primate genome comparisons. We even see significant gene ontology term enrichments for breakpoint regions of ancestral nodes close to the root of the phylogeny. Additionally, our analysis of transposable elements has revealed a significant accumulation of LINE-1 elements in mammalian breakpoint regions. In summary, CYNTENATOR is a flexible and scalable tool for the identification of conserved gene orders across multiple species over long evolutionary distances. © 2010 Rödelsperger.

News Article | December 18, 2015
Site: phys.org

Ohler, a computer scientist, focused on speech recognition programs during his studies. He made use of statistical procedures to filter out relevant information from the background "noise pollution" surrounding the data and thus identify words accurately. The mathematical methods used for this purpose, which include the Fourier transform, have been indispensable to modern data processing for some time. Astrophysicists investigating spectra in the light from distant stars or developers working on speech recognition for mobile phones face the same challenge: "noisy" signals need to be interpreted as accurately as possible. Now Ohler is applying these filtering methods to molecular biology. With his colleagues from the Berlin Institute for Medical Systems Biology (BIMSB) at the MDC he has developed and tested RiboTaper. The program filters the relevant information out of certain sequencing data to determine whether one of the cellular protein factories - ribosomes - is actually active on the RNA. RiboTaper is based on a laboratory procedure that was developed several years ago in the United States. It is called Ribo-seq and is used to identify the part of a gene that encodes a protein. This is important, as the theory that all genes encoded in the DNA contain a "construction manual" for a protein is not entirely accurate. Thousands of genes that have been mapped in the genome in recent years are indeed transcribed in the RNA, but it is not known whether they contain small, protein-coding sections. Overall, only a small part of the genome is responsible for producing proteins. The lion's share of the DNA has regulatory functions. Furthermore, from cell to cell, different genes are sometimes up and sometimes down regulated, or are shut down. How can we find out which genes in which cells actually produce protein and which do not? The answer can be found by looking at the ribosomes and the construction manual that they work from. Ribo-seq helps with this, because this wet lab procedure in effect "freezes" the ribosomes in their place on the RNA strand. The RNA is the construction manual transmitted from the genes. Everything except ribosome and the associated RNA are digested using biochemical tools. This allows the molecular biologists to determine which instructions the ribosomes are working with. The problem is that the data obtained with Ribo-seq is "noisy." There are tiny remnants of DNA, RNA and proteins that occur naturally and are dismantled in each cell. Furthermore, one never knows exactly whether the ribosomes are really active, and produce proteins, at the identified point on the RNAs, or whether they are, in effect, just waiting for another signal. The dry lab method RiboTaper should help to fill this information gap. It can be used to clarify the roles of DNA, RNA and ribosomes much more precisely. "We know, for example, that a specific ribosome usually covers some 29 RNA building blocks, or nucleotides," says Ohler. "And we also know that the ribosome moves along the RNA in intervals of three nucleotides." This creates a periodic pattern, which bioinformaticians can search for in all the data. "This then shows us the points on the RNA where something significant is happening," says Ohler. You can begin to imagine what this is like if you think of a kitchen that has been gutted by fire. Forensics investigate the kitchen and find recipe sheets, sugar, eggs and flour. But was the cake ready when the kitchen was on fire? Or were only the ingredients for the dough ready? What did the cook intend to bake? Using Ribo-seq in combination with RiboTaper, molecular biology forensics is much closer to finding out the secret of the cellular kitchen. Ohler explains: "With RiboTaper we can hunt down smaller proteins in previously poorly studied genes and help to clear up conflicting data interpretations." Ohler also sees another advantage: "Sequencing devices are now available in many laboratories, but only a few centers also have access to a good mass spectrometer. With RiboTaper we can draw conclusions about which transcripts are actively translated into proteins." To test the new procedure, Ohler put his samples to the test and had the RiboTaper data checked by his MDC colleague Matthias Selbach, using mass spectrometry. Since a number of groups at the MDC are already using Ribo-seq, RiboTaper may be able to assist them in their interpretation in exciting new ways. Ohler's laboratory collaborated with colleagues from the BIMSB on this study, including research groups led by Markus Landthaler, Benedikt Obermayer and Matthias Selbach. More information: Lorenzo Calviello et al. Detecting actively translated open reading frames in ribosome profiling data, Nature Methods (2015). DOI: 10.1038/nmeth.3688

Rembold M.,University of Cologne | Ciglar L.,Genome Biology Unit | Omar Yanez-Cuna J.,Research Institute of Molecular Pathology IMP | Zinzen R.P.,Genome Biology Unit | And 8 more authors.
Genes and Development | Year: 2014

The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila ~25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. Here we demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, we identified 106 genes that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of "Snail-activated" genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twistmediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, we show that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which we demonstrate is essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development. © 2014 Rembold et al.

Pham T.-H.,University of Regensburg | Benner C.,University of California at San Diego | Lichtinger M.,University of Regensburg | Lichtinger M.,University of Birmingham | And 5 more authors.
Blood | Year: 2012

Cellular differentiation is orchestrated by lineage-specific transcription factors and associated with cell type-specific epigenetic signatures. In the present study, we used stage-specific, epigenetic "fingerprints" to deduce key transcriptional regulators of the human monocytic differentiation process. We globally mapped the distribution of epigenetic enhancer marks (histone H3 lysine 4 monomethylation, histone H3 lysine 27 acetylation, and the histone variant H2AZ), describe general properties of marked regions, and show that cell type-specific epigenetic "fingerprints" are correlated with specific, de novo-derived motif signatures at all of the differentiation stages studied (ie, hematopoietic stem cells, monocytes, and macrophages). We validated the novel, de novo-derived, macrophage-specific enhancer signature, which included ETS, CEBP, bZIP, EGR, E-Box and NF-κB motifs, by ChIP sequencing for a subset of motif corresponding transcription factors (PU.1, C/EBPβ, and EGR2), confirming their association with differentiation-associated epigenetic changes. We describe herein the dynamic enhancer landscape of human macrophage differentiation, highlight the power of genome-wide epigenetic profiling studies to reveal novel functional insights, and provide a unique resource for macrophage biologists. © 2012 by The American Society of Hematology.

Marguerat S.,University College London | Schmidt A.,University of Basel | Codlin S.,University College London | Chen W.,Berlin Institute for Medical Systems Biology | And 3 more authors.
Cell | Year: 2012

Data on absolute molecule numbers will empower the modeling, understanding, and comparison of cellular functions and biological systems. We quantified transcriptomes and proteomes in fission yeast during cellular proliferation and quiescence. This rich resource provides the first comprehensive reference for all RNA and most protein concentrations in a eukaryote under two key physiological conditions. The integrated data set supports quantitative biology and affords unique insights into cell regulation. Although mRNAs are typically expressed in a narrow range above 1 copy/cell, most long, noncoding RNAs, except for a distinct subset, are tightly repressed below 1 copy/cell. Cell-cycle-regulated transcription tunes mRNA numbers to phase-specific requirements but can also bring about more switch-like expression. Proteins greatly exceed mRNAs in abundance and dynamic range, and concentrations are regulated to functional demands. Upon transition to quiescence, the proteome changes substantially, but, in stark contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume. © 2012 Elsevier Inc.

Karanam K.,Harvard University | Kafri R.,Harvard University | Loewer A.,Harvard University | Loewer A.,Berlin Institute for Medical Systems Biology | Lahav G.,Harvard University
Molecular Cell | Year: 2012

DNA double-strand breaks are repaired by two main pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). The choice between these pathways depends on cell-cycle phase; however the continuous effect of cell cycle on the balance between them is still unclear. We used live cell imaging and fluorescent reporters for 53BP1, Rad52, and cell cycle to quantify the relative contribution of NHEJ and HR at different points of the cell cycle in single cells. We found that NHEJ is the dominant repair pathway in G1 and G2 even when both repair pathways are functional. The shift from NHEJ to HR is gradual, with the highest proportion of breaks repaired by HR in mid S, where the amount of DNA replication is highest. Higher proportions of HR also strongly correlate with slower rates of repair. Our study shows that the choice of repair mechanism is continuously adjusted throughout the cell cycle and suggests that the extent of active replication, rather than the presence of a sister chromatid influences the balance between the two repair pathways in human cells. © 2012 Elsevier Inc.

Munger S.C.,Duke University | Munger S.C.,The Jackson Laboratory | Natarajan A.,Duke University | Looger L.L.,Howard Hughes Medical Institute | And 3 more authors.
PLoS Genetics | Year: 2013

In vertebrates, primary sex determination refers to the decision within a bipotential organ precursor to differentiate as a testis or ovary. Bifurcation of organ fate begins between embryonic day (E) 11.0-E12.0 in mice and likely involves a dynamic transcription network that is poorly understood. To elucidate the first steps of sexual fate specification, we profiled the XX and XY gonad transcriptomes at fine granularity during this period and resolved cascades of gene activation and repression. C57BL/6J (B6) XY gonads showed a consistent ∼5-hour delay in the activation of most male pathway genes and repression of female pathway genes relative to 129S1/SvImJ, which likely explains the sensitivity of the B6 strain to male-to-female sex reversal. Using this fine time course data, we predicted novel regulatory genes underlying expression QTLs (eQTLs) mapped in a previous study. To test predictions, we developed an in vitro gonad primary cell assay and optimized a lentivirus-based shRNA delivery method to silence candidate genes and quantify effects on putative targets. We provide strong evidence that Lmo4 (Lim-domain only 4) is a novel regulator of sex determination upstream of SF1 (Nr5a1), Sox9, Fgf9, and Col9a3. This approach can be readily applied to identify regulatory interactions in other systems. © 2013 Munger et al.

Li N.,TU Berlin | You X.,TU Berlin | Chen T.,TU Berlin | Mackowiak S.D.,Berlin Institute for Medical Systems Biology | And 8 more authors.
Nucleic Acids Research | Year: 2013

MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5′ tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences. © The Author(s) 2013. Published by Oxford University Press.

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