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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. Source

Rodelsperger C.,Charite - 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. Source

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. Source

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. Source

News Article
Site: http://phys.org/biology-news/

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

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