Rosetta Inpharmatics

Seattle, WA, United States

Rosetta Inpharmatics

Seattle, WA, United States
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Casellas J.,Autonomous University of Barcelona | Casellas J.,University of California at Davis | Gularte R.J.,Autonomous University of Barcelona | Gularte R.J.,University of California at Davis | And 8 more authors.
Genetics | Year: 2012

Transmission ratio distortion (TRD) is the departure from the expected genotypic frequencies under Mendelian inheritance. This departure can be due to multiple physiological mechanisms during gametogenesis, fertilization, fetal and embryonic development, and early neonatal life. Although a few TRD loci have been reported in mouse, inheritance patterns have never been evaluated for TRD. In this article, we developed a Bayesian binomial model accounting for additive and dominant deviation TRD mechanisms. Moreover, this model was used to perform genome-wide scans for TRD quantitative trait loci (QTL) on six F2 mouse crosses involving between 296 and 541 mice and between 72 and 1854 genetic markers. Statistical significance of each model was checked at each genetic marker with Bayes factors. Genome scans revealed overdominance TRD QTL located in mouse chromosomes 1, 2, 12, 13, and 14 and additive TRD QTL in mouse chromosomes 2, 3, and 15, although these results did not replicate across mouse crosses. This research contributes new statistical tools for the analysis of specific genetic patterns involved in TRD in F2 populations, our results suggesting a relevant incidence of TRD phenomena in mouse with important implications for both statistical analyses and biological research. © 2012 by the Genetics Society of America.


Stec E.,Merck And Co. | Locco L.,Merck And Co. | Szymanski S.,Merck And Co. | Bartz S.R.,RNA Therapeutics | And 9 more authors.
Journal of Biomolecular Screening | Year: 2012

Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR). © 2012 Society for Laboratory Automation and Screening.


Sietsema K.E.,University of California at Los Angeles | Meng F.,Merck And Co. | Yates N.A.,Merck And Co. | Hendrickson R.C.,Merck And Co. | And 5 more authors.
Biomarkers | Year: 2010

Proteomics was utilized to identify novel potential plasma biomarkers of exercise-induced muscle injury. Muscle injury was induced in nine human volunteers by eccentric upper extremity exercise. Liquid chromatographymass spectrometry identified 30 peptides derived from nine proteins which showed significant change in abundance post-exercise. Four of these proteins, haemoglobin α chain, haemoglobin β chain, α1-antichymotrypsin (ACT) and plasma C-1 protease inhibitor (C1 Inh), met the criterion for inclusion based on changes in at least two distinct peptides. ACT and C1 Inh peptides peaked earlier post-exercise than creatine kinase, and thus appear to provide new information on muscle response to injury. © 2010 Informa UK Ltd.


Wang M.L.,Fred Hutchinson Cancer Research Center | Wang M.L.,University of Washington | Walsh R.,Rosetta Inpharmatics | Robinson K.L.,Fred Hutchinson Cancer Research Center | And 6 more authors.
Cell Cycle | Year: 2011

Cancer cells exhibit the ability to proliferate indefinitely, but paradoxically, overexpression of cellular oncogenes in primary cells can result in a rapid and irreversible cell cycle arrest known as oncogene-induced senescence (OIS). However, we have shown that constitutive overexpression of the oncogene c-MYC in primary human foreskin fibroblasts results in a population of cells with unlimited lifespan; these immortalized cells are henceforth referred to as iMYC. Here, in order to further elucidate the mechanisms underlying the immortalization process, a gene expression signature of 3 independently established iMYC cell lines compared with matched early passage c-MYC overexpressing cells was derived. Network analysis of this "iMYC signature" indicated that a large fraction of the downregulated genes were functionally connected and major nodes centered around the TGFβ, IL-6 and IGF-1 signaling pathways. Here, we focused on the functional validation of the alteration of TGFβ response during c-MYC-mediated immortalization. The results demonstrate loss of sensitivity of iMYC cells to activation of TGFβ signaling upon ligand addition. Furthermore, we show that aberrant regulation of the p27 tumor suppressor protein in iMYC cells is a key event that contributes to loss of response to TGFβ. These findings highlight the potential to reveal key pathways contributing to the self-renewal of cancer cells through functional mining of the unique gene expression signature of cells immortalized by c-MYC. © 2011 Landes Bioscience.


Dews M.,Children's Hospital of Philadelphia | Fox J.L.,Children's Hospital of Philadelphia | Fox J.L.,University of Pennsylvania | Hultine S.,Children's Hospital of Philadelphia | And 16 more authors.
Cancer Research | Year: 2010

c-Myc stimulates angiogenesis in tumors through mechanisms that remain incompletely understood. Recent work indicates that c-Myc upregulates the miR-17∼92 microRNA cluster and downregulates the angiogenesis inhibitor thrombospondin-1, along with other members of the thrombospondin type 1 repeat superfamily. Here, we show that downregulation of the thrombospondin type 1 repeat protein clusterin in cells overexpressing c-Myc and miR-17∼92 promotes angiogenesis and tumor growth. However, clusterin downregulation by miR-17∼92 is indirect. It occurs as a result of reduced transforming growth factor-β (TGFβ) signaling caused by targeting of several regulatory components in this signaling pathway. Specifically, miR-17-5p and miR-20 reduce the expression of the type II TGFβ receptor and miR-18 limits the expression of Smad4. Supporting these results, in human cancer cell lines, levels of the miR-17∼92 primary transcript MIR17HG negatively correlate with those of many TGFβ-induced genes that are not direct targets of miR-17∼92 (e.g., clusterin and angiopoietin-like 4). Furthermore, enforced expression of miR-17∼92 in MIR17HGlow cell lines (e.g., glioblastoma) results in impaired gene activation by TGFβ. Together, our results define a pathway in which c-Myc activation of miR-17∼92 attenuates the TGFβ signaling pathway to shut down clusterin expression, thereby stimulating angiogenesis and tumor cell growth. ©2010 AACR.


Wang C.-Y.,University of Wisconsin - Madison | Stapleton D.S.,University of Wisconsin - Madison | Schueler K.L.,University of Wisconsin - Madison | Rabaglia M.E.,University of Wisconsin - Madison | And 7 more authors.
Journal of Lipid Research | Year: 2012

Nonalchoholic fatty liver disease (NAFLD) is the most common cause of liver dysfunction and is associated with metabolic diseases, including obesity, insulin resistance, and type 2 diabetes. We mapped a quantitative trait locus (QTL) for NAFLD to chromosome 17 in a cross between C57BL/6 (B6) and BTBR mouse strains made genetically obese with the Lepob/ob mutation. We identified Tsc2 as a gene underlying the chromosome 17 NAFLD QTL. Tsc2 functions as an inhibitor of mammalian target of rapamycin, which is involved in many physiological processes, including cell growth, proliferation, and metabolism. We found that Tsc2+/- mice have increased lipogenic gene expression in the liver in an insulin-dependent manner. The coding single nucleotide polymorphism between the B6 and BTBR strains leads to a change in the ability to inhibit the expression of lipogenic genes and de novo lipogenesis in AML12 cells and to promote the proliferation of Ins1 cells. This difference is due to a different affinity of binding to Tsc1, which affects the stability of Tsc2. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.


Kuppers D.A.,Fred Hutchinson Cancer Research Center | Hwang H.C.,Fred Hutchinson Cancer Research Center | Hwang H.C.,PhenoPath Laboratories | Jackson A.L.,Rosetta Inpharmatics | And 4 more authors.
PLoS ONE | Year: 2011

Mice lacking the p27Kip1 Cdk inhibitor (Cdkn1b) exhibit increased susceptibility to lymphomas from the Maloney murine leukemia virus (M-MuLV), and exhibit a high frequency of viral integrations at Xpcl1 (Kis2), a locus on the X-chromosome. Xpcl1 encodes miR-106a~363, a cluster of microRNAs that are expressed in response to adjacent retroviral integrations. We report the first large-scale profile of microRNA expression in MuLV-induced lymphomas, in combination with microarray gene expression analysis. The source material was T-cell lymphomas induced by M-MuLV in p27Kip1 knockout mice and normal thymus. Surprisingly, the overall levels of miRNA expression were equivalent in lymphomas and normal thymus. Nonetheless, the expression of specific microRNAs was altered in tumors. The miR-106a~363 miRNA were over-expressed in lymphomas, particularly those with viral integrations at the Xpcl1 locus. In contrast, p27Kip1 deletion itself was associated with a different pattern of microRNA expression. Gene expression was dramatically altered in lymphomas, yet paralleled data from T-cell lymphomas induced by other mechanisms. Genes with altered expression in association with the p27Kip1 null genotype were of similar functional classes to those associated with Xpcl1 integration, but with the opposite pattern of expression. Thus, the effect of p27Kip1 deletion may be to oppose an anti-oncogenic effect of Xpcl1 rather than enhancing its oncogenic functions. A subset of miR-106a~363 target genes was consistently reduced in lymphomas with Xpcl1 integrations, particularly genes with cell cycle and immune functions. We identify four predicted target genes of miR-106a~363 miRNA, including N-Myc (Mycn), and the TGF-beta receptor (Tgfbr2) using 3'UTR reporter assays. Still, bioinformatic miRNA target predictions were poor predictors of altered gene expression in lymphomas with Xpcl1 integration. Confirmation of miR-106a~363 gene targeting relevant to the tumor phenotype requires in vivo validation, because only a subset of predicted targets are consistently reduced in tumors that overexpress miR-106a~363. © 2011 Kuppers et al.


Sotillo E.,Children's Hospital of Philadelphia | Laver T.,University of Pennsylvania | Mellert H.,University of Pennsylvania | Schelter J.M.,Rosetta Inpharmatics | And 7 more authors.
Oncogene | Year: 2011

Downregulation of microRNA-34a by Myc is known to be essential for tumorigenesis and improve tumor-cell survival. Conversely, upregulation of miR-34a by p53 is thought to enhance its acetylation and activity and contribute to the pro-apoptotic effects of this tumor suppressor. We sought to determine whether restoration of miR-34a levels in B-lymphoid cells with Myc overexpression would aid therapeutic apoptosis. Unexpectedly, delivery of miR-34a, which doesn't target p53 directly, severely compromised steady-state p53 levels. This effect was preceded and mediated by direct targeting of Myc, which sustained p53 protein levels via the Arf-Hdm2 pathway. As a result, in the presence of Myc, miR-34a inhibited p53-dependent bortezomib-induced apoptosis as efficiently as anti-p53 small interfering RNA. Conversely, inhibition of miR-34a using antisense RNA sensitized lymphoma cells to therapeutic apoptosis. Thus, in tumors with deregulated Myc expression, miR-34a confers drug resistance and could be considered a therapeutic target. © 2011 Macmillan Publishers Limited All rights reserved.


Fraser H.B.,Rosetta Inpharmatics | Fraser H.B.,Stanford University | Babak T.,Rosetta Inpharmatics | Babak T.,Merck And Co. | And 7 more authors.
PLoS Genetics | Year: 2011

The idea that most morphological adaptations can be attributed to changes in the cis-regulation of gene expression levels has been gaining increasing acceptance, despite the fact that only a handful of such cases have so far been demonstrated. Moreover, because each of these cases involves only one gene, we lack any understanding of how natural selection may act on cis-regulation across entire pathways or networks. Here we apply a genome-wide test for selection on cis-regulation to two subspecies of the mouse Mus musculus. We find evidence for lineage-specific selection at over 100 genes involved in diverse processes such as growth, locomotion, and memory. These gene sets implicate candidate genes that are supported by both quantitative trait loci and a validated causality-testing framework, and they predict a number of phenotypic differences, which we confirm in all four cases tested. Our results suggest that gene expression adaptation is widespread and that these adaptations can be highly polygenic, involving cis-regulatory changes at numerous functionally related genes. These coordinated adaptations may contribute to divergence in a wide range of morphological, physiological, and behavioral phenotypes. © 2011 Fraser et al.


Yang R.,Merck And Co. | Castriota G.,Merck And Co. | Chen Y.,Merck And Co. | Cleary M.A.,Rosetta Inpharmatics | And 10 more authors.
International Journal of Obesity | Year: 2011

Objective:To investigate the impact of reduced adipocyte fatty acid-binding protein 4 (FABP4) in control of body weight, glucose and lipid homeostasis in diet-induced obese (DIO) mice.Methods:We applied RNA interference (RNAi) technology to generate FABP4 germline knockdown mice to investigate their metabolic phenotype.Results:RNAi-mediated knockdown reduced FABP4 mRNA expression and protein levels by almost 90% in adipocytes of standard chow-fed mice. In adipocytes of DIO mice, RNAi reduced FABP4 expression and protein levels by 70 and 80%, respectively. There was no increase in adipocyte FABP5 expression in FABP4 knockdown mice. The knockdown of FABP4 significantly increased body weight and fat mass in DIO mice. However, FABP4 knockdown did not affect plasma glucose and lipid homeostasis in DIO mice; nor did it improve their insulin sensitivity.Conclusion:Our data indicate that robust knockdown of FABP4 increases body weight and fat mass without improving glucose and lipid homeostasis in DIO mice. © 2011 Macmillan Publishers Limited. All rights reserved.

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