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Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-β pathway and reveal microRNA regulation of TGFBR2

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Silence 2011年第1期2卷 1-20页

作者： Schultz, Nikolaus Marenstein, Dina R. De Angelis, Dino A. Wang, Wei-Qing Nelander, Sven Jacobsen, Anders Marks, Debora S. Massagué, Joan Sander, Chris Computational Biology Program Memorial Sloan-Kettering Cancer Center New York NY United States Cancer Biology and Genetics Program Memorial Sloan-Kettering Cancer Center New York NY United States High-Throughput Screening Core Facility Memorial Sloan-Kettering Cancer Center New York NY United States Cancer Center Sahlgrenska University of Gothenburg Gothenburg Sweden Department of Systems Biology Harvard Medical School Boston MA United States

Background: RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in ***: We assayed how strongly single siRNAs targeting each of 6,000 genes affect the nuclear translocation of a green fluorescent protein (GFP)-SMAD2 reporter fusion protein. Surprisingly, we found no novel TGF-β pathway members, but we did find dominant off-target effects. All siRNA hits, whatever their intended direct target, reduced the mRNA levels of two known upstream pathway components, the TGF-β receptors 1 and 2 (TGFBR1 and TGFBR2), via micro (mi)RNA-like off-target effects. The scale of these off-target effects was remarkable, with at least 1% of the sequences in the unbiased siRNA library having measurable off-target effects on one of these two genes. It seems that relatively minor reductions of message levels via off-target effects can have dominant effects on an assay, if the pathway output is very dose-sensitive to levels of particular pathway components. In search of mechanistic details, we identified multiple miRNA-like sequence characteristics that correlated with the off-target effects. Based on these results, we identified miR-20a, miR-34a and miR-373 as miRNAs that inhibit TGFBR2 ***: Our findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-β receptor is regulated by multiple miRNAs. We also conclude that the risk of obtaining misleading results in siRNA screens using large libraries with single-assay readout is substantial. Control and rescue experiments are essential in the interpretation of such screens, and improvements to t

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Author Correction: Progressive plasticity during colorectal cancer metastasis

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Nature 2025年第8047期637卷 E28页

作者： Andrew Moorman Elizabeth K Benitez Francesco Cambuli Qingwen Jiang Ahmed Mahmoud Melissa Lumish Saskia Hartner Sasha Balkaran Jonathan Bermeo Simran Asawa Canan Firat Asha Saxena Fan Wu Anisha Luthra Cassandra Burdziak Yubin Xie Valeria Sgambati Kathleen Luckett Yanyun Li Zhifan Yi Ignas Masilionis Kevin Soares Emmanouil Pappou Rona Yaeger T Peter Kingham William Jarnagin Philip B Paty Martin R Weiser Linas Mazutis Michael D'Angelica Jinru Shia Julio Garcia-Aguilar Tal Nawy Travis J Hollmann Ronan Chaligné Francisco Sanchez-Vega Roshan Sharma Dana Pe'er Karuna Ganesh Computational and Systems Biology Program Memorial Sloan Kettering Cancer Center New York NY USA. Molecular Pharmacology Program Memorial Sloan Kettering Cancer Center New York NY USA. Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program New York NY USA. New York Genome Center New York NY USA. Pharmacology Program Weill Cornell Graduate School New York NY USA. Department of Medicine Memorial Sloan Kettering Cancer Center New York NY USA. Case Western Reserve University Cleveland OH USA. Department of Pathology and Laboratory Medicine Memorial Sloan Kettering Cancer Center New York NY USA. Department of Surgery Memorial Sloan Kettering Cancer Center New York NY USA. Tri-Institutional PhD Program in Computational Biology and Medicine New York NY USA. Bristol Myers Squibb Princeton NJ USA. Computational and Systems Biology Program Memorial Sloan Kettering Cancer Center New York NY USA. peerd@***. Howard Hughes Medical Institute Chevy Chase MD USA. peerd@***. Molecular Pharmacology Program Memorial Sloan Kettering Cancer Center New York NY USA. ganeshk@***. Department of Medicine Memorial Sloan Kettering Cancer Center New York NY USA. ganeshk@***.

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Fluorescent imaging for in situ measurement of drug target engagement and cell signaling pathways

Fluorescent imaging for in situ measurement of drug target e...

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Microscopy Histopathology and Analytics, Microscopy 2020

作者： McMahon, Nathan Solanki, Allison Jones, Jocelyn Kwon, Sunjong Chang, Young-Hwan Chin, Koei Nederlof, Michel Gray, Joe Gibbs, Summer L. Department of Biomedical Engineering Computational Biology Program Oregon Health and Science University PortlandOR97201 United States Knight Cancer Institute Computational Biology Program Oregon Health and Science University PortlandOR97201 United States Center for Spatial Systems Biomedicine Computational Biology Program Oregon Health and Science University PortlandOR97201 United States Quantitative Imaging Systems PittsburghPA15238 United States

Successful targeted cancer treatment continues to elude modern medicine and its many therapeutic strategies. Herein, we propose a fluorescent imaging technique to measure drug localization and subsequent cell signalin... 详细信息

ISBN: (纸本)9781943580743

关键词： Cell signaling

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Array of micromechanical mass sensors enables high-throughput single-cell growth-rate measurements

Array of micromechanical mass sensors enables high-throughpu...

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2016 Solid-State Sensors, Actuators and Microsystems Workshop, Hilton Head 2016

作者： Olcum, Selim Cermak, Nathan Manalis, Scott R. Koch Institute for Integrative Cancer Research United States Program in Computational and Systems Biology Massachusetts Institute of Technology CambridgeMA02139 United States

ISBN: (纸本)9781940470023

Technologies that rapidly assess cell growth would be transformative for many applications, including drug susceptibility testing for pathogens and cancer cells. Resonant micromechanical mass sensors can precisely measure growth, but are limited to measuring one cell at a time. Here, we demonstrate an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with multiple resonant mass sensors distributed along its length, weighing each cell repeatedly over the small fraction of a cell cycle it spends traversing this channel. Because multiple cells traverse this channel at the same time, we obtain growth rates up to 60 cells/h, while retaining a resolution of 0.2 pg/h for mammalian cells. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessing cellular responses to antibiotics or antimicrobial peptides within minutes. © 2016 TRF.

关键词： Growth kinetics

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Interleukin-10 production by innate lymphoid cells restricts intestinal inflammation in mice

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Mucosal Immunology 2025年

作者： Li, Jing Jacobse, Justin Pilat, Jennifer M. Kaur, Harsimran Gu, Weihong Kang, Seung Woo Rusznak, Mark Huang, Hsin-I Barrera, Julio Oloo, Pauline A. Roland, Joseph T. Hawkins, Caroline V. Pahnke, Andrew P. Khalil, Marian Washington, M.Kay Wilson, Keith T. Williams, Christopher S. Peebles, R.Stokes Konnikova, Liza Choksi, Yash A. Hammer, Gianna Elena Lau, Ken S. Goettel, Jeremy A. Department of Pathology Microbiology and Immunology Vanderbilt University Medical Center Nashville TN United States Department of Medicine Division of Gastroenterology Hepatology and Nutrition Vanderbilt University Medical Center Nashville TN United States Willem-Alexander Children's Hospital Department of Pediatrics Leiden University Medical Center Leiden Netherlands Veterans Affairs Tennessee Valley Healthcare System Nashville 37212 TN United States Program in Cancer Biology Vanderbilt University School of Medicine Nashville TN United States Program in Chemical and Physical Biology Vanderbilt University School of Medicine Nashville TN United States Department of Pediatrics Yale Medical School New Haven 06520 CT United States Department of Cell and Developmental Biology Vanderbilt University School of Medicine Nashville TN United States Department of Internal Medicine Vanderbilt University Medical Center Nashville TN United States Department of Pathology Division of Microbiology and Immunology University of Utah Salt Lake City 84112 UT United States Department of Surgery Vanderbilt University Medical Center Nashville TN United States Epithelial Biology Center Vanderbilt University Medical Center Nashville TN United States Center for Mucosal Inflammation and Cancer Vanderbilt University Medical Center Nashville TN United States Department of Immunobiology Yale School of Medicine New Haven 06520 CT United States Center for Systems and Engineering Immunology Yale School of Medicine New Haven 06520 CT United States Human and Translational Immunology Program Yale School of Medicine New Haven 06520 CT United States Program in Translational Biomedicine Yale School of Medicine New Haven 06520 CT United States Department of Obstetrics Gynecology and Reproductive Sciences Yale School of Medicine New Haven 06520 CT United States Center for Computational Systems Biology Vanderbilt University Nashville TN United States Vanderbilt Institut

Interleukin-10 (IL-10) is an immunomodulatory cytokine critical for intestinal immune homeostasis. IL-10 is produced by various immune cells but IL-10 receptor signaling in intestinal CX₃CR1⁺ mononuclear phagocytes is necessary to prevent spontaneous colitis in mice. Here, we utilized fluorescent protein reporters and cell-specific targeting and found that Rorc-expressing innate lymphoid cells (ILCs) produce IL-10 in response to anti-CD40-mediated intestinal inflammation. Deletion of Il10 specifically in Rorc-expressing ILCs led to phenotypic changes in intestinal macrophages and exacerbated both innate and adaptive immune-mediated models of experimental colitis. The population of IL-10⁺ producing ILCs shared markers with both ILC2 and ILC3 with nearly all ILC3s being of the NCR⁺ subtype. Interestingly, Ccl26 was enriched in IL-10⁺ ILCs and was markedly reduced in IL-10-deficient ILC3s. Since CCL26 is a ligand for CX₃CR1, we employed RNA in situ hybridization and observed increased numbers of ILCs in close proximity to Cx3cr1-expressing cells under inflammatory conditions. Finally, we generated transgenic Rorc^tdTomato reporter mice that faithfully marked RORγt⁺ cells that could rescue disease pathology and aberrant macrophage phenotype following adoptive transfer into mice with selective Il10 deficiency in ILC3s. These results demonstrate that IL-10 production by a population of ILCs functions to promote immune homeostasis in the intestine possibly via direct effects on intestinal macrophages. © 2025 The Author(s)

关键词： CCL26 CX3CR1 IBD IL-10 ILC3

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Worm Perturb-Seq: massively parallel whole-animal RNAi and RNA-seq

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Nature Communications 2025年第1期16卷 1-21页

作者： Hefei Zhang Xuhang Li Shivani Nanda Albertha J. M. Walhout Dongyuan Song Jingyi Jessica Li Onur Yukselen Alper Kucukural Manuel Garber Department of Systems Biology University of Massachusetts Chan Medical School Worcester MA USA Bioinformatics Interdepartmental Ph.D. Program University of California Los Angeles CA USA Department of Statistics and Data Science Department of Biostatistics Department of Computational Medicine and Department of Human Genetics University of California Los Angeles CA USA Via Scientific Inc. Cambridge MA USA Department of Genomics and Computational Biology University of Massachusetts Chan Medical School Worcester MA USA

Transcriptomes provide highly informative molecular phenotypes that, combined with gene perturbation, can connect genotype to phenotype. An ultimate goal is to perturb every gene and measure transcriptome changes, however, this is challenging, especially in whole animals. Here, we present ‘Worm Perturb-Seq (WPS)’, a method that provides high-resolution RNA-sequencing profiles for hundreds of replicate perturbations at a time in living animals. WPS introduces multiple experimental advances combining strengths of Caenhorhabditis elegans genetics and multiplexed RNA-sequencing with a novel analytical framework, EmpirDE. EmpirDE leverages the unique power of large transcriptomic datasets and improves statistical rigor by using gene-specific empirical null distributions to identify DEGs. We apply WPS to 103 nuclear hormone receptors (NHRs) and find a striking ‘pairwise modularity’ in which pairs of NHRs regulate shared target genes. We envision the advances of WPS to be useful not only for C. elegans, but broadly for other models, including human cells.

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Multiplex gene editing models of del(7q) reveal combined CUX1 and EZH2 loss drives clonal expansion and drug resistance

Blood Neoplasia

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Blood Neoplasia 2025年第2期2卷 100083-100083页

作者： Matthew R. M. Jotte Angela Stoddart Tanner C. Martinez Raven Moten Yuqing Xue Molly K. Imgruet Hunter Blaylock Henna S. Nam Bonnie Hu Jermaine Austin Ningfei An Saira Khan Sandeep K. Gurbuxani Megan E. McNerney Interdisciplinary Scientist Training Program The University of Chicago Medicine Chicago IL Department of Pathology The University of Chicago Medicine Chicago IL Committee on Cancer Biology The University of Chicago Medicine Chicago IL Department of Pediatrics The University of Chicago Medicine Chicago IL Committee on Immunology The University of Chicago Medicine Chicago IL Committee on Genetics Genomics and Systems Biology The University of Chicago Medicine Chicago IL The University of Chicago Medicine Comprehensive Cancer Center The University of Chicago Medicine Chicago IL

Abstract Loss of all or part of chromosome 7 [−7/del(7q)] is recurrent in myeloid neoplasms and associated with a poor response to chemotherapy. Chromosome 7–encoded genes driving drug resistance and the consequences of combinatorial 7q tumor suppressor gene loss have remained unclear, the latter question largely because of the challenges of modeling aneuploidy. Here, we use in silico data mining to uncover 7q genes involved in chemotherapy resistance. We establish murine models of del(7q) clonal hematopoiesis and drug resistance with multiplex CRISPR-Cas9 (CRISPR–associated protein 9)–mediated inactivation of 4 genes, Cux1 , Ezh2 , Kmt2c , and Kmt2e . Postgenotoxic exposure, combined deficiency of Cux1 and Ezh2 preferentially promotes clonal myeloid expansion in vivo, with compounding defects in DNA damage recognition and repair. Human acute myeloid leukemia cell lines similarly illustrate central roles for CUX1 and EZH2 loss in survival and DNA damage resolution after chemotherapy exposure. Transcriptome analysis reveals combined Cux1 and Ezh2 loss recapitulates gene signatures of -7 patients and defective DNA damage response pathways, to a greater extent than single gene loss. This work reveals a genetic interaction between CUX1 and EZH2 , and sheds light on how −7/del(7q) contributes to leukemogenesis and drug resistance characteristic of these adverse-risk neoplasms. These data support the concept of 7q as a contiguous gene syndrome region, in which combined loss of multiple gene drives pathogenesis. Furthermore, our CRISPR-based approach may serve as a framework for interrogating other recurrent aneuploid events in cancer.

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A specific form of cPRC1 containing CBX4 is co-opted to mediate oncogenic gene repression in diffuse midline glioma

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Molecular Cell 2025年

作者： Eimear Lagan Dáire Gannon Ademar Jesus Silva Peter Bibawi Anthony M. Doherty Darragh Nimmo Rachel McCole Craig Monger Giovani Luiz Genesi Aurelie Vanderlinden James A. Innes Charlotte L.E. Jones Lu Yang Bryan Chen Guido van Mierlo Pascal W.T.C. Jansen Chinmayi Pednekar Alexander Von Kriegsheim Kieran Wynne Francisco J. Sánchez-Rivera Gerard L. Brien Smurfit Institute of Genetics Trinity College Dublin Dublin 2 Ireland Cancer Research UK Edinburgh Centre Institute of Genetics and Cancer University of Edinburgh Edinburgh UK MRC Human Genetics Unit Institute of Genetics and Cancer The University of Edinburgh Edinburgh UK Department of Neurology Perelman School of Medicine University of Pennsylvania Philadelphia PA 19104 USA Epigenetics Program Perelman School of Medicine Philadelphia PA 19104 USA Abramson Cancer Center Perelman School of Medicine Philadelphia PA 19104 USA Department of Systems Biology Beckman Research Institute City of Hope Duarte CA 91010 USA Department of Molecular Biology Faculty of Science Radboud Institute for Molecular Life Sciences Oncode Institute Radboud University Nijmegen 6525 GA Nijmegen the Netherlands Systems Biology Ireland University College Dublin Belfield Dublin 4 Ireland Koch Institute for Integrative Cancer Research Massachusetts Institute of Technology Boston MA USA Department of Biology Massachusetts Institute of Technology Cambridge MA USA

Diffuse midline glioma (DMG) is a fatal childhood brain tumor characterized primarily by mutant histone H3 (H3K27M). H3K27M causes a global reduction in Polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3). Paradoxically, PRC2 is essential in DMG cells, although the downstream molecular mechanisms are poorly understood. Here, we have discovered a specific form of canonical PRC1 (cPRC1) containing CBX4 and PCGF4 that drives oncogenic gene repression downstream of H3K27me3 in DMG cells. Via a novel functional region, CBX4 preferentially associates with PCGF4-containing cPRC1. The characteristic H3K27me3 landscape in DMG rewires the distribution of cPRC1 complexes, with CBX4/PCGF4-cPRC1 accumulating at H3K27me3-enriched CpG islands. Despite comprising <5% of cPRC1 in DMG cells, the unique repressive functions of CBX4/PCGF4-cPRC1 are essential for DMG growth. Our findings link the altered distribution of H3K27me3 to imbalanced cPRC1 function, which drives oncogenic gene repression in DMG, highlighting potential therapeutic opportunities for this incurable childhood brain cancer.

关键词： H3K27M diffuse midline glioma diffuse intrinic pontine glioma Polycomb PRC1 CBX4 PCGF4 H3K27me3 PRC2 EZH2

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Annotation of gene promoters by integrative data-mining of ChIP-seq Pol-II enrichment data

Annotation of gene promoters by integrative data-mining of C...

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8th Asia-Pacific Bioinformatics Conference, APBC 2010

作者： Gupta, Ravi Wikramasinghe, Priyankara Bhattacharyya, Anirban Perez, Francisco A. Pal, Sharmistha Davuluri, Ramana V. Center for Systems and Computational Biology Molecular and Cellular Oncogenesis Program Wistar Institute Philadelphia PA United States Graduate Group in Genomics and Computational Biology Department of Genetics University of Pennsylvania Philadelphia PA United States

Background: Use of alternative gene promoters that drive widespread cell-type, tissue-type or developmental gene regulation in mammalian genomes is a common phenomenon. Chromatin immunoprecipitation methods coupled with DNA microarray (ChIP-chip) or massive parallel sequencing (ChIP-seq) are enabling genome-wide identification of active promoters in different cellular conditions using antibodies against Pol-II. However, these methods produce enrichment not only near the gene promoters but also inside the genes and other genomic regions due to the non-specificity of the antibodies used in ChIP. Further, the use of these methods is limited by their high cost and strong dependence on cellular type and context. Methods: We trained and tested different state-of-art ensemble and meta classification methods for identification of Pol-II enriched promoter and Pol-II enriched non-promoter sequences, each of length 500 bp. The classification models were trained and tested on a bench-mark dataset, using a set of 39 different feature variables that are based on chromatin modification signatures and various DNA sequence features. The best performing model was applied on seven published ChIP-seq Pol-II datasets to provide genome wide annotation of mouse gene promoters. Results: We present a novel algorithm based on supervised learning methods to discriminate promoter associated Pol-II enrichment from enrichment elsewhere in the genome in ChIP-chip/seq profiles. We accumulated a dataset of 11,773 promoter and 46,167 non-promoter sequences, each of length 500 bp, generated from RNA Pol-II ChIP-seq data of five tissues (Brain, Kidney, Liver, Lung and Spleen). We evaluated the classification models in building the best predictor and found that Bagging and Random Forest based approaches give the best accuracy. We implemented the algorithm on seven different published ChIP-seq datasets to provide a comprehensive set of promoter annotations for both protein-coding and non-coding genes in

关键词： Mammals

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Examining Dynamic Emergent Properties of the DNA Sensing Pathway

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IFAC-PapersOnLine 2018年第19期51卷 112-113页

作者： Gregg, Robert W. Sarkar, Saumendra Shoemaker, Jason E. Department of Chemical and Petroleum Engineering University of Pittsburgh United States Cancer Virology Program University of Pittsburgh Cancer Institute United States Department of Microbiology and Molecular Genetics University of Pittsburgh School of Medicine United States McGowan Institute for Regenerative Medicine Pittsburgh PA United States Department of Computational and Systems Biology University of Pittsburgh United States

The DNA sensing pathway is a component of the innate immune system that recognizes DNA in the cytosol of cells. Its main purpose after DNA detection is to promote interferon production, which acts as a “warning signal” to neighboring cells and subsequently induces anti-viral and pro-inflammatory proteins. DNA that activates this pathway can originate from pathogens (herpes simplex virus, tuberculosis) or leakage from the nucleus (observed in cancer cells). Unfortunately, the regulatory factors underlying the DNA sensing pathway are poorly understood. Without a model describing the dynamics of the DNA sensing pathway, little progress can be made toward understanding its role with pathogens, cancer, and autoimmunity. To investigate the dynamics of the DNA sensing pathway, we constructed an ordinary differential equation (ODE) model parameterized from compiled literature data. With the established model, we performed several in silico knock-down experiments and observed sustained pathway activation without the presence of a negative feedback mechanism for DNA degradation and robustness to perturbations. This model provides a foundation to predict how pharmaceutical interventions could modulate the DNA sensing pathway to desired phenotypes. © 2018

关键词： DNA

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