Identification of Gene Positioning Factors Using High-Throughput Imaging Mapping

Genomes are arranged non-randomly in the 3D space of the cell nucleus. Here, we have developed HIPMap, a high-precision, high-throughput, automated fluorescent in situ hybridization imaging pipeline, for mapping of the spatial location of genome regions at large scale. High-throughput imaging position mapping (HIPMap) enabled an unbiased siRNA screen for factors involved in genome organization in human cells. We identify 50 cellular factors required for proper positioning of a set of functionally diverse genomic loci. Positioning factors include chromatin remodelers, histone modifiers, and nuclear envelope and pore proteins. Components of the replication and post-replication chromatin re-assembly machinery are prominently represented among positioning factors, and timely progression of cells through replication, but not mitosis, is required for correct gene positioning. Our results establish a method for the large-scale mapping of genome locations and have led to the identification of a compendium of cellular factors involved in spatial genome organization.

[1]  E. Bertolino,et al.  Transcriptional repression mediated by repositioning of genes to the nuclear lamina , 2008, Nature.

[2]  Dimos Gaidatzis,et al.  Step-Wise Methylation of Histone H3K9 Positions Heterochromatin at the Nuclear Periphery , 2012, Cell.

[3]  Timothy J Mitchison,et al.  Small‐Molecule Screening and Profiling by Using Automated Microscopy , 2005, Chembiochem : a European journal of chemical biology.

[4]  V. Corces,et al.  CTCF: Master Weaver of the Genome , 2009, Cell.

[5]  Roland Eils,et al.  Global Chromosome Positions Are Transmitted through Mitosis in Mammalian Cells , 2003, Cell.

[6]  W. D. Laat,et al.  A Decade of 3c Technologies: Insights into Nuclear Organization References , 2022 .

[7]  Lani F. Wu,et al.  Multidimensional Drug Profiling By Automated Microscopy , 2004, Science.

[8]  Ty C. Voss,et al.  DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes , 2012, Nature.

[9]  Robert S Illingworth,et al.  Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization , 2014, Genes & development.

[10]  Tom Misteli,et al.  Spatial genome organization in the formation of chromosomal translocations. , 2007, Seminars in cancer biology.

[11]  Job Dekker,et al.  Organization of the Mitotic Chromosome , 2013, Science.

[12]  Prabhakar R. Gudla,et al.  Disease-specific gene repositioning in breast cancer , 2009, The Journal of cell biology.

[13]  Andrew Smellie,et al.  Visualization and Interpretation of High Content Screening Data , 2006, J. Chem. Inf. Model..

[14]  Prabhakar R. Gudla,et al.  Automatic segmentation and supervised learning‐based selection of nuclei in cancer tissue images , 2012, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[15]  Wendy A Bickmore,et al.  Chromatin Motion Is Constrained by Association with Nuclear Compartments in Human Cells , 2002, Current Biology.

[16]  G. Coetzee,et al.  Biological Implications and Regulatory Mechanisms of Long-range Chromosomal Interactions , 2013, The Journal of Biological Chemistry.

[17]  Manolis Kellis,et al.  Constitutive nuclear lamina–genome interactions are highly conserved and associated with A/T-rich sequence , 2013, Genome research.

[18]  Thomas Cremer,et al.  Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages , 2003, The Journal of cell biology.

[19]  Helena Fidlerová,et al.  Chromatin position in human HepG2 cells: Although being non-random, significantly changed in daughter cells , 2009, Journal of structural biology.

[20]  L. Wessels,et al.  Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions , 2008, Nature.

[21]  Roy Riblet,et al.  Subnuclear Compartmentalization of Immunoglobulin Loci During Lymphocyte Development , 2002, Science.

[22]  Wouter Meuleman,et al.  Chromatin Position Effects Assayed by Thousands of Reporters Integrated in Parallel , 2013, Cell.

[23]  Hui Ling Chen,et al.  CTCF Mediates Interchromosomal Colocalization Between Igf2/H19 and Wsb1/Nf1 , 2006, Science.

[24]  Tom Misteli,et al.  Locus-specific and activity-independent gene repositioning during early tumorigenesis , 2008, The Journal of cell biology.

[25]  T. Misteli,et al.  Gene deregulation and spatial genome reorganization near breakpoints prior to formation of translocations in anaplastic large cell lymphoma , 2009, Proceedings of the National Academy of Sciences.

[26]  O. Cuvier,et al.  Roles of chromatin insulator proteins in higher-order chromatin organization and transcription regulation , 2011, Nucleus.

[27]  Michael R. Speicher,et al.  The new cytogenetics: blurring the boundaries with molecular biology , 2005, Nature Reviews Genetics.

[28]  Wendy A Bickmore,et al.  The spatial organization of the human genome. , 2013, Annual review of genomics and human genetics.

[29]  Robert S. Illingworth,et al.  Chromatin decondensation is sufficient to alter nuclear organization in embryonic stem cells , 2014, Science.

[30]  Jesse R. Dixon,et al.  Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions , 2012, Nature.

[31]  P. Flicek,et al.  Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation. , 2010, Molecular cell.

[32]  E. Cabuy,et al.  Primary laminopathy fibroblasts display altered genome organization and apoptosis , 2007, Aging cell.

[33]  Ian X. Y. Leung,et al.  Intra- and inter-chromosomal interactions correlate with CTCF binding genome wide , 2010, Molecular systems biology.

[34]  Aki Minoda,et al.  Double-Strand Breaks in Heterochromatin Move Outside of a Dynamic HP1a Domain to Complete Recombinational Repair , 2011, Cell.

[35]  Benjamin D. Pope,et al.  Space and time in the nucleus: developmental control of replication timing and chromosome architecture. , 2010, Cold Spring Harbor symposia on quantitative biology.

[36]  T. Misteli.  Beyond the Sequence: Cellular Organization of Genome Function , 2011 .

[37]  K. Sandhu,et al.  Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions , 2006, Nature Genetics.

[38]  Wendy A. Bickmore,et al.  The Radial Positioning of Chromatin Is Not Inherited through Mitosis but Is Established De Novo in Early G1 , 2004, Current Biology.

[39]  D. Spector,et al.  A genetic locus targeted to the nuclear periphery in living cells maintains its transcriptional competence , 2008, The Journal of cell biology.

[40]  T. Misteli,et al.  In vitro generation of human cells with cancer stem cell properties , 2011, Nature Cell Biology.

[41]  Michael S. Becker,et al.  Spatial Organization of the Mouse Genome and Its Role in Recurrent Chromosomal Translocations , 2012, Cell.

[42]  T. Xie,et al.  Identification of Genes That Promote or Antagonize Somatic Homolog Pairing Using a High-Throughput FISH–Based Screen , 2012, PLoS genetics.

[43]  T. Misteli,et al.  Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus , 2006, Journal of Cell Science.

[44]  Elizabeth Kerr,et al.  Recruitment to the Nuclear Periphery Can Alter Expression of Genes in Human Cells , 2008, PLoS genetics.

[45]  T. Misteli,et al.  Transformation resistance in a premature aging disorder identifies a tumor-protective function of BRD4. , 2014, Cell reports.

[46]  M. Fornerod,et al.  Characterization of the Drosophila melanogaster genome at the nuclear lamina , 2006, Nature Genetics.

[47]  Tom Misteli,et al.  The Meaning of Gene Positioning , 2008, Cell.

[48]  G. Schroth,et al.  Cohesin-mediated interactions organize chromosomal domain architecture , 2013, The EMBO journal.

[49]  A. Pombo,et al.  Gene positioning. , 2010, Cold Spring Harbor perspectives in biology.

[50]  A. Pombo,et al.  Intermingling of Chromosome Territories in Interphase Suggests Role in Translocations and Transcription-Dependent Associations , 2006, PLoS biology.

[51]  T. Misteli,et al.  The emerging role of nuclear architecture in DNA repair and genome maintenance , 2009, Nature Reviews Molecular Cell Biology.

[52]  Jesse R. Dixon,et al.  Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells , 2013, Proceedings of the National Academy of Sciences.

[53]  V. Sartorelli,et al.  The histone chaperone Spt6 coordinates histone H3K27 demethylation and myogenesis , 2013, The EMBO journal.

[54]  I. Amit,et al.  Comprehensive mapping of long range interactions reveals folding principles of the human genome , 2011 .

[55]  A. Fisher,et al.  Nuclear repositioning marks the selective exclusion of lineage‐inappropriate transcription factor loci during T helper cell differentiation , 2004, European journal of immunology.

[56]  Tom Misteli,et al.  Spatial proximity of translocation-prone gene loci in human lymphomas , 2003, Nature Genetics.