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.
National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Sigal
S
Shachar
High Throughput Imaging Facility (HiTIF), National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Ty C
TC
Voss
High Throughput Imaging Facility (HiTIF), National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Gianluca
G
Pegoraro
National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Nicholas
N
Sciascia
National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address: mistelit@mail.nih.gov.
Tom
T
Misteli
D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics*; D004261:DNA Replication; D005796:Genes; D005821:Genetic Techniques; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods