Chromatin Insulator Function and Nuclear Organization

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Abstract

Cell based assays for insulator body localization are currently being utilized and/or developed to answer basic mechanistic questions. Localization of insulator bodies can be determined by standard indirect immunofluorescence using antibodies specific for gypsy insulator proteins. In addition, eight independent stable cell lines that express a fluorescent insulator protein have been produced and clonally selected. Prior to immunofluorescence or monitoring of GFP signal, cells are first subjected to dsRNA knockdown for transcripts of interest to determine whether the proteins they encode are required for maintenance of insulator bodies. Candidates of interest are factors involved in RNA silencing, which themselves are subject to degradation using this method, as well as novel factors found to copurify with insulator proteins.[unreadable] [unreadable] As a complimentary assay to insulator body localization, we are utilizing transcriptional reporter cell lines under the control of two distinct insulator sequences. Cell lines harboring either gypsy or Fab-8 insulator sequence in between the OpIE2 enhancer and a minimal promoter driving the enhanced green fluorescent protein gene (EGFP) are examined by fluorescence activated cell sorting (FACS) following dsRNA knockdown of transcripts of interest (Ciavatta et al., 2007). Unlike immunofluorescence, FACS analysis is quantitative and indicates the full range of insulator activities in the cell population.[unreadable] [unreadable] Stably transfected cells expressing a GFP-tagged insulator protein can be used to identify novel factors required for insulator body integrity using high throughput genome wide dsRNA screening. With the assistance of the Drosophila RNAi Screening Center at Harvard Medical School, a library of 21,000 dsRNAs corresponding to all annotated Drosophila genes will be applied to the cells using automated liquid handling. Automated imaging will be utilized to identify RNAs that no longer support GFP reporter localization to 20-25 nuclear foci indicating disruption of insulator body formation. This screen should identify factors required specifically for proper insulator localization as well as general nuclear organization.