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Imaging Gene Expression

Volume 1042 of the series Methods in Molecular Biology pp 3-12

Date:

High-Throughput Fluorescence-Based Screen to Identify Factors Involved in Nuclear Receptor Recruitment to Response Elements

  • Tina B. MirandaAffiliated withLaboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health
  • , Ty C. VossAffiliated withLaboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health
  • , Gordon L. HagerAffiliated withLaboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health

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Abstract

The glucocorticoid receptor is an inducible transcription factor which plays important roles in many ­physiological processes. Upon activation, GR interacts with regulatory elements and modulates the expression of genes. Although GR is widely expressed in multiple tissues, its binding sites within chromatin and the genes it regulates are tissue specific. Many accessory proteins and cofactors are thought to play a role in dictating GR’s function; however, mechanisms involved in targeting GR to specific sites in the genome are not well understood. Here we describe a high-throughput fluorescence-based method to identify factors involved in GR loading at response elements. This screen utilizes a genetically engineered cell line that contains 200 repeats of a glucocorticoid response promoter and expresses GFP-tagged GR. Upon treatment with corticosteroids, GFP–GR forms a steady-state distribution at the promoter array, and its concentration at this focal point can be quantitatively determined. This system provides a novel approach to identify activities important for GR loading at its response element using siRNA libraries to target factors that enhance or inhibit receptor localization.

Key words

Nuclear receptor Glucocorticoid receptor siRNA screen Chromatin