Identification of chromatin modifiers involved in the recruitment of the glucocorticoid receptor to response elements by a high-throughput fluorescence-based screen

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	NCI	EPIGEN/TRANS/CHROM-14

Authors

• T Miranda
• T Voss
• L Schiltz
• G Hager

Abstract

Recent studies have indicated that nucleoprotein structure plays an important role in directing binding of the glucocorticoid receptor to specific sites in chromatin. To better understand how chromatin modulating activities affect the ability of GR to bind to glucocorticoid response elements (GREs), we have conducted an image based high-throughput screen to identify chromatin modifiers important for GR loading at GREs. Direct GR binding to response elements in living cells can be visualized using a cell line containing 200 repeats of a hormone responsive promoter, and expressing GFP-GR. Upon treatment with dexamethasone these cells form a steady state distribution of GFP-GR at the promoter arrays, and this localized binding appears as a focal structure in the nucleus of hormone treated cells. We have developed computer algorithms that automatically detect these structures, and determine quantitatively the levels of GFP-GR concentrated at the amplified response elements. Multiple robotic systems have been used to reverse transfect these cells with a siRNA library targeting 300 different chromatin modifiers. Using this assay, we have identified multiple chromatin modifiers that affect array formation when suppressed with siRNAs from the chromatin library.

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