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Genomic Analysis of Glucocorticoid-regulated Promoters in Murine T-lymphoma Cells

Lu Chen^*,, Celeste Finnerty^*, William C. Gustafson^*,, Craig R. Bush, Ping Chi^*, Huiping Guo^*, Bruce Luxon^,, Alan P. Fields^*,|| and E. Aubrey Thompson^*,

^* Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555
Bioinformatics Program, University of Texas Medical Branch, Galveston, Texas 77555
^|| Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555

We have undertaken a high-throughput analysis to identify targets of glucocorticoid regulation in P1798 murine T-lymphoma cells. G1/S-arrested cultures were treated for 8 hours with 0.1 µM dexamethasone (dex) in the presence and absence of 1 µg/ml cycloheximide. Untreated cultures and cultures exposed to cycloheximide alone were prepared as controls. RNA was isolated and gene expression analyzed using Affymetrix MG-U74A oligonucleotide arrays (Gene Chips®). Three independent experiments were performed. The data were analyzed using a variety of statistical and analytical approaches in order to identify primary transcriptional targets of the glucocorticoid receptor. We identified 44 genes that increase by > 2-fold in both dex-treated and dex + cycloheximide-treated cultures (relative to control and cycloheximide-treated cultures) in three replicate experiments. Statistical analysis of control data indicate that the probability that a given probeset would, as a result of random error, increase > 2-fold both in the presence and absence of cycloheximide in two independent experiments is approximately 7 x 10^-9. We have retrieved from the Celera mouse genomic sequence 8 kb of promoter sequence, spanning 4 kb either side of the 5'-end of the cDNA from eight of the induced genes. These sequences were analyzed for potential glucocorticoid receptor binding sites. Five of these genes contain the sequence ACAnnnTGTnCT within 4 kb of the presumptive transcriptional start site. Eight control genes were selected at random and analyzed for the sequence ACAnnnTGTnCT. Two control genes had such sequences within 4 kb of the transcriptional start site.

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