Genomic analysis of glucocorticoid-regulated promoters in murine T-lymphoma cells.
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 microM dexamethasone (dex) in the presence and absence of 1 microg/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.
Chen
Lu
L
Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Finnerty
Celeste
C
Gustafson
William C
WC
Bush
Craig R
CR
Chi
Ping
P
Guo
Huiping
H
Luxon
Bruce
B
Fields
Alan P
AP
Thompson
E Aubrey
EA
D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005938:Glucocorticoids / Q000494:pharmacology*; D016399:Lymphoma, T-Cell / Q000235:genetics*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D011965:Receptors, Glucocorticoid / Q000502:physiology; D014407:Tumor Cells, Cultured
D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D004247:DNA