Genomic analysis of glucocorticoid-regulated promoters in murine T-lymphoma cells
Lu Chen1, Celeste Finnerty, William C Gustafson
1Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Abstract:
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.
Insights
This study identifies 44 direct glucocorticoid-regulated genes in T-lymphoma cells using high-throughput gene expression analysis. Five of these genes contain potential glucocorticoid receptor binding sites in their promoters.
Area of Science:
- Molecular Biology
- Genomics
- Cell Biology
Background:
- Glucocorticoids are potent regulators of gene expression with broad physiological effects.
- Understanding direct targets of glucocorticoid action is crucial for deciphering their roles in cellular processes and disease.
Purpose of the Study:
- To identify primary transcriptional targets of the glucocorticoid receptor in P1798 murine T-lymphoma cells.
- To analyze promoter regions of induced genes for potential glucocorticoid receptor binding sites.
Main Methods:
- High-throughput gene expression analysis using Affymetrix oligonucleotide arrays.
- Treatment of G1/S-arrested cells with dexamethasone and cycloheximide.
- Bioinformatic analysis of promoter sequences for specific DNA motifs.
Main Results:
- Identified 44 genes with > 2-fold induction by glucocorticoids, independent of new protein synthesis.
- Statistical analysis confirmed the significance of the identified gene set.
- Five of eight analyzed induced genes contained a potential glucocorticoid receptor binding site (ACAnnnTGTnCT) in their promoter regions.
Conclusions:
- Dexamethasone directly regulates a specific set of genes in T-lymphoma cells.
- The identified genes represent direct targets of glucocorticoid receptor signaling.
- The presence of putative binding sites suggests a direct mechanism of regulation for a subset of these genes.
