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

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 M dexamethasone (dex) in the presence and absence of 1 g/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 Ͼ 2-fold in both dex-treated and dex ϩ 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 Ͼ 2-fold both in the presence and absence of cycloheximide in two independent experiments is approximately 7 ϫ 10 Ϫ9 . We have retrieved from the Celera mouse genomic sequence 8 kb of promoter sequence, spanning 4 kb either side of the 5Ј-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.

Involvement of small G protein RhoB in the regulation of proliferation, adhesion and migration by dexamethasone in osteoblastic cells

“…However, the transcription inhibitor—actinomycin D couldn’t block totally the upregulation of RhoB mRNA expression by Dex, indicating that the possibility of transcriptional regulation still exists. Using Affymetrix oligonucleotide array, Chen et al declared a putative GRE (ACAATATGTAC) in the promoter region (-3721 to -3711 bp) of murine RhoB gene, which may be responsible for Dex-induced murine RhoB mRNA expression [39]. Although there have been no direct data so far that identified this half site of GRE as essential nucleotides for GC-induced RhoB transcript, the possibility that Dex mediates RhoB mRNA expression via its modulation in transcriptional level still can’t be excluded.…”

11β-Hydroxysteroid dehydrogenase type 1 is an important regulator at the interface of obesity and inflammation

“…A number of studies have been performed to identify GR␣ target genes at a larger scale, including some with bias towards primary target genes [88,[93][94][95]. In order to compile these target genes we took advantage of a bioinformatic tool, the Ingenuity Pathways Analysis (www.ingenuity.com), which allows for assessing and building comprehensive molecular networks based on an extensive knowledge database.…”

Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes

“…To localize precisely the GRE sequences within the fragments implicated by ChIP scanning and functional assays, we identified and mutated sequences that contain at least six of eight conser ved nucleotides of the consensus GR-binding site (ACANNNTGTTNT) (26). In this article, we focus on the four sites found within the GILZ GR-binding fragment (Fig.…”