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. 2003 Nov 25;100(24):13845-50.
doi: 10.1073/pnas.2336092100. Epub 2003 Nov 14.

VSports app下载 - Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor

Inez Rogatsky  1 , Jen-Chywan Wang, Mika K Derynck, Daisuke F Nonaka, Daniel B Khodabakhsh, Christopher M Haqq, Beatrice D Darimont, Michael J Garabedian, Keith R Yamamoto
Affiliations

Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor

Inez Rogatsky et al. Proc Natl Acad Sci U S A. .

Abstract (V体育ios版)

The glucocorticoid receptor (GR) activates or represses transcription depending on the sequence and architecture of the glucocorticoid response elements in target genes and the availability and activity of interacting cofactors. Numerous GR cofactors have been identified, but they alone are insufficient to dictate the specificity of GR action. Furthermore, the role of different functional surfaces on the receptor itself in regulating its targets is unclear, due in part to the paucity of known target genes VSports手机版. Using DNA microarrays and real-time quantitative PCR, we identified genes transcriptionally activated by GR, in a translation-independent manner, in two human cell lines. We then assessed in U2OS osteosarcoma cells the consequences of individually disrupting three GR domains, the N-terminal activation function (AF) 1, the C-terminal AF2, or the dimer interface, on activation of these genes. We found that GR targets differed in their requirements for AF1 or AF2, and that the dimer interface was dispensable for activation of some genes in each class. Thus, in a single cell type, different GR surfaces were used in a gene-specific manner. These findings have strong implications for the nature of gene response element signaling, the composition and structure of regulatory complexes, and the mechanisms of context-specific transcriptional regulation. .

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Figures (VSports在线直播)

Fig. 3.
Fig. 3.
Differential effects of AF1, AF2, and dimer interface disruptions on GR activation. (A) Generation of U2OS cells expressing mutant rGR variants. Diagrammed are AF1:30iiB (E219K/F220L/W234R), dim:A477T, and AF2:E773R GR constructs stably introduced into U2OS cells. Cells were cultured in the absence or presence of dex for 2 h, and total cell extracts were analyzed by immunoblotting with antibodies to GR and extracellular signal-regulated kinase (ERK) as an internal control for loading. (B) GR target genes are differentially responsive to mutations in the GR AF1/AF2 and dimer interface. U2OS cells expressing WT or mutant GR were cultured in the absence or presence of dex for 2 h, and total RNA was analyzed by real-time PCR as described in Fig. 2. (Upper) Absolute level of hormonal induction over the untreated control in each clone. (Lower) Percentage induction of each gene by mutant GR relative to that of WT. Genes are arranged by the relative sensitivity to the AF1 disruption, from hIAP (most sensitive) to I6PK (completely insensitive). At least two independent clones for each GR mutant were tested with similar results. (C) Introduction of WT GR into mutant clones partially restores hormonal responsiveness. U2OS cells expressing dim or AF2 mutants were transiently transfected with increasing amounts of WT GR cDNA, and the expression of GILZ, IGFPB1, and ladinin was examined by real-time PCR.
Fig. 1.
Fig. 1.
Expression profiling of glucocorticoid-induced genes. U2OS-rGR, -hGR, and A549 cells were cultured in the absence or presence of 100 nM dex with 10 μg/ml cycloheximide, as shown, for indicated times. Total RNA was isolated, amplified, labeled, and subjected to microarray hybridization (see Materials and Methods). Each test hybridization contained Cy5-labeled RNA from dex-treated cells vs. Cy3-labeled RNA from vehicle-treated cells. Matching reference control lanes are hybridizations of RNA isolated from cells cultured in the absence of dex only, labeled with Cy3 and Cy5. A hierarchical clustering algorithm was used to identify genes induced in both cells lines (A) and those that are induced in U2OS only (B) or A549 cells only (C). Genes that were subsequently tested for induction in U2OS-rGR cells by real-time PCR are in bold. (D)(Upper) Microarray data were analyzed by significance analysis of microarrays (SAM). Solid line represents genes for which the observed and expected relative differences from the reference are identical and that would therefore be found to be “regulated” by chance. At a Δ= 1.02 (limited by dashed lines), 72 genes in U2OS cells are found to be significantly activated (46 genes, red) or repressed (26 genes, green), with a false discovery rate of 0.91. (Lower) Log-transformed ratio of medians values for these genes and their accession numbers are shown. The order of lanes is the same throughout.
Fig. 2.
Fig. 2.
Ongoing transcription but not translation is required for the induction of GR targets. U2OS-rGR cells were either pretreated for 1 h with 5 μg/ml actinomycin D, after which dex was added to 100 nM as indicated and incubation continued for 2 h (A); or treated with 100 nM dex for 2 h in the absence or presence of 10 μg/ml cycloheximide (B). Total RNA was harvested, reverse-transcribed, and subjected to real-time PCR with primer pairs to indicated genes. The data were transformed by the standard ∂∂Ct method, with amplification of ribosomal Rpl19 RNA used as an internal control for normalization.
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References

    1. Ito, M. & Roeder, R. G. (2001) Trends Endocrinol. Metab. 12, 127–134. - PubMed
    1. Levine, M. & Tjian, R. (2003) Nature 424, 147–151. - "VSports" PubMed
    1. Yamamoto, K. R., Darimont, B. D., Wagner, R. L. & Iniguez-Lluhi, J. A. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 587–598. - PubMed
    1. Lefstin, J. A. & Yamamoto, K. R. (1998) Nature 392, 885–888. - PubMed
    1. Rogatsky, I., Luecke, H. F., Leitman, D. C. & Yamamoto, K. R. (2002) Proc. Natl. Acad. Sci. USA 99, 16701–16706. - PMC - PubMed

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