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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 24, 2008 |
| Title |
The role of RIP140 in retinoid mediated signaling |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Cell-and context-specific activities of nuclear receptors may in part be due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We have shown previously that silencing of RIP140 enhances RA-induced differentiation and enhances the induction of model RA target genes in human embryonal carcinoma cells (EC). Through use of microarray technology we sought to elucidate in a de novo fashion the global role of RIP140 in RA-dependent signaling. RIP140-dependent gene expression was largely consistent with RIP140 functioning to limit RAR signaling. Few if any genes were regulated in a manner to support a role for RIP140 in “active repression”. Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140, suggesting that RIP140 may discriminate between different classes of RA target genes. RIP140 silencing also accelerated RA target gene activation and sensitized EC cells to low doses of RA. Together the data suggests that the RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation. RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy.
Keywords: Drug treatment with siRNA
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| Overall design |
The current investigation sought to determine the global effects of RIP140 on RA-dependent and RA–independent gene expression. A dose of siRNA of 90 nM was chosen. Twelve independent hybridizations were performed from mRNA isolated from 12 independent samples. One scrambled and two independent RISC-free siRNA transfections and transfections with three distinct RIP140 siRNAs were performed in NT2/D1 cells. Each siRNA treatment was further treated with either 10 μM RA or DMSO control for 24 hours. Hence, the design consisted of four groups, the three siRNA controls treated with vehicle, Group 1; the siRNA controls treated with RA, Group 2; siRNA to RIP140 treated with vehicle, Group 3; and siRNA to RIP140 treated with RA, Group 4 .
The treatments were done in the presence of normal sera, since charcoal absorption of sera removes many small hydrophobic ligands to the nuclear receptor family in addition to retinoids. This measure was taken with the aim to perhaps identify target genes of other nuclear receptors including orphan receptors that could be regulated by RIP140 in the presence of low levels of their cognate ligands. RA treatment of 10 μM for 24 hours was chosen since previous experience with NT2/D1 cells indicated that a reasonably sized set of altered genes would be obtained. In addition, NT2/D1 cells commit to RA-induced differentiation by 48 hours and RIP140 repression enhances and accelerates the effects of RA in these cells. Therefore, the 24-hour time point is well-within the range of the RA commitment “window” and a larger percentage of direct RA-target genes could be expect to be uncovered compared to later time points.
Based on the current understanding of the properties of RIP140, a number of potential outcomes were anticipated: 1) RIP140 may limit RA target gene activation by negative-feedback inhibition upon RA addition, 2) RIP140 may participate in “active repression” of gene expression upon RA addition, 3) an unexpected outcome that RIP140 may participate in the activation of a subset of target genes, 4) RIP140 may regulate the transcription of target genes of other nuclear receptors or even non-nuclear receptor transcription factors.
Total RNA was purified using RNeasy columns (Qiagen). Hybridizations were performed according to Affymetrix guidelines at the Dartmouth College Microarray Shared Resource using an Affymetrix GeneChip Workstation. Biotin-labeled cRNA was generated from 5 μg of total RNA and hybridized to the Human Genome (HG) U133 Plus 2.0 chip A total of 12 hybridizations were performed comprising 12-independent biologic samples organized into the four groups of three.
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| Contributor(s) |
Heim KE, White-Branco KA, Spinella MJ |
| Citation(s) |
17880687 |
| Submission date |
Apr 11, 2007 |
| Last update date |
Mar 25, 2019 |
| Contact name |
Michael Spinella |
| E-mail(s) |
spinella@illinois.edu
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| Phone |
603 707 7847
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| Organization name |
University of Illinois
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| Department |
Comparative Biosciences
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| Street address |
2001 South Lincoln Ave
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| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61802 |
| Country |
USA |
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| Platforms (1) |
| GPL570 |
[HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array |
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| Samples (12)
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| GSM181589 |
Group 1 replicate scramble siRNA no RA |
| GSM181754 |
Group 1 replicate RISC-free siRNA from Dharmacon no RA |
| GSM181755 |
Group 1 replicate 2 RISC-free siRNA from Dharmacon no RA |
| GSM181756 |
Group 3 siRNA#1 for RIP140 no RA |
| GSM181757 |
Group 3 siRNA#2 for RIP140 no RA |
| GSM181758 |
Group 3 siRNA#3 for RIP140 no RA |
| GSM181759 |
Group 2 scramble siRNA from Dharmacon treated with RA |
| GSM181760 |
Group 2 RISC-free siRNA from Dharmacon treated with RA |
| GSM181761 |
Group 4 siRNA#1 for RIP140 with RA |
| GSM181762 |
Group 4 siRNA#2 for RIP140 with RA |
| GSM181763 |
Group 4 siRNA#3 for RIP140 with RA |
| GSM181764 |
Group 2 RISC-free replicate siRNA with RA |
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| Relations |
| BioProject |
PRJNA100483 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE7500_RAW.tar |
62.9 Mb |
(http)(custom) |
TAR (of CEL, CHP) |
| Processed data provided as supplementary file |
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