Figure 1.
Identification of a RORE in the SOX4 promoter.
A) Schematic representing the SOX4 gene. The single exon gene is shown in the diagram the untranslated regions are also shown. The area where significant RORα occupancy was detected in the ChIP-microarray screen is indicated above the gene. The putative RORE is shown below the gene structure and alignment illustrates absolute conservation between xenopus, mouse and human sequences. The RORE is indicated by the underlined sequence. Alignment of the putative SOX4 RORE with the prototypic RORE from the prototypic ROR target gene, BMAL1. Right Panel: Screen shot from genome browser indicating regions generating signal from the ChIP/chip study on both SOX4 and the positive control ARNTL (BMAL1). B) Chromatin immunoprecipitation assessing the occupancy of RORα at the SOX4 promoter. IgG was used as a negative control and RNA polII was used as a positive control. C) Cotransfection assay where a luciferase reporter under the direction of the SOX4 promoter was transfected into HEK 293 cells along with a vector directing the expression of RORα. Inclusion of RORα results in stimulation of luciferase expression. The second panel demonstrates that when the RORE is mutated in the SOX4 promoter, which inhibits the ability of RORα to bind, RORα no longer has the ability to activate transcription of this reporter. WT, indicates wild type and MT, indicates mutant. Empty expression vector was included in the control wells. D) Adenoviral overexpression of RORα in HepG2 cells results in stimulation of SOX4 mRNA expression relative to the LacZ adenovirus control. Suppression of expression of RORα using siRNA results in a reduction of SOX4 mRNA. *, indicates p<0.05. E) Western blot illustrating that overexpression of RORα results in increased p53 protein while suppression of RORα expression results in decreased p53 protein expression. F) Analysis of the effect of RORα overexpression on the half-life of p53. HEK293 cells overexpressing either LacZ (control) or RORα were treated with cycloheximide for various amounts of time (0, 10, 60, 90 min) and p53 protein was assessed by western analysis and normalized to tubulin expression. Densitometry was used to assess expression and was signal was normalized to tubulin and the normalized relative (to time 0) expression is indicated below the blots. p53 displayed a half-life of 22±6 min (mean±S.E.) in the absence of RORα and a half-life >90 min with RORα overexpressed.
Figure 2.
A.) Overexpression of RORα in HepG2 cells results in increased expression of p53 target genes including p21, BAX, and PUMA. In control cells where the RORα adenovirus was not included, a control LacZ adenovirus was used. *, indicates p<0.05. Experiments shown in the lower panels were performed identical to the upper panels with the exception of inclusion of siRNA treatments as indicated. B) Cell cycle analysis of control HepG2 cells infected with LacZ adenovirus (top) or RORα adenovirus (bottom). C) Analysis of the number of cells in various stages of the cell cycle in control or RORα overexpressing HepG2 cells. D) Cell cycle analysis of HepG2 cells overexpression RORα (adenovirus treatment) after treatment with either control siRNA or p53 siRNA. E) Analysis of the number of MCF-7 breast cancer cells in sub-G1 infected with control adenovirus (LacZ) or RORα adenovirus. *, indicates p<0.05.
Figure 3.
The RORα agonist, SR1078, increases SOX4 expression and p53 function.
A) Chemical structure of SR1078. B) Treatment of HepG2 cells with SR1078 leads to an increase in SOX4 and REV-ERBα mRNA expression. C) Treatment of HepG2 cells with SR1078 leads to increased p53 protein levels. + indicates 1 µM and ++ indicates 5 µM SR1078. D) Treatment of HepG2 cells with SR1078 leads to increased expression of p53 target genes, p21 and PUMA. Experiments shown in the lower panels were performed identical to the upper panels with the exception of inclusion of siRNA treatments as indicated. E) Cell cycle analysis of control HepG2 cells treated with vehicle control or SR1078. Note the substantial increase in cells in sub-G1 following SR1078 treatment, 0.9% vs 9.4% indicated on the graph. F) Cell cycle analysis of HepG2 cells treated with either control siRNA or RORα siRNA in the presence of vehicle control or SR1078. G) Cell cycle analysis of HepG2 cells treated with either control siRNA or p53 siRNA in the presence of vehicle control or SR1078. *, indicates p<0.05.