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. 2011 Jun;26(3):187-99.
doi: 10.1177/0748730411401579.

Modulation of clock gene expression by the transcriptional coregulator receptor interacting protein 140 (RIP140)

Ariel H B Poliandri  1 , Joshua J Gamsby, Mark Christian, Michael J Spinella, Jennifer J Loros, Jay C Dunlap, Malcolm G Parker
Affiliations

Modulation of clock gene expression by the transcriptional coregulator receptor interacting protein 140 (RIP140)

Ariel H B Poliandri et al. J Biol Rhythms. 2011 Jun.

Abstract

Circadian rhythms are generated in central and peripheral tissues by an intracellular oscillating timing mechanism known as the circadian clock. Several lines of evidence show a strong and bidirectional interplay between metabolism and circadian rhythms. Receptor interacting protein 140 (RIP140) is a coregulator for nuclear receptors and other transcription factors that represses catabolic pathways in metabolic tissues. Although RIP140 functions as a corepressor for most nuclear receptors, mounting evidence points to RIP140 as a dual coregulator that can repress or activate different sets of genes. Here, we demonstrate that RIP140 mRNA and protein levels are under circadian regulation and identify RIP140 as a modulator of clock gene expression, suggesting that RIP140 can participate in a feedback mechanism affecting the circadian clock. We show that the absence of RIP140 disturbs the basal levels of BMAL1 and other clock genes, reducing the amplitude of their oscillations. In addition, we demonstrate that RIP140 is recruited to retinoid-related orphan receptor (ROR) binding sites on the BMAL1 promoter, directly interacts with RORα, and increases transcription from the BMAL1 promoter in a RORα-dependent manner. These results indicate that RIP140 is not only involved in metabolic control but also acts as a coactivator for RORα, influencing clock gene expression.

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Figures

Figure 1.
Figure 1.
Circadian expression of RIP140. (A) Western blots for RIP140 and β-actin, representative of 2 independent experiments. (B) Levels of RIP140 mRNA, measured by real-time PCR, and protein (measured by densitometry of A). Points represent mean ± SEM (n = 4). Figure representative of 3 independent experiments. Cells were synchronized by 50% serum pulse as described in Materials and Methods. (C) mRNA levels for BMAL1, RIP140, and Rev-erbα after depletion of BMAL1 using a specific siRNA for BMAL1 and levels of Rev-erbα and RIP140 mRNA after depletion of Rev-erbα using a specific siRNA for Rev-erbα. U2OS cells were transfected with siRNA, and mRNA was collected 48 hours after transfection. Bars represent mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. Student t test (n = 6). Figure representative of 3 independent experiments. (D) Levels of BMAL1, fatty acid synthase (FAS), sterol regulatory element-binding protein 1c (SREBP1c), and phospho enol pyruvate carboxykinase (PEPCK) mRNA, superimposed on RIP140 protein levels. Points represent mean ± SEM of 4 biological replicates.
Figure 2.
Figure 2.
RIP140 absence reduces the basal expression of clock genes. (A) mRNA level of different clock genes in the liver and anterior hypothalamus (AHT) of wild-type (WT) and RIP140 knockout (KO) mice. Samples were collected at ZT2 (2 hours after lights are turned on), and mRNA was extracted and quantified by RT-QPCR. Bars represent mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. Student t test (n = 6). (B) mRNA levels of clock genes in HepG2 human liver cell lines depleted of RIP140 quantified by RT-QPCR. RIP140 knockdown cells (shRIP) were generated by stably transfecting HepG2 cells with a vector expressing RIP140-specific shRNA. HepG2 cells constitutively expressing a nonspecific scrambled shRNA (scr) were used as a control. (Inset) Western blot showing RIP140 expression in the different cell lines. Bars represent mean ± SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. Student t test. (C) mRNA levels of clock genes in HuH7 human liver cells transiently transfected with a RIP140 expressing vector or equal amounts of an eGFP expressing vector. Bars represent mean ± SEM (n = 3). *p < 0.05 and **p < 0.01 versus control. Student t test.
Figure 3.
Figure 3.
Clock gene expression and circadian rhythms in RIP140 knockout (KO) mouse embryonic fibroblasts (MEFs). The MEFs were generated as described in Materials and Methods by crossing RIP140 KO heterozygous animals. (A) Level of expression of clock genes in unsynchronized MEFs generated from 2 wild-type (WT1/2) and 2 knockout (KO1/2) embryos measured by real-time PCR. Bars represent mean ± SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 versus WT1. One-way ANOVA followed by Student Newman-Keuls post hoc multiple comparison test. Figure representative of 3 independent experiments. (B) Daily oscillations in the mRNA levels of BMAL1, CRY1, and PER1 in synchronized WT and KO MEFs measured by real-time PCR. Cells were synchronized by 50% serum pulse as described in Materials and Methods. (C) RIP140 KO MEFs were stably transfected with the BMAL1 promoter driving luciferase. Seven individual clones (clone 1-7) were assayed for rhythmicity. After synchronization by serum pulse, luciferase activity was measured in a real-time luminometer for 6 days. τ and φ were calculated as described in Materials and Methods (N = 7).
Figure 4.
Figure 4.
RIP140 activates the BMAL1 promoter. HuH7 cells were cotransfected with a reporter vector containing an intact (A and B, BMAL1-Luc) or a ROREs mutant (C, 2xmtROREBMAL1-Luc) BMAL1 promoter with RORA or RORC and increasing concentrations of RIP140 expression vectors. (D) HuH7 cells were cotransfected with a BMAL1 reporter and siRNA duplexes _targeting RIP140, RORA, Rev-erbα, or a noncoding (siNC) oligo as control. (E) Cotransfection of a BMAL1 reporter with RIP140 and siRNA oligos _targeting RORA. (F) Cotransfection of a BMAL1 reporter with RORA and siRNA oligos _targeting RIP140. (G) Knockdown of RIP140, RORA, and Rev-erbα was confirmed by RT-QPCR. Figures representative of at least 3 independent experiments. Bars represent mean ± SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. Two-way ANOVA followed by Student Newman-Keuls multiple comparison test.
Figure 5.
Figure 5.
Rev-erbα antagonizes the effect of RIP140. (A) HuH7 cells were cotransfected with a BMAL1 reporter, siRNA duplexes _targeting Rev-erbα (siRev-erbα), or a noncoding oligo (siNC) as control and increasing amounts of a RIP140 expressing vector. (B) Cotransfection of a BMAL1 reporter with an expression vector for RIP140 and increasing concentrations of a Rev-erbα expression vector. Figures representative of 3 independent experiments. Bars represent mean ± SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control. ΔΔp < 0.01 and ΔΔΔp < 0.001 versus siRev-erbα (A) or RIP140 (B). Two-way ANOVA followed by Student Newman-Keuls multiple comparison test.
Figure 6.
Figure 6.
RIP140 is recruited to ROR binding elements and interacts with RORA. (A) Chromatin immunoprecipitation assay for RIP140 and RORA performed with nuclear extracts of unsynchronized HepG2 cells. Purified DNA from precipitated chromatin was amplified by real-time QPCR using primers encompassing the ROR binding elements on the BMAL1 or CRY1 genes. Distal regions of these genes lacking ROR binding elements were used as control. Bars represent mean ± SEM of 3 independent experiments. (B) Sequential chromatin immunoprecipitation assay (rechip). DNA immunoprecipitated with an anti-RIP140 antibody was then immunoprecipitated with an anti-RORA antibody. DNA purified after the second precipitation was amplified by real-time QPCR using primers encompassing the ROR binding elements on the BMAL1 gene. A distal region of this gene lacking ROR binding elements was used as control. Bars represent mean ± SEM (n = 3). (C) HEK293 cells were transiently transfected with V5-tagged RIP140 and HA-tagged RORA expression vectors. Cells cotransfected with YFP expression vector and empty vector or GFP- and HA-tagged RORA expression vectors were used as controls. Total cell lysates were immunoprecipitated using anti-V5, anti-HA, or anti-GFP antibodies, and Western blots were performed using specific RIP140, HA, or GFP antibodies.
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