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Rasd1 Interacts with Ear2 (Nr2f6) to Regulate Renin Transcription Jen Jen Tan1,2, Shufen Angeline Ong1, Ken-Shiung Chen1*

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Rasd1 Interacts with Ear2 (Nr2f6) to Regulate Renin Transcription Jen Jen Tan1,2, Shufen Angeline Ong1, Ken-Shiung Chen1* Tan et al. BMC Molecular Biology 2011, 12:4 http://www.biomedcentral.com/1471-2199/12/4 RESEARCHARTICLE Open Access Rasd1 interacts with Ear2 (Nr2f6) to regulate renin transcription Jen Jen Tan1,2, Shufen Angeline Ong1, Ken-Shiung Chen1* Abstract Background: The Rasd1 protein is a dexamethasone induced monomeric Ras-like G protein that oscillates in the suprachiasmatic nucleus (SCN). Previous studies have shown that Rasd1 modulates multiple signaling cascades. However, it is still unclear exactly how Rasd1 carries out its function. Studying protein-protein interactions involving Rasd1 may provide insights into its biological functions in different contexts. Results: To further explore the molecular function of Rasd1, we performed a yeast two-hybrid screen and identified Ear2, a negative regulator of renin transcription, as an interaction partner of Rasd1. We validated the interaction in vitro and in transfected COS-7 cells. We further confirmed the interaction of endogenous Rasd1 and Ear2 from HEK293T cell and mouse brain extract. Rasd1 inhibited transcriptional repression by Ear2 on a renin promoter-luciferase reporter construct both in the presence and absence of all-trans-retinoic acid. Moreover, real- time RT-PCR showed upregulation of endogenous renin transcription in As4.1 cells over-expressing Rasd1. We demonstrated that the ligand binding domain of Ear2 is required for physical and functional interaction between the two proteins. In addition, we demonstrated that shRNA-mediated knockdown of Rasd1 results in further repression of Ear2-mediated renin transcription, whereas induction of Rasd1 by dexamethasone counteracts the effects of shRNA-mediated Rasd1 knockdown. Finally, our study showed that Rasd1 missense mutations not only attenuate their physical interaction with Ear2 but also abolish their ability to counteract repression of renin transcription mediated by Ear2. Conclusions: Our study provides evidence for physical and functional interactions between Rasd1 and Ear2. The results suggest that their interactions are involved in renin transcriptional regulation. These findings not only reveal a novel role for Rasd1-medated signaling but also provide the basis for potential intervention of renin expression. Background enhancer was identified ~2.6 kb upstream of the mouse The renin-angiotensin system plays a major physiological renin gene, and this enhancer is homologous to a role in the control of blood pressure, fluid volume and sequence ~12 kb upstream of the human renin gene electrolyte balance. A functional renin-angiotensin sys- [6-8]. The transcriptional enhancer contains several tran- tem is also essential for the normal development of the scription factor binding sites that have both excitatory mammalian renal system [1]. Renin, an aspartyl protease, and inhibitory regulatory functions [9-12]. is the rate-limiting enzyme in the renin-angiotensin enzy- One protein that has been identified to bind to and matic cascade which leads to the production of angioten- regulate the renin enhancer is Ear2 [13]. It was deter- sin II (Ang II), a vasoactive peptide and major effector mined that Ear2 negatively regulates renin expression by molecule in the renin-angiotensin system [2,3]. Renin competing with retinoic acid receptor/ retinoid X recep- gene expression is largely regulated at the transcriptional tor (RAR/RXR) for binding to the retinoic acid response level, although post transcriptional regulation has also elements (RARE) on the renin enhancer [13]. Ear2 is an been reported [4,5]. A potent classical transcriptional orphan nuclear hormone receptor that belongs to the chicken ovalbumin upstream promoter-transcription * Correspondence: [email protected] factors (COUP-TF) gene family [14]. COUP-TFs have 1School of Biological Sciences, Department of Genomics and Genetics, been shown to bind to a number of variable direct and Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore indirect repeats with different spacings between the Full list of author information is available at the end of the article © 2011 Tan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tan et al. BMC Molecular Biology 2011, 12:4 Page 2 of 17 http://www.biomedcentral.com/1471-2199/12/4 repeats [15] to affect a large plethora of genes [16-19]. Table 1 Proteins identified from the yeast two-hybrid COUP-TFs have been proposed to inhibit transactivation screen of other nuclear receptors through multiple mechanisms, No Protein/ Gene Name Genbank including competitively binding to regulatory elements, Gene Accession No. competitively binding to RXR, mediating active repres- 1 Cenpb Mus musculus centromere protein B NM_007682.2 sion via direct binding to regulatory elements, and med- 2 Gnb1 Mus musculus guanine nucleotide NM_008142.3 iating transrepression of another nuclear receptor binding protein, beta 1 without binding to DNA itself [14,20-22]. Nuclear hor- 3 Nr2f6 Mus musculus nuclear receptor NM_010150.2 mone receptors have the ability to bind directly to DNA subfamily 2, group F, member 6 and regulate the expression of specific target genes; 4 Plscr1 Mus musculus phospholipid NM_011636.2 scramblase 1 therefore, they are extremely crucial to the development, 5 Sh3gl2 Mus musculus SH3-domain GRB2-like 2 NM_019535.2 homeostasis and metabolism of an organism [16,20,21, 23-25]. Ear2 is expressed in tissues of all major systems, 6 Supt16 h Mus musculus suppressor of Ty 16 NM_033618.2 homolog and its expression has been implicated in the regulation 7 Trp53bp2 Mus musculus transformation related NM_173378.2 of gene expression for normal embryo development protein 53 binding protein 2 [26,27]. Ear2 knockout mice are viable and fertile, but 8 Ywhah Mus musculus tyrosine 3- NM_011738.1 they possess circadian and nociception defects and monooxygenase/tryptophan 5- abnormal locus coeruleus (LC) development [25]. monooxygenase activation protein, eta Using a yeast two-hybrid system, we identified Ear2 as polypeptide an interacting protein of Rasd1. Rasd1 is a G-protein that belongs to the RAS superfamily of small GTPases. It was first discovered as a dexamethasone-inducible between Rasd1 and Ear2, we conducted an in vitro bind- gene in the AtT-20 pituitary cell line [28]. Rasd1 is an ing study utilizing transfected COS-7 cells. GST-Ear2 oscillating gene specific to the suprachiasmatic nucleus was immobilized on GSH-linked beads. The GST fusion (SCN) [29]. Rasd1 modulates several signaling cascades protein containing full-length Ear2 interacted with HA- and has several known functions. It interacts with tagged full-length Rasd1 from the lysate of transfected neuronal nitric oxide synthase via CAPON to enhance COS-7 cells (Figure 1A, lane 1), whereas GST itself physiological nitric oxide signaling [30]. Rasd1 is also showed no such interaction (Figure 1A, lane 2). crucial in the regulation of the responsiveness of the To test whether Rasd1 and Ear2 interact in intact circadian clock to external stimuli [31]. In this study, we mammalian cells, we performed co-transfection and demonstrate that the interaction between Rasd1 and co-precipitation experiments. pHisHA-Rasd1 was Ear2 regulates renin transcription. Our findings reveal a co-transfected together with pGST-Ear2 into COS-7 novel regulatory role of Rasd1 and a novel regulatory cells. As a control, pHisHA-Rasd1 was co-trans- mechanism for Ear2-mediated renin transcription. fected with pxJGST into COS-7 cells. GST-Ear2 or GST from transfected COS-7 cell lysate was Results immobilized on GSH-linked magnetic particles. Pre- Identification of Ear2 as a Rasd1 interacting protein-To cipitation of GST-Ear2 resulted in the co-precipita- identify the proteins that are associated with Rasd1, we tion of HisHA-Rasd1 (Figure 1B, lane 1), whereas conducted a yeast two-hybrid screen. A mouse brain precipitation of GST-tag alone did not co-precipitate cDNA library was screened against full length Rasd1. All HisHA-Rasd1 (Figure 1B, lane 2). This demonstrates surviving colonies after nutritional selection were that Rasd1 and Ear2 form a physiological complex in cul- screened for LacZ expression using b-galactosidase. tured cells. Additionally, a co-immunoprecipitation assay Table 1 lists the proteins that were identified. Ear2 is was carried out using lysate derived from COS-7 co-trans- the protein for which the highest number of indepen- fected with pHisHA-Rasd1 and pGST-Ear2. As a control, dent clones was obtained, with a total of three clones experiments were also conducted using lysate derived identified. To confirm the specificity of interaction from COS-7 cells co-transfected with pHisHA-Rasd1 and between Rasd1 and Ear2, a separate yeast two-hybrid pxJGST. Immunoprecipitation of GST-Ear2 with analysis was performed by mating yeast containing the anti-GST antibody resulted in the co-precipitation of Ear2 cDNA plasmid with yeast that already carried HisHA-Rasd1 (Figure 1C, lane 1). There was no non-spe- Rasd1. This was followed by nutritional selection and cific interaction between GST tag and HisHA-Rasd1 b-galactosidase assay. Ear2 again showed positive inter- (Figure 1C, lane 2). actions with Rasd1 (not shown). Interaction of endogenous Rasd1 and Ear2 was identi- Rasd1 and Ear2 interact in vitro and in living cells- fied in HEK293T cells and the mouse brain-Theexis- To confirm the specificity of biochemical interaction tence of endogenous Rasd1-Ear2 complexes was further Tan et al. BMC Molecular Biology 2011, 12:4 Page 3 of 17 http://www.biomedcentral.com/1471-2199/12/4 Figure 1 Rasd1 and Ear2 interact in vitro and in living cells.(A)In vitro binding of GST-Ear2 and HisHA-Rasd1. COS-7 cells were transfected with plasmids expressing the indicated constructs. Cell lysates containing HisHA-Rasd1 was incubated with immobilized GST-Ear2 or GST, and specifically bound HisHA-Rasd1 was eluted by heating at 95°C for 10 minutes, and detected by Western blotting with Anti-HA antibody.
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