Rasd1 Modulates the Coactivator Function of NonO Figure 2. Effects of Rasd1 and NonO in the cAMP-signaling pathway in HEK293T cells were studied using reporter gene assay. (A) NonO- (2 mg) and Rasd1- (or Rasd1 mutants) (2 mg) expressing plasmids were co-transfected in HEK293T cells. Immunofluorescence was performed 2 days after transfection. Cells were transfected with different amounts of His-Rasd1-expressing vector (0, 0.5, 1 and 2 mg) along with reporter vector and CREB-expression vector. Two days later, cells were induced with forskolin (20 mM) for 4 hours before harvest. Luciferase assays were subsequently performed on the cell lysates. The results show that luciferase expression was reduced by 40% upon transfection of pHis-Rasd1 (compare bars I and IV). The suppression of luciferase expression by Rasd1 is dosage-dependent, as increasing amounts of pHis-Rasd1 resulted in further down-regulation of luciferase expression (compare bars II and III; and bars III and IV). (B) Parallel experiments were conducted by transfecting different amounts of pNonO-V5 (0, 0.1, 0.2, 1 and 2 mg) into HEK293T cells. The presence of NonO in HEK293T cells results in a slight up-regulation of luciferase expression of up to 130% (compare bars I and V), and this up-regulation is dosage-dependent (Compare bars II and IV; bars II and V). Next, pNonO-V5 and pHis-Rasd1 were co-transfected in HEK293T cells to determine if the proteins cooperate to influence CREB’s transactivation functions. Different amounts of pHis-Rasd1 (0, 0.5, 1 and 2 mg) were co-transfected with pNonO-V5 (2 mg), and cells were treated with forskolin before harvest. We observed that luciferase expression was reduced by 80% in the presence of both NonO and Rasd1 (compare bars I and VIII). The activation effect observed when NonO was transfected alone was abolished upon co-transfection of NonO with Rasd1 in the cells (compare bars V with VIII). The suppressive effect on luciferase expression was further enhanced in the presence of NonO and Rasd1 as compared to that of transfection of Rasd1 alone (compare (A) bar IV and (B) bar VIII). Representative Western blots were included to show the protein expressions of Rasd1 and NonO. Actin was included as a loading control. Rasd1 is detected with anti-Xpress; NonO is detected with anti-V5; and actin is detected with anti-actin. doi:10.1371/journal.pone.0024401.g002 results support the finding that CREB regulates only a certain cohort of CRE-containing promoters in different cell lines, perhaps due to a difference in the expression of co-regulators. Therefore, in subsequent studies conducted with HEK293T cells, we focus on the effect of NonO and Rasd1 on the transcription of the endogenous NR4A1, NR4A2 and FOS. We observed that induction of NR4A1 and NR4A2 gene expression by forskolin was abolished upon co-transfection of pNonO-V5 and pGST-Rasd1 (compare Figure 5A, bars II and IV, for the respective genes). However, the induction of FOS gene expression by forskolin was not affected by the presence of NonO and Rasd1 (compare Figure 5A, bars IV of FOS, NR4A1 and NR4A2). These results suggest that Rasd1 and NonO regulate a subset of the CREB target genes in HEK293T cells. The expression of negative control YWHAH was not affected by Rasd1 and NonO and/or forskolin (Figure 5A, bars I–IV). To determine if Rasd1 is able to regulate transcription of CREB target genes on its own, we performed experiments to knockdown endogenous NONO in HEK293T cells. NONO-knockdown was able to reduce the transcriptional activity of CREB target genes to levels comparable to that observed in uninduced HEK293 cells (compare Figure 5B, bars I with III of each gene). Transcription levels of these genes were restored to levels similar to that in cells induced with forskolin alone when NONO-knockdown cells were transfected with NonO-expressing plasmid (compare Figure 5B, bars III with IV of each gene), suggesting that NonO has a direct effect on the regulation of genes containing the CRE-responsive element. pGST- Rasd1 was transfected in NONO-knockdown HEK293T cells, and the presence of Rasd1 had no effect on the transcript levels of the CREB target genes (compare Figure 5B, bars III with V). These results suggest that Rasd1 requires NonO to regulate the transcription of CREB target genes. Next, we investigated the influence of over-expression of Rasd1 and NonO on the transcriptional regulation of NR4A1, FOS and NR4A2 in NONO- knockdown cells. A similar trend to the results in Figure 5A, bar IV, was observed upon co-transfection of Rasd1- and NonO-expressing plasmids in NONO-knockdown cells (Figure 5B, bar VI of NR4A1 and NR4A2). Expression levels of FOS remained unaffected by the presence of Rasd1 (compare Figure 5B, bars VI of FOS with NR4A1 and NR4A2). Hence, Rasd1 and NonO cooperate to regulate a subset of CREB target genes including NR4A1 and NR4A2. Physical presence of Rasd1 and NonO at the CRE-site of NR4A2 promoter is required for repression of NR4A2 transcription ChIP was carried out using forskolin-induced HEK293T lysates transfected with pGST-Rasd1. Anti-NONO was incubated with the lysates, and PCR was performed on the chromatin co- immunoprecipitated along with NONO. Compared to cells transfected with pGST (negative control), there was an increase in the amount of NONO bound to the CRE-site of the NR4A2 promoter only in cell lysates transfected with pGST-Rasd1 (compare Figure 5C, Lanes 2 and 3). A similar experiment was conducted on HEK293T cells transfected with either pHis-Rasd1 or pHis-Del-NLS (mutant that does not interact with NonO to serve as negative control) to determine if increased binding of NONO to the NR4A2 promoter could be due to the presence of Rasd1 at the target promoter. Neither Rasd1 nor the mutant, Del- NLS, was observed to be at the CRE-site of the FOS promoter (Figure 5D, Lanes 5 and 6). This seemed reasonable, as Rasd1 was unable to work with NonO to suppress the transcription of FOS. In the case of the NR4A2 promoter, only Rasd1 was able to co- immunoprecipitate the NR4A2 promoter (Figure 5D, Lane 2). The results suggest that binding of Rasd1and NonO to the CRE-site of the NR4A2 promoter is required for the repression of its transcription. Discussion In this study, we have identified NonO as a novel binding partner of Rasd1 via in vitro affinity-based assay, and this interaction is validated using pulldown and co-immunoprecipita- tion assays. We then studied the roles of Rasd1 and NonO in the cAMP pathway. Our findings show that co-localisation of Rasd1 and NonO in the nucleus is associated with the repression of a subset of CREB target genes. This process involves the GTP hydrolysis activity of Rasd1 and requires interaction of Rasd1 with full-length NonO at the CRE-site of the target promoter. We propose that Rasd1 modulates the function of NonO to down- regulate CREB target genes, NR4A1 and NR4A2. Our results show that deletion of the putative bipartite nuclear localisation sequence located at the C-terminal portion of Rasd1 does not deter Rasd1 from entering the nucleus, implying that Rasd1 enters the nucleus by other means. Small molecular weight proteins of less than 60 kDa or 9 nm in diameter are able to enter the nucleus via the nuclear pore complex by passive diffusion [37], which is one possible mechanism employed by Rasd1, whose molecular weight is 32 kDa, to enter the nucleus. NonO contains a bipartite nuclear localisation signal, and Rasd1 may bind to NonO to facilitate its entry into the nucleus. Our results indicated that the lack of NonO’s NLS prevented accumulation of Rasd1 in the nucleus, which suggests that NonO may play a role in retaining Rasd1 in the nucleus. In addition, studies using Rasd1 mutants show that GDP-bound Rasd1 resulted in cytoplasmic localisation of NonO. This implies that nuclear retention of Rasd1 by NonO is PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24401