A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway

Abstract

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the "coactivator trap," to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1-3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54(nrb)) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.

Fig. 1.

Coactivator trap identifies TORC 1, 2, or 3 functional interactions with NONO (p54^nrb). Shown are transient transfection assays with GAL4 UAS::luciferase reporter in HEK293T cells cotransfected with GAL4-cDNA library. (a) Heat map representation of transcriptional activities increased (red) or repressed (green) at least 10-fold by any TORC compared with pCMV-SPORT6 vector control. For comparison, the coactivators CBP and p300 expression constructs are also mapped. (b) Changes in transcriptional activity of GAL4 fusion proteins induced by cotransfection with TORCs 1, 2, or 3 plotted against a sorted rank of transcriptional activity of fusion constructs from left to right. (c) Changes in transcriptional activity of GAL4 fusion proteins induced by cotransfection with either p300 or CBP plotted against a sorted rank of transcriptional activity of fusion constructs. (d) Transient transfection assays with GAL4 UAS::luciferase reporter in HEK293T cells cotransfected with GAL4-NONO and indicated coactivators (n = 3 wells, mean ± SEM).

Fig. 2.

cAMP signaling stimulates TORC–NONO complex formation. (a) (Left) Endogenous NONO coimmunoprecipitates with TORC2. TORC2 was immunoprecipitated by using affinity-purified 638A, 3363, or 3364 α-TORC2 antibody (TORC-IP) or beads alone (Control-IP) from cell lysates stimulated with forskolin (+) or vehicle (−) and were immunoblotted (IB) with α-NONO or α-p65/NFκB. (Right) Pre-IP of NONO and p65/NFκB protein (2% of input) is shown. (b) (Left) FRET analysis of interaction of TORC and NONO. Shown are representative pseudocolored fluorescence images of cells cotransfected with CFP-TORC and GFP-NONO expression plasmids and treated with DMSO (control) or forskolin and IBMX for 1 h. (Scale bar, 10 μm.) (Right) Percentage of overlap in fluorescence energy of CFP–TORC and GFP–NONO expression plasmids in control and forskolin-treated cells (n = 5 experiments; mean ± SEM; each experiment was the average of 10 measurements). (c) (Left) FRET/GFP ratio as captured in pseudocolored fluorescence images of transfected cells after control and forskolin treatment for 1 h. (Scale bars, 10 μm.) Shown are FRET efficiency (Center) and the distance between CFP donor and GFP acceptor (Right) between CFP–TORC and GFP–NONO in control and forskolin-treated cells (n = 5 experiments; mean ± SEM). Data are from at least five regions of interest (ROI) per treatment group from three independent experiments.

Fig. 3.

NONO plays essential roles in cAMP-dependent transcription. Shown is the effect of NONO knockdown by siRNA on the cAMP-responsive EVX1 luciferase (Top) versus control IFN luciferase (Middle) reporters after forskolin or TNFα stimulation, respectively, in HEK293T cells. (Bottom) Western blot showing amounts of protein in cells treated with siRNA (n = 3 wells; mean ± SEM).

Fig. 4.

NONO recruits RNA pol II to cAMP-responsive promoters. NONO is present at a cAMP-responsive promoter along with CREB and TORC2. (a) ChIP of the NR4A2 and GAPDH promoters from HEK293T cells using anti-CREB (Left), anti-TORC2 (Center), anti-NONO (Right), or anti-GAL4-specific antisera as a negative control. Preimmunoprecipitation (Pre-IP) control DNA is also shown. (b) Quantification of precipitated NR4A2 promoter by TaqMan real-time (qRT) PCR (n = 3 experiments; mean ± SEM; each experiment was the average of three measurements). Occupancy of the target protein on the NR4A2 promoter is expressed relative to the GAPDH promoter. (c) RNA from HEK293T cells transfected with siRNAs for CREB, TORC2, NONO, or NS control was quantified by quantitative RT-PCR using YWHAH, NR4A2, and FOS mRNA-specific TaqMan probes. Fold changes in endogenous YWHAH, NR4A2, and FOS mRNA levels after treatment with forskolin or DMSO vehicle for 45 min are graphed. NONO siRNA abolishes cAMP-dependent transcriptional activation but not constitutive activity of YWHAH (n = 3 wells; mean ± SEM). (d) Knockdown of NONO protein attenuates RNA pol II recruitment to cAMP-responsive promoters. ChIP assay of YWHAH, NR4A2, FOS, and GAPDH promoters from HEK293T cells using anti-RNA pol II antibody. Fold occupancy of RNA pol II on the target promoter is expressed relative to the GAPDH promoter (n = 3 experiments; mean ± SEM).