Molecular mechanism of nur77 gene expression and downstream target genes in the early stage of forskolin-induced differentiation in PC12 cells

Forskolin promotes neuronal differentiation of PC12 cells via the PKA-CREB-dependent signaling pathway. Activation of PKA by forskolin phosphorylates CREB, which then binds to CRE sites in numerous gene promoters. However, it is unclear which gene contains the CRE sites responsible for forskolin-induced neuronal differentiation. In this study, we investigated how an immediate early gene, nur77, which has CRE sites in the promoter region, contributes to the early stage of differentiation of forskolin-treated PC12 cells. After treatment with forskolin, expression of Nur77 was upregulated within 1 hr. In addition, knockdown of nur77 inhibited neurite outgrowth induced by forskolin. We also revealed that the specific four CRE sites near the transcriptional start site (TSS) of nur77 were strongly associated with phosphorylated CREB within 1 hr after treatment with forskolin. To analyze the roles of these four sites, reporter assays using the nur77 promoter region were performed. The results showed that nur77 expression was mediated through three of the CRE sites, −242, −222, and −78, and that −78, the nearest of the three to the TSS of nur77, was particularly important. An analysis of neuronal markers controlled by Nur77 after A-CREB-Nur77-Synapsin1 signaling pathway plays a pivotal role in differentiation of forskolin-induced PC12 cells.

www.nature.com/scientificreports www.nature.com/scientificreports/ Nur77 is an orphan nuclear receptor that is also referred to as NGFI-B, TR3 and Nr4a1. Nur77 is a member of the Nur77 family, which also contains the orphan nuclear transcription factors Nurr-1 and Nor-1. Nur77 was originally identified as a protein that is rapidly induced by nerve growth factors in PC12 cells 36,37 and by serum in fibroblasts 38 . Recent studies have shown that Nur77 expression is regulated by CRE-binding proteins. The nur77 gene contains four CRE sites (TGCGTCA; previously defined as the AP1 (activator protein 1) element) upstream of the transcription start site (TSS) 26,39,40 . These four CRE sites have been suggested to bind CREB protein 25,26,37,41 , and binding of transcription factors at the four CRE sites in the nur77 promoter may play an important role in the early stage of forskolin-induced neuronal differentiation. However, the detailed mechanisms of nur77 transcription in the early phase of neurite extension are largely unknown, and the relevance of nur77 and its transcription factors in neural differentiation is not understood.
In this study, we investigated the mechanism underlying regulation of nur77 transcription during the early phase of neurite extension induced by forskolin in PC12 cells. We found that the four CRE sites upstream of the TSS of nur77 are associated with phosphorylated CREB (P-CREB) within 1 hr after treatment with forskolin. We also found that the −242, −222 and −78 CRE sites, and especially −78, play particularly important roles. To identify the critical molecules regulated by Nur77 during forskolin-induced neurite extension, Nur77 regulation of proteins that serve as neuronal differentiation markers was analyzed. The findings showed that Nur77 regulated one such protein, Synapsin 1, but did not influence β-tubulin III or NeuroD, although it was reported that β-tubulin III or NeuroD was expressed under the Nur77 regulation. These results suggest that the PKA-CREB-Nur77-Synapsin1 signaling pathway is essential for forskolin-induced differentiation of PC12 cells, including neurite extension.

Nur77 is involved in neurite outgrowth induced by forskolin in PC12 cells. To confirm if forskolin
has a role in neurite outgrowth, the lengths of neurites were measured after treatment with forskolin in PC12 cells. Neurite lengths of PC12 cells treated with 10 µM forskolin for 24 hr were significantly greater than those from untreated cells (Fig. 1A,B and see Supplementary Table S1 on line) as reported previously 42,43 .
Previous studies have shown that expression of nur77 is significantly increased by db-cAMP in PC12 cells, and that Nur77 is essential for early stage of differentiation in neurons and Schwann cells 20,34,35,44 . To investigate whether the increased expression of Nur77 is mainly responsible for neurite outgrowth that occurs from 0 to 24 hr after 10 µM forskolin treatment, expression of nur77 gene and Nur77 induced by forskolin was examined using qPCR and immunoblotting analysis ( Fig. 2A-C). The peak expression of nur77 gene and Nur77 after forskolin treatment was reached at 1-4 hr. These data suggest that expression of nur77 gene and Nur77 are induced at 0-4 hr after 10 µM forskolin treatment.
Binding of CREB with CRE sites near the TSS of nur77 is responsible for forskolin-induced Nur77 activation and neurite outgrowth. To analyze the details of the function of Nur77 in the early period of forskolin-treated). For analysis of neurite outgrowth, cells (more than 200 /well) were randomly photographed using a KEYENCE microscope. The lengths of neurite were measured using BZ-H1C software. ***P < 0.001 (untreated vs. forskolin-treated cells by Kolmogorov-Smirnov test).
forskolin treatment, knockdown experiments were performed using siRNA against nur77 mRNA. First, it was confirmed that knockdown of nur77 mRNA inhibited neurite outgrowth after treatment with forskolin. Neurite length in cells treated with siRNA against nur77 mRNA in the presence of forskolin was significantly lower than that in cells treated with negative universal control siRNA (Fig. 3A,B and see Supplementary Table S2 on line). These results indicate that upregulation of Nur77 is required for differentiation of PC12 cells by 10 µM forskolin.
CRE sites near the TSS are known to be heavily involved in regulation of transcription. In PC12 cells, we have shown that the CRE site-containing nur77 promoter region near the TSS is associated with acetylated Lys14 of histone H3 after treatment with db-cAMP or HDAC (Histone Deacetylase) inhibitors TSA and K350; and that expression of Nur77 induced by db-cAMP is regulated by the PKA-CREB pathway 20,34,35 . Based on these findings, it is likely that CRE sites upstream of the TSS of nur77 and CREB are important for nur77 expression.
To explore the role of these CREs sites and CREB in the early stage of forskolin-induced differentiation of PC12 cells, immunoblotting analysis using anti-phosphorylated CREB antibody was first performed. P-CREB was first observed 1 hr after treatment with 10 µM forskolin, similar to expression of Nur77, and phosphorylation reached a peak at 1-4 hr and then declined to the basal level after 4 hr (Fig. 4A,B). To examine the contribution of P-CREB to 10 µM forskolin-induced nur77 expression mediated by CRE sites, a ChIP assay using anti-P-CREB and CREB antibody was carried out. This assay revealed that the nur77 promoter region containing the CRE sites ( Fig. 4C) was particularly associated with P-CREB within 1 hr after treatment with 10 µM forskolin (Fig. 4D). The ChIP assay also showed that the nur77 promoter region was associated with CREB with or without 10 µM forskolin treatment (Fig. 4D). Taken together, these results suggest that binding of P-CREB to the CRE sites of the nur77 promoter plays a particularly important role in mediating transcriptional activation of nur77. www.nature.com/scientificreports www.nature.com/scientificreports/ To analyze the details of the role of CREB in the early period of 10 µM forskolin induced nur77 gene expression and neurite outgrowth, knockdown experiments were performed using siRNA against creb mRNA. First, we analyzed the relationship between nur77 gene expression and CREB. After treatment with creb siRNA for 24 hr in the presence or absence of forskolin, nur77 gene reduced strongly compared with cells treated with a negative control siRNA (Fig. 4E). Next, we confirmed that neurite length in cells treated with siRNA against creb mRNA in the presence of forskolin was significantly lower than that in cells treated with negative control siRNA ( To analyze the role of the nur77 promoter in forskolin-induced neurite outgrowth, luciferase reporter plasmids were constructed with the four CRE sites deleted one by one (Fig. 5A). Strong Nur77-related luciferase activities were observed with the WT and −242 plasmid with or without treatment with forskolin. In contrast, the −222, −78 and −49 plasmids had marked reduction of luciferase activity with treatment of forskolin, and −78 and −49 had reduced luciferase activity without forskolin (Fig. 5A). With plasmid −49, luciferase activity was completely abolished. Therefore, the regulatory elements for nur77 expression appear to be present in the promoter region between −242 and −78.
We next examined which of these three CRE sites in the promoter play significant roles in nur77 expression. As shown in Fig. 5B, luciferase activity was measured using promoter assay plasmids in which mutations were interpolated in one or two or all of the three distal CRE sites. Plasmids with a point mutation at any one of three CRE sites −242, −222 or −78 caused a reduction in luciferase activity under forskolin-treated condition, but particularly marked reduction occurred with the −78 mutant. In contrast, under untreated conditions, the luciferase activity was markedly reduced only with the −78 mutant. With mutants at two or three CRE sites, luciferase activity was completely abolished when a mutation at the −78 CRE site was included (Fig. 5B). These results suggest  for the indicated times. Cells were then subjected to ChIP assays with an antibody against P-CREB and CREB as described in Methods. The nur77 gene promoter region was detected by qPCR. **P < 0.01 compared with 0 hr (P-CREB), ## P < 0.01 compared to 0 hr (CREB). € Cells transfected with creb siRNA or ncRNA were treated with or without 10 μM forskolin for 1 hr. creb and gapdh mRNA were detected by qPCR. creb mRNA were normalized against gapdh mRNA. **P < 0.01 compared with ncRNA. (F) After co-transfection with creb siRNA or ncRNA containing a GFP-expression plasmid for 48 hr, cells were treated with or without 10 μM forskolin for 24 hr. Phase contrast (Phase) and GFP-expression (GFP) images were detected and photographed. Scale bar: 50 (2020) 10:6325 | https://doi.org/10.1038/s41598-020-62968-y www.nature.com/scientificreports www.nature.com/scientificreports/ that the regulatory elements of nur77 expression are present in the promoter region between −242 and − 78, and particularly at −78.
The Nur77-Synapsin signaling pathway is required for forskolin-dependent neuronal differentiation. The effect of Nur77 on neuron-specific markers was analyzed in neurite outgrowth induced by forskolin. We first examined if the neuron-specific marker, Synapsin1 as a synaptic marker 45 , β-tubulin III as a neuronal marker of newly generated neurons [46][47][48] or NeuroD as a neuronal marker for the early phase of the neuronal lineage 48 , was upregulated with treatment of 10 µM forskolin for 24 hr. qPCR and immunoblotting detected Synapsin1 and β-tubulin III, but not NeuroD (Fig. 6A-C). After treatment with nur77 siRNA for 24 hr in the presence or absence of forskolin, immunoblotting showed strong reduction of Synapsin1 compared with cells treated with a negative control siRNA. In contrast, the β-tubulin III level was not altered by treatment with nur77 siRNA (Fig. 6D-F).
To analyze the details of the role of Synapsin1 in the early period of 10 µM forskolin treatment, knockdown experiments were performed using siRNA against synapsin1 mRNA. We examined that neurite length in cells treated with siRNA against synapsin1 mRNA in the presence of forskolin was significantly lower than that in cells treated with negative control siRNA ( These results suggest that the Nur77-Synapsin1 signaling pathway, but not β-tubulin III or NeuroD, has an important role in differentiation of forskolin-treated PC12 cells.

Discussion
Therapeutic agents for nerve injury must have two main characteristics of passage through the BBB and induction of neuronal differentiation. Forskolin has these properties and is also a natural product, which may reduce side effects. Therefore, this compound may be a useful drug for nerve injury. Determination of the intracellular mechanism of differentiation induced by forskolin is needed as part of the risk assessment for this potential drug. In this study, we showed that Nur77 has an important role in forskolin-induced differentiation, and we found that binding of P-CREB to the nur77 promoter region and expression of Nur77 occurred within 1 hr of forskolin treatment. In particular, we suggest that binding of P-CREB at the −242, −222 and −78 CRE sites among the four CRE sites in the nur77 promoter region near the TSS is essential for forskolin-induced nur77 gene activity.
The CRE sites near the TSS of nur77 have previously been shown to be important in nur77 transcription, but this study is the first to show that binding of CREB at specific CRE sites has a pivotal role in the early stage of forskolin-induced neurite outgrowth. Three of the four CRE sites in the nur77 promoter between −274 and −69 from the TSS have previously been shown to have important roles in response to extracellular stimuli 25,40,41 . Similarly, our data showed that the three CRE sites at −242, −222 and −78 play important roles in forskolin-induced differentiation in PC12 cells (Fig. 5B). These results suggest that these three CRE sites have especially pivotal roles in nur77 expression. To understand the details of the mechanism of Nur77 expression, it will be important to know which transcription factors bind to these three CRE sites. It has recently been reported that binding of CREB to these CRE sites is enhanced by 8 Br-cAMP, PMA, EGF and TNF treatment 25,41 .
CREB plays an important role in differentiation and is also an essential mediator of Ca 2+ -activated Nur77 expression in PC12 cells and binds to the endogenous nur77 promoter region in vivo. Moreover, CRE sites in the nur77 promoter contain the central CpG that is critical for CREB binding 25,26,49 . Our previous data showed that the PKA-CREB-Nur77 pathway is important in neurite outgrowth in the db-cAMP-induced mechanism in PC12 cells 20 . Binding of Jun family proteins to CRE sites in the nur77 promoter is also enhanced by 8Br-cAMP 25 . One such protein, JunD, is an atypical member of the AP1 family that can act as both a transcriptional activator and repressor, and is also activated by an elevated level of intracellular cAMP 50 . c-jun, another Jun family member, is a master regulator of neurite outgrowth and axonal regeneration in nerve injury 51,52 . However, there is no direct evidence that binding of CREB or Jun proteins during the upregulation of Nur77 expression is involved in the differentiation, and it is uncertain of there is involvement of gene regulators other than the CREB or Jun family. Further detailed studies are required in this area.
IEGs other than Nur77 are also involved in differentiation. For example, Egr1 is an IEG that induces neuronal differentiation and causes neurite outgrowth in N2 neuroblastoma cells 53 . The induction of Egr1 via the MAPK-ERK pathway has been linked to neuronal differentiation and plasticity. Two other nur77 family genes, nurr1 and nor-1, have also been shown to act as IEGs, and are correlated with neuronal differentiation. nurr1 is required for the development and survival of dopaminergic neurons 54,55 , and upregulation of nurr1 in neuronal precursor cells promotes undifferentiated cells into mature and functional dopaminergic neurons 56 . Consistent with this, nurr1-deficient mice exhibit abnormal development of midbrain dopaminergic neurons 54 . nor-1 also plays a critical role in neuronal survival and axonal guidance in the developing murine hippocampus 57 , and is also involved in regulating neuronal differentiation during extension of processes in cultured fetal rat brain cells 58 . These findings indicate that Nur77 family members are involved in several aspects of neuronal differentiation in the developing nervous system.
Similarly to nur77, the nurr1 and nor-1 genes also have DNA motifs near the TSS that bind with transcriptional factors, such as CREB, with the transcriptional activity that is increased by phosphorylated PKA. The nurr1 μm. (G) Histograms of neurite lengths of forskolin-treated cells (open bars) pretreated with creb siRNA (gray bars) and ncRNA (hatched bars). Cells pretreated with siRNA or ncRNA in the presence of a GFP-expression plasmid for 48 hr were then treated with forskolin for a further 24 hr. Cells were cultured without (closed bars: untreated) or with 10 μM forskolin (open, hatched and gray bars). For analysis of neurite outgrowth, cells (more than 200 cells /well) were randomly photographed. ***P < 0.001 (creb siRNA vs. ncRNA).

Scientific RepoRtS |
(2020) 10:6325 | https://doi.org/10.1038/s41598-020-62968-y www.nature.com/scientificreports www.nature.com/scientificreports/ promoter contains a TGACG motif near the TSS, and the nor-1 promoter has three CRE sites (TGACGTAG, TGGCGTCA, and TGACGTCT) near the TSS 59 . This suggests that the same transcription factor, CREB, binds to CRE sites in these Nur77 family members and may participate in neuronal differentiation. Therefore, the Nur77 family may have a common molecular mechanism in neural differentiation. However, Nur77 family members also show some differences in transcriptional activities, despite having highly homologous amino acid sequences 41,60 . These differences are thought to be due to the involvement of DNA binding domains other than the CRE sites. For example, nur77 and nor-1 contain binding sites for MEF2 (MEF2 response elements, MRE) near the TSS and the CRE sites in their promoter regions 26,61 . Early work suggested that MEF2 modulates CREB-dependent Nur77 expression by acting as a repressor in quiescent cells, while CREB is necessary for Ca 2+ -activated Nur77 expression 26 . Therefore, to define the relationship between the mechanism of nur77 gene expression in neuronal differentiation and the DNA motifs near the TSS, a detailed analysis of the CRE sites and the relationships between other DNA binding motifs around these sites and the relevant transcription factors is required.
In this study, we also found that the Nur77 upregulated Synapsin1, but not β-tubulin III or NeuroD, as a key step during forskolin-induced neurite outgrowth in PC12 cells. This finding provides direct evidence that Nur77 is involved in the differentiation of PC12 cells. Synapsin is an abundant pre-synaptic phosphoprotein associated with the cytoplasmic side of synaptic vesicles that promotes actin polymerization and triggers cell differentiation [62][63][64][65] . Synapsin1 is a useful indicator of PC12 cell differentiation 66 , and post-translational modifications of Synapsin1 by PKA contribute to the regulation of neurotransmitter release 67 . These results lead us to speculate on the following model (Fig. 7). Under basal conditions, CREB binds to the promoter region of nur77 via the −78 CRE site (Fig. 7A). If forskolin activates the PKA-CREB pathway, CREB is phosphorylated and binds to CRE sites upstream of the TSS in nur77. Subsequently, Nur77 expression is induced via binding of P-CREB at the −244, −222, and −78 CRE sites of the nur77 promoter. Induced Nur77 may then upregulate the expression of Synapsin1 and cause differentiation of neurons (Fig. 7B). However, it is unclear if Synapsin1 is directly upregulated by www.nature.com/scientificreports www.nature.com/scientificreports/ Nur77 or regulated by a transcription factor controlled by Nur77. An answer to this questions requires further analysis of the molecular mechanism of the Nur77-Synapsin1 signaling pathway in forskolin-induced neuronal differentiation.
In this study, we also revealed that NeuroD was not involved in forskolin-induced neurite outgrowth of PC12 cells. However, previous study demonstrated that Bcl-2 regulates neurite outgrowth through the Bmp4 -Tbx3 -NeuroD1 cascade in H19-7 cells 68 . In addition, it has been known that Nur77 is localized to the mitochondria, inducing the release of Cytochrome C and Caspase 3 through interaction with Bcl-2. This molecular interaction causes apoptosis in following apoptosis stimulation 69,70 . These findings suggest that the function or the expression of NeuroD may be regulated by Nur77 in neurite outgrowth that does not involve in the PKA-CREB pathway. Furthermore, we demonstrated that β-tubulin III was not regulated by Nur77 in forskolin-induced neurite outgrowth of PC12 cells. On the other hand, Damodaran et al. have reported that phosphorylation of CREB by PKA and a member of the CaMK family, CaMKII play a crucial role in the altered axonal transport by modulating the transcription of β-tubulin gene 71 . In addition, it has also been shown that CaMKI pathway contributes to cAMP-induced Nur77 expression in MA-10 Leydig cells 72 . These findings suggest that Nur77 may regulate the expression of tubulin in neurite outgrowth induced by activators of the PKA-CREB pathway. There are numerous genes that are regulated by Nur77 in neural differentiation, and their regulatory mechanisms are still unclear. Therefore, we need more detailed analyses about the downstream molecules of Nur77 in future. The findings in this study reveal that the PKA-CREB-Nur77 pathway is essential for forskolin-induced differentiation of PC12 cells. We also showed that expression of nur77 is initiated by binding of CREB to the CRE sites −242, −222 and −78, and especially −78, in the nur77 promoter region near the TSS. We also found that Nur77 upregulated Synapsin1, a neuronal differentiation marker. Further detailed studies are needed to define the molecular mechanisms of Nur77-mediated differentiation induced by forskolin. Measurement of neurite outgrowth. Cells were seeded in 24-well plates at 1.5 × 10 4 cells/cm 2 . After 16 hr, the medium was replaced with DMEM in the presence or absence of 10 µM forskolin (Sigma, MO, USA) supplemented with 1% (v/v) FBS. The cells were then further incubated for 24 hr and fixed with 4% paraformaldehyde for 30 min. For analysis of neurite outgrowth, cells (more than 200 /well) were randomly photographed using a KEYENCE microscope (Biozero BZ-9100, Osaka, Japan). Neurite lengths were measured using the BZ-H1C software. All measurements were made in duplicate. Differences between groups were compared by Kolmogorov-Smirnov test, with P < 0.001 considered to denote a significant difference.
Cells were seeded onto 6-well plates at 1.0 × 10 5 cell/cm 2 and incubated for 16 hr, and then transfected with 100 nM nur77 siRNA and 5 nM creb and synapsin1 siRNA or with ncRNA using Lipofectamine ® RNAiMAX (Life Technologies). After 48 hr, the medium was replaced with 10 µM forskolin in DMEM supplemented with 1% (v/v) FBS, and the cells were incubated for a further 1 hr and 24 hr. Then, the cells were used for RNA extraction or immunoblot analysis. For measurement of neurite lengths of PC12 cells transfected with siRNAs directed against target mRNAs, the cells were plated onto 24-well plates at 1.5 × 10 4 cells/cm 2 and incubated for 16 hr. We then co-transfected siRNAs (100 nM) against target mRNAs with 0.125 nM pCX-EGFP using Lipofectamine ® RNAiMAX (Life Technologies).
After 48 hr, the cells were treated with 10 µM forskolin in DMEM supplemented with 1% (v/v) FBS and incubated for another 24 hr, and then fixed with 4% (w/v) paraformaldehyde for 30 min. For analysis of neurite outgrowth, GFP-positive cells (more than 200 /well) were randomly photographed and measured 24 hr after initial treatment with forskolin. All measurements were made in duplicate.

RNA extraction and Quantitative real-time PCR (qPCR).
Total RNA was collected with ISOGEN (Nippongene, Toyama, Japan) and extracted with phenol-chloroform. cDNA was then synthesized using the ReverTraAce ® qPCR RT Kit (TOYOBO, Osaka, Japan). 25 ng of cDNA was used as template for StepOnePlus TM real-time PCR system (Applied Biosystems, USA) using THUNDERBIRD ® Probe qPCR Mix (TOYOBO, Osaka, Japan). www.nature.com/scientificreports www.nature.com/scientificreports/ The data were analyzed with delta delta Ct method and normalized to the amounts of gapdh RNA expression in each sample. PCR primers used were those of TaqMan ® gene expression assay kits for nur77 (Assay ID: Rn00666994_g1), creb (Assay ID: Rn06140207_g1), synapsin1 (Assay ID: Rn00569468_m1), tubulin, beta 3 class III (Assay ID: Rn01431594_m1), neurod1 (Assay ID: Rn00824571_s1), and gapdh (Assay ID: Rn01775763_g1). All measurements were made in triplicate. Data are presented as the mean ± SEM (n = 3). Statistical significance for each time was determined by Student's t-test. **p < 0.01. Immunoblot analysis. Cells were seeded onto 6 cm dishes at 1.0 × 10 5 cell/cm 2 . After 16 hr, the medium was replaced with DMEM supplemented with 1% (v/v) FBS, and the cells were then treated with 10 µM forskolin for 0-24 hr, respectively. Immunoblot analysis were performed as described previously 73 . The first antibody (anti-phospho CREB (Ser133) antibody (Merck), anti-CREB antibody (Cell Signaling Technology, Danvers, MA, USA), anti-Nur77 antibody (Abcam, Cambridge, UK), anti-Synapsin1 antibody (Abcam), anti-NeuroD antibody (Cell Signaling Technology), anti-β-tubulin III antibody (Sigma) or anti-GAPDH antibody (Merck)) was loaded onto the membrane after blocking with 5% skimmed-milk (Nakarai, Kyoto, Japan), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (MBL CO., LTD., Nagoya, Japan). The bands were detected by ECL Select Western Blotting Detection System Reagent or ECL Prime Western Blotting Detection System Reagent and visualized with an Image Quant LAS 4000 (all GE Healthcare Life Sciences, Little Chalfont, UK). All measurements were made in triplicate. Quantitative analysis was performed by determining the immunofluorescence intensity of the target protein(s) using ImageJ. Data are presented as the mean ± SEM (n = 3). Statistical significance for each time was determined by Student's t-test. *p < 0.05.
Chromatin immunoprecipitation (ChIP) assay. ChIP assays were performed as described previously 20 with some modifications. The diluted chromatin of 5 × 10 6 cells was incubated with anti-phospho CREB (Ser133) antibody (Merck), anti-CREB antibody (Cell Signaling Technology) or normal rabbit IgG (Cell Signaling Technology) for 12-16 hr at 4 °C. Immune complexes were bound to Protein G sepharose beads preblocked with salmon sperm DNA and bovine serum albumin for 120 min at 4 °C. The beads were washed once each with low-salt wash buffer (0.1% (w/v) SDS, 1% (v/v) Triton-X100, 2 mM EDTA, 150 mM NaCl, and 20 mM Tris-HCl at pH 8.0), high-salt wash buffer (500 mM NaCl wash buffer), LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% (v/v) deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl at pH 8.0), and twice with TE buffer (10 mM Tris-HCl at pH 8.0 and 1 mM EDTA). Immune complexes bound to Protein G beads were incubated overnight at 65 °C and treated with 100 µg/ml proteinase K for 1 hr at 56 °C before extraction once with phenol/chloroform. The DNA was then precipitated with ethanol containing glycogen as a carrier and resuspended in 20 µl of dH 2 O. DNA was analyzed by qPCR using following PCR probes (sense: GAT CAA ACA ATC CGC GCT CCC anti-sense: CAC CTC TTA AGC GCT CCG TGA, and TaqMan ® MGB probe: TAT GGC CAA AGC TC). The ChIP enriched DNA levels were then normalized to input DNA. All measurements were made in duplicate. Data are presented as the mean ± SEM (n = 3). Statistical significance was determined by Student's t-test. **p < 0.01, ## p < 0.01.

Luciferase assays.
A promoter region of the rat nur77 gene (WT) was cloned from rat genome DNA using Site-directed mutagenesis of CRE sites within the −242, −222 and −78 nur77 promoters was also achieved using the KOD-Plus Mutagenesis Kit (TOYOBO). Promoter fragments containing substituted nucleotide sequences were generated by PCR using the following primers: TGA TGG AAC CCC GCG TGC GT (−242 promoter forward primer), ACG CAG GGA GCG CGG ATT G (−242 promoter reverse primer), TGC GCG CGC AGA CAT TCC AGG C (−222 promoter forward primer), ACG CAC GCG GGG TTC CAT TGA C (−222 promoter reverse primer), TGG TGG CGC CCC CGC CCC TCT C (−78 promoter forward primer), and ACG CAC ACG GCC CGG CGA GC (−78 promoter reverse primer). The nucleotide sequences of all inserts were confirmed using a 3130xl Genetic Analyzer (Thermo Fisher Scientific, MA, USA).
Plasmids with the appropriate nur77 promoters were transfected with the phRL-TK plasmid (Promega) using Lipofectamine 2000 (Life Technologies). After 48 hr, transfected cells were then established as untreated or stimulated cells for 1 hr with 10 µM forskolin. After stimulation, the cells were lysed and the activities of firefly and Renilla luciferase were measured using the Dual-luciferase reporter assay system (Promega). Firefly luciferase activities were normalized to the Renilla control. All measurements were made in duplicate. Data are presented as the mean ± SEM (n = 3). Statistical significance for each time was determined by Student's t-test. **p < 0.01, ## p < 0.01 and *p < 0.05.