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Originally published In Press as doi:10.1074/jbc.M604409200 on September 5, 2006

J. Biol. Chem., Vol. 281, Issue 45, 34617-34629, November 10, 2006
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Neuronal PAS Domain Protein 1 Is a Transcriptional Repressor and Requires Arylhydrocarbon Nuclear Translocator for Its Nuclear Localization*

Christina H. L. Teh, Kevin K. Y. Lam, Chin C. Loh, Jia M. Loo, Tie Yan, and Tit Meng Lim1

From the Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Kent Ridge, Singapore 117542

Received for publication, May 9, 2006 , and in revised form, August 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal PAS domain protein 1 (NPAS1), a basic helix-loop-helix-PAS transcription factor expressed in the central nervous system, has been suggested to be involved in neuronal differentiation. However, relatively little is known about the molecular mechanism underlying the role of NPAS1 during development. In this study we set out to characterize the different domains within NPAS1. We showed that the nuclear localization of NPAS1 is dependent on the presence of ARNT. In addition, the transcriptional potential of ARNT is not required for this localization. In the absence of ARNT, NPAS1 is excluded from the nucleus, and this exclusion is due to the presence of a nuclear export signal within the N terminus of NPAS1. The interaction between NPAS1 and ARNT is via their N termini. We found no transactivation domain within NPAS1; instead, we mapped out at least three repression domains within NPAS1, suggesting that NPAS1 acts as a repressor. Furthermore, our experiments showed that NPAS1 is able to repress the transactivation functions of ARNT and ARNT2. We suggest that NPAS1 is guided into the nucleus by ARNT via the ARNT nuclear localization signal, and NPAS1 can override the activation function of adjacent transcription factors, providing a mechanism by which NPAS1 may inhibit transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The class of basic helix-loop-helix (bHLH)2 PAS (Per, ARNT, Sim) proteins are transcription factors that are known to play important roles in several physiological functions that include xenobiotic metabolism (the dioxin or Ah receptor (AhR)) (1), maintenance of circadian rhythms (Clock) (2), hypoxic responses such as angiogenesis and erythropoiesis (hypoxia-inducible factor (HIF)-{alpha} and HIF 1{alpha}-like factor) (3-5), neurogenesis (Single-minded) (6, 7), and embryonic tubulogenesis (Trachealess) (8). Each member of this family consists of an N-terminal basic region that binds DNA, the HLH and PAS domains for dimerization and specificity of interacting partner, and a highly divergent C-terminal region (9, 10). Many of these proteins form heterodimers with another bHLH-PAS domain protein, the AhR nuclear translocator (ARNT) (11), which is ubiquitously expressed. The basic region of each subunit then contacts a half-site of the asymmetric E-box (12, 13) to regulate transcription (14-16). Besides ARNT, another protein ARNT2, which is highly similar to ARNT, is shown to be able to interact with the partners of ARNT (17). However, Arnt2 expression is more restricted to the brain and kidneys, which differs from that of Arnt, suggesting that ARNT2 may play different roles from ARNT (18).

Neuronal PAS domain protein 1 (NPAS1/MOP5) is a bHLH-PAS domain protein that is found to be expressed only in certain regions of the brain (19). NPAS1 mRNA expression is first detected at embryonic day 15 of mouse development, shortly after organogenesis of the brain. NPAS1 is found to be highly homologous to NPAS3, whose expression is also exclusively in the brain (20). Recent studies reveal that laboratory mice lacking NPAS1 and NPAS3 exhibit a spectrum of behavioral and neurochemical abnormalities. In addition, adult brain tissues from NPAS3- and NPAS1/NPAS3-deficient mice showed a distinct reduction in reelin (21). These results suggest that NPAS1 may play a role during neuronal differentiation.

Although NPAS1 knock-out mice showed behavioral abnormalities that may be related to schizophrenia, relatively little is known about the mechanism of action by NPAS1, except that it forms a heterodimer with ARNT and negatively regulates the expression of erythropoietin (22), a factor that promotes production of neuronal progenitors in the central nervous system (23, 24). To better understand the role of NPAS1, we set out to characterize the different domains of NPAS1. In this study we report that NPAS1 requires the presence of either ARNT or ARNT2 for translocation into the nucleus, and the N terminus prevents nuclear localization in the absence of ARNT or ARNT2. Similar to other bHLH-PAS domain proteins, NPAS1 interacts with the N terminus of ARNT via its N-terminal domain. Interestingly, we did not find any transactivation domain (TAD) in NPAS1; instead, we identified three non-overlapping repression domains. We also found that NPAS1 is able to suppress the transactivation function of ARNT and ARNT2, further supporting previous findings that NPAS1 plays a negative transcriptional regulatory role.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of Mouse NPAS1 cDNA—Mouse NPAS1 cDNA was amplified from RNA isolated from MN9D cells. MN9D is a mouse dopaminergic neuronal cell line derived from the fusion of rostral mesencephalic neurons from embryonic C57BL/6J mice with the N18TG2 neuroblastoma cells (25). The full-length NPAS1 cDNA was first sequenced in pGEMTEasy vector and subcloned into an expression plasmid (pcDNA3.1(-)_fl NPAS1_GFP) to encode a GFP epitope-C-terminal tagged mouse NPAS1 protein. To generate the N terminus (aa 1-354) and C terminus (aa 348-594) deletion mutants of NPAS1 (pcDNA3.1(-)_NPAS1(N)_GFP and pcDNA3.1(-)_NPAS1(C) _GFP), cDNAs encoding the N terminus or C terminus region of NPAS1 were obtained using pcDNA3.1(-)_fl NPAS1_GFP as a template with the primers 5'-CGTCGATCTAGAACCATGGCGACCCCCTATCCC-3' and 5'-GTCGACCTCGAGGCTTTGGCGGATCCTGGTTGC-3' or 5'-CGTCGATCTAGAACCATGGCAACCAGGATCCGCCAAAGCCAT-3' and 5'-CCCGGGCTCGAGGTCTCCCTTCCGCTGCA-3', respectively. To generate NPAS1 with a mutated NES, a QuikChange mutagenesis kit (Stratagene) was used.

Construction of Plasmids—pcDNA3.1(+) plasmids containing either full-length Arnt or Arnt2 cDNAs were kind gifts from Dr. Jacques Michaud (Research Center, Hopital Sainte-Justine, Montreal, Canada) and Dr. Chen-ming Fan (Department of Embryology, Carnegie Institution of Washington, Baltimore, MD), respectively. Mouse full-length ARNT, ARNT(N) (aa 1-354), or ARNT(C) (aa 348-594) were PCR-amplified and cloned into BamHI and XhoI of pXJ40_FLAG or pXJ40_HA. Murine ARNT2 protein was cloned into HindIII and SmaI of pXJ40_FLAG. To generate ARNT{Delta}b, two rounds of PCR were carried out with the following sets of primers overlapping at the region to be deleted. The primers used were 5'-TACGCAGGATCCATGGCGGCGACTACAGCT-3' with 5'-CATCTTGTTCCGTCGCAAAAATTTAGT-3' and 5'-TTGCGACGGAACAAGATGGCTCGAAAACCAGACAAGCTA-3' with 5'-GGTCGACTCGAGCTATTCGGAAAAGGGGGG-3'. The two PCR products were then used as template for a second round of PCR with the outlying primers to join the two fragments. The final amplicon ARNT{Delta}b was then digested with restriction enzymes BamHI and XhoI and ligated into pXJ40_FLAG vector. The encoded murine ARNT and deletion mutants, ARNT2, or ARNT{Delta}b have the FLAG or HA epitope linked to the N terminus.

For yeast hybrid work expression plasmids encoding a LexA DNA binding domain (DBD) epitope-tagged version of full-length NPAS1, NPAS1 N terminus, and NPAS1 C terminus were constructed as follows. The pcDNA3.1(-)_fl NPAS1_GFP plasmid was used as template with the following sets of primers: 5'-CGTCGACCATGGATGGCGACCCCCTATCCC-3' and 5'-CCCGGGCTCGAGGTCTCCCTTCCGCTGCA-3' or 5'-CGTCGACCATGGATGGCGACCCCCTATCCC-3' and 5'-GTCGACCTCGAGGCTTTGGCGGATCCTGGTTGC-3' or 5'-CGTCGACCATGGGCAACCAGGATCCGCCAAAGCCAT-3' and 5'-GTCGACCTCGAGTCAGTCTCCCTTCCGCTGCACCCT-3' to generate the fl NPAS1, NPAS1(N), or NPAS1(C) inserts, respectively. The amplified DNA products were digested with the restriction enzymes NcoI and XhoI and ligated into pLexA vector (Clontech). Expression plasmids encoding a LexA DBD epitope-tagged version of full-length (pLexA_fl ARNT), N terminus (aa 1-579) (pLexA_ARNT(N)), or C terminus (aa 578-771) (pLexA_ARNT(C)) of the ARNT protein were constructed as follows. The pcDNA3.1(+)_fl ARNT plasmid was used as a template with the following sets of primers: 5'-TGGCTGGAATTCGCAGAGAATTTCAGGAAT-3' and 5'-GGTCGACTCGAGCTATTCGGAAAAGGGGGG-3' or 5'-TGGCTGGAATTCGCAGAGAATTTCAGGAAT-3' and 5'-CCTGAACTCGAGTGCCGGCCGGGGGTTAGG-3' or 5'-TGGCTGGAATTCGCAGAGAATTTCAGGAAT-3' and 5'-GGTCGACTCGAGCTATTCGGAAAAGGGGGG-3' to generate the fl ARNT, ARNT(N), and ARNT(C) inserts, respectively. The amplified DNA products were digested with the restriction enzymes EcoRI and XhoI and ligated into pLexA vector.

For mammalian hybrid work, expression plasmids encoding a GAL4 DBD epitope-tagged version of full-length NPAS1, NPAS1 N terminus, and NPAS1 C terminus were constructed as follows. The pcDNA3.1(-)_fl NPAS1_GFP plasmid was used as a template with the following sets of primers: 5'-CGTCGACCCGGGCATGGCGACCCCCTATCCC-3' and 5'-GTCGACAAGCTTTCAGTCTCCCTTCCGCTG-3' or 5'-CGTCGACCCGGGCATGGCGACCCCCTATCCC-3' and 5'-GTCGACAAGCTTGCTTTGGCGGATCCTGGT-3' or 5'-CGTCGACCCGGGGGCAACCAGGATCCGCCAA-3' and 5'-GTCGACAAGCTTTCAGTCTCCCTTCCGCTG-3 to generate the fl ARNT, ARNT(N), and ARNT(C) inserts, respectively. The amplified DNA products were digested with the restriction enzymes SmaI and HindIII and ligated into pM vector (Clontech). Expression plasmids encoding a GAL4 DBD epitope-tagged version of full-length (pM_fl ARNT), N terminus (pM_ARNT(N)), or C terminus (pM_ARNT(C)) of the ARNT protein were constructed as follows. pLexA_fl ARNT, pLex-A_ARNT(N), and pLexA_ARNT(C) were digested with restriction enzymes EcoRI and SalI, and the inserts were subcloned into pM vector.

Culture and Transfection of Mammalian Cells—The murine clonal MN9D cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, streptomycin (100 µg/ml), and penicillin (100 units/ml) (26, 27). HEK293 cells, embryonic kidney cells of human origin, were grown in RPMI media supplemented with 10% fetal bovine serum, streptomycin (100 µg/ml), and penicillin (100 units/ml). Both cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO2.

For transient transfection, cells were grown to the appropriate confluency and transfected with Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. After 48 h the cells were collected and processed for the respective experiments.

Immunocytochemistry—Cells grown in chamber slides were fixed in ice-cold 3% paraformaldehyde in PBS for 30 min followed by permeabilization with 2% Triton X-100 in PBS. The cells were then blocked in 5% goat serum in PBS for 1 h followed by incubation with the rabbit FLAG antibodies (Sigma) in dilution buffer (2% goat serum in PBS) for 1 h at room temperature at a dilution ratio 1:200. After this, three washes were carried out before incubating the cells with rhodamine-coupled goat anti-rabbit IgG (Santa Cruz Technologies) in dilution buffer (1:250) for 1 h at room temperature. The cells were again washed three times and stained with Hoescht stain (Sigma) for 5 min before the images were acquired.


Figure 1
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  		FIGURE 1.
Effect of ARNT and ARNT2 on cellular localization of NPAS1. MN9D cells (A) or HEK293 cells (B) were co-transfected with expression plasmids coding for GFP-tagged NPAS1 and FLAG (i), FLAG-tagged ARNT (ii) or FLAG-tagged ARNT2 (iii). NPAS1_GFP fusion protein (green) was found to be localized in the cytoplasm when it was co-expressed with the empty FLAG plasmid (i). When co-expressed with FLAG-tagged ARNT (ii) or FLAG-tagged ARNT2 (iii), there was a translocation of NPAS1 into the nucleus. The nuclear localization of ARNT (2) was determined by staining with FLAG antibody and detected with rhodamine-conjugated secondary antibody. Cells were stained with Hoechst stain to show the position of the nuclei. Images on the far left were taken under phase contrast to show the outline of the cells. The results are representative of three independent experiments. Each scale bar represents 50 µm. FITC, fluorescein isothiocyanate.

 


Figure 2
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  		FIGURE 2.
Leptomycin B has no effect on subcellular localization of NPAS1. MN9D cells were transfected with expression plasmids coding for NPAS1_GFP. After 1 day the transfected cells were treated with 50 ng/ml leptomycin B, and the cells were fixed at 0, 1, and 8 h, respectively. NPAS1_GFP (fluorescein isothiocyanate (FITC)) was found to be excluded from the nucleus in all cases. The results are representative of three independent experiments. The scale bar represents 50 µm.

 
Isolation of Proteins from Mammalian Cells—Transfected cells were washed three times with PBS and lysed with the lysis buffer (100 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, and 1% Triton X-100). Total cell lysates were collected and incubated overnight at -20 °C. Cell debris were removed by centrifugation at 12,000 x g for 5 min at 4 °C. Protein concentration was determined by the Bio-Rad Bradford assay kit (Bio-Rad).

Western Blot Analysis—Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were probed with the respective primary antibodies (Santa Cruz Technologies) and the corresponding horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Technologies). Proteins were visualized with an ECL detection kit (Pierce).

Immunoprecipitation—Cells transfected with expression plasmids were lysed in 1 ml of lysis buffer as described above. Lysates were directly analyzed either as whole-cell lysates or used in affinity precipitation with M2 anti-FLAG-agarose beads (Sigma-Aldrich). Briefly, the lysate and beads were incubated overnight at 4 °C with gentle rotation. The beads were then washed 5 times with 2% Triton X-100 in PBS (for HA_ARNT pulldown) or just PBS (for HA_NPAS1 pulldown). Samples were run in SDS-PAGE gels followed by Western blotting.

Yeast Work—All yeast cultures were grown in the appropriate synthetic dropout media (BIO101) at 30 °C with shaking at 280 rpm. For transformation, 1 ml of overnight culture of strain EGY48 yeast cells was harvested by centrifugation at 5000 rpm for 1 min in a tabletop centrifuge. The supernatant was discarded, and the yeast pellet was resuspended in 95 µl of ONE-STEP buffer (0.2 N LiCl, 40% polyethylene glycol 3550, pH 5.0, and 100 mM dithiothreitol). The suspension was then transferred into an Eppendorf tube containing ~300 ng of plasmid DNA and 50 µg of Herring sperm DNA. The whole mixture was then incubated at 45 °C for 30 min. The yeast suspension was then plated out on the appropriate synthetic dropout agar plates for 3-5 days at 30 °C.

Five independent yeast clones were selected for each transformation set and grown overnight in selective medium. The yeast cultures were then used for beta-galactosidase assay with yeast beta-galactosidase assay kit (Pierce) according to the microplate assay protocol (stopped) in the instruction manual.

CAT and beta-Galactosidase Assay in Mammalian Cells— Cells were transfected with the respective pM and/or pcDNA3.1(-)_GFP effector plasmids together with a reporter plasmid, pG5CAT (Clontech), and an internal control plasmid pSV40_beta-gal (Promega). After 48 h, the cells were processed and tested for CAT and beta-galactosidase activities using the CAT enzyme-linked immunosorbent assay (ELISA) and beta-galactosidase ELISA kit, respectively (Roche Applied Science).


Figure 3
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  		FIGURE 3.
The N terminus of NPAS1 is excluded from the nucleus in the absence of ARNT or ARNT2. A, schematic representation of the two NPAS1 deletion mutant constructs used to test for subcellular localization. Solid boxes represent the bHLH region, and hatched areas represent the PAS A and PAS B regions. B, MN9D cells were either co-transfected with expression plasmids coding for GFP-tagged NPAS1(N) and FLAG (i) or GFP-tagged NPAS1(C) and FLAG (ii). NPAS1(N)_GFP fusion protein was found to be excluded from the nucleus, whereas NPAS1(C)_GFP fusion protein was evenly distributed throughout the whole cell. FITC, fluorescein isothiocyanate. C, NPAS1(N)_GFP was found both in the nucleus and cytoplasm in the presence of ARNT or ARNT2 overexpression. MN9D cells were co-transfected with expression plasmids coding for NPAS1(N)_GFP and FLAG (i), FLAG_ARNT (ii), or FLAG_ARNT2 (iii). Upon co-expression with FLAG_ARNT or FLAG_ARNT2 protein, NPAS1(N)_GFP was localized both to the nucleus and cytoplasm. The nuclear localization of ARNT (2) was determined by staining with FLAG antibody and detected with rhodamine-conjugated secondary antibody. The results are representative of three independent experiments. Each scale bar represents 50 µm.

 
Dual Luciferase Assay—Dual luciferase assay was carried out using the Promega kit. Transfected cells in 24-well format were washed three times with ice-cold PBS and lysed in 100 µl of 1x passive lysis buffer. The cell lysate was then centrifuged at 15,000 x g at 4 °C for 5 min to remove the cell debris. A luminometer was programmed to perform a reading every 5 s. In a luminometer tube, 100 µl of LAR II was added followed by 20 µl of cell lysate. The tube was then placed in the luminometer, and reading was initiated. Several readings (for firefly luciferase activity) were taken until the readings started to decrease before the tube was removed, and 100 µl of Stop & Glo reagent was added and mixed by shaking. The tube was then placed back into the luminometer for further readings until the readings started to decrease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
It has been separately reported that NPAS1 is localized to the nucleus of interneurons in the mouse (21) and in SHSY5Y, a neuronal cell line (22). However, in both papers, there was no mention of the mechanism for this nuclear localization of NPAS1. We examined if NPAS1 is constitutively localized to the nucleus by overexpressing the protein in different cell lines. When NPAS1 was tagged to GFP (NPAS1_GFP) and expressed in MN9D cells, the fusion protein was found to be excluded from the nucleus (Fig. 1Ai). Similar results were observed in a non-neuronal cell line, HEK293 (Fig. 1Bi). These suggest that NPAS1 is not constitutively localized to the nucleus.

A phylogenetic analysis reported previously revealed that NPAS1 is a mouse homolog of Drosophila Trachealess (28), and nuclear localization of Trachealess requires the presence of its interacting partner, Tango (the ortholog of mammalian Arnt) (29). We then went on to determine if NPAS1 also uses a partner for nuclear localization. When NPAS1_GFP was co-expressed with FLAG-tagged ARNT or ARNT2 (FLAG_ARNT or FLAG_ARNT2), NPAS1_GFP was found to be localized to the nucleus (Fig. 1, A, ii and iii). The same results were observed when HEK293 cells were used (Fig. 1, B, ii and iii), suggesting that nuclear translocation of NPAS1 by ARNT or ARNT2 is not cell type-specific. In the control experiment, the native GFP protein was evenly distributed between the cytoplasm and nucleus when co-expressed with FLAG_ARNT or FLAG_ARNT2 in both cell lines (results not shown), indicating that the nuclear translocation is specific to NPAS1 and not the GFP tag. Therefore, the localization of NPAS1 in the nucleus requires the presence of ARNT or ARNT2.


Figure 4
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  		FIGURE 4.
Mutation of NES within NPAS1 allows its accumulation in the nucleus. A, a nuclear export signal is predicted in the N terminus of NPAS1, from aa 310-317. All the leucine and isoleucine residues were mutated to alanine residues to generate NPAS1mNES mutant, as indicated. Solid boxes represent the bHLH region, and hatched areas represent the PAS A and PAS B regions. B, MN9D and HEK293 cells were transfected with plasmids overexpressing NPAS1mNES_GFP. Cells were stained with Hoechst stain to show the position of nuclei. NPAS1mNES_GFP was localized to both the nucleus and cytoplasm. The results are representative of three independent experiments. Each scale bar represents 50 µm.

 
We then proceeded to elucidate the mechanism underlying the nuclear localization of NPAS1 by ARNT. NPAS1 may be constitutively localized in the cytoplasm and requires ARNT for entry into the nucleus, or alternatively, NPAS1 may shuttle between the nucleus and cytoplasm but requires ARNT for it to be retained in the nucleus. To determine whether there is an active nuclear export of NPAS1 into the cytoplasm, leptomycin B was added to cells overexpressing NPAS1_GFP. Leptomycin B is an unsaturated, branched-chain fatty acid and has been found to be a specific inhibitor of proteins containing nuclear export signal. The suggested inhibition mechanism involves the direct binding of leptomycin B to CRM1, a protein involved in the nuclear export machinery, thereby blocking the binding of CRM1 to proteins containing the nuclear export signal (30, 31). As shown in Fig. 2, NPAS1_GFP was still localized to the cytoplasm upon the addition of leptomycin B. This suggests that NPAS1 is constitutively localized to the cytoplasm and does not shuttle between the nucleus and cytoplasm.

We then determined the region within NPAS1 that is important for the nuclear exclusion of NPAS1. The N terminus (aa 1-354), consisting of the bHLH and PAS domains, and the C-terminal (aa 348-594) domain of NPAS1 were individually fused upstream of GFP to generate NPAS1(N)_GFP and NPAS1(C)_GFP fusion protein, respectively (Fig. 3A). When co-expressed with FLAG, NPAS1(N)_GFP was found to be excluded from the nucleus (Fig. 3Bi). However, upon co-expression with FLAG_ARNT or FLAG_ARNT2, NPAS1(N)_GFP was found to be localized to the nucleus (Fig. 3C). On the other hand, NPAS1(C)_GFP was found in both the cytoplasm and nucleus when co-expressed with FLAG (Fig. 3Bii). These results suggest that the N terminus of NPAS1 excludes it from the nucleus in the absence of ARNT overexpression.

Using a nuclear export signal prediction program, the N terminus of NPAS1 was found to contain a NES, LSLGLTIL, at aa 310-317. To determine whether this NES plays a role in excluding NPAS1 from the nucleus, site-directed mutagenesis was performed on the NES. Because basic amino acids are known to be crucial for NES function, all the leucine and isoleucine residues within the NES of NPAS1 were mutated to alanine (Fig. 4A), and the subcellular localization of the mutated NPAS1 was determined. In transfected MN9D and HEK293 cells, the fusion protein was found to be localized to the nuclei, indicating that the nuclear exclusion of NPAS1 is due to this NES (Fig. 4B).

It has been reported that NPAS1 and ARNT are able to interact with each other both in vitro and in vivo (22). However, the exact domains where they interact have not been mapped out. Because it is known that bHLH-PAS domain proteins interact via their N termini, we decided to test if NPAS1 also interacts with ARNT via its N terminus. From our unpublished data we know that overexpression of NPAS1 is more efficient in HEK293 cells compared with other cell lines. Therefore, we decided to overexpress FLAG_fl NPAS1_GFP, FLAG_NPAS1(N), and FLAG_NPAS1(C) individually with HA_fl ARNT in HEK293 cells and use agarose conjugated to FLAG Ab for the pulldown assay. As shown in Fig. 5A, only fl NPAS1 and NPAS1(N), but not NPAS1(C), were able to pull down fl ARNT. This result suggests that NPAS1 interacts with ARNT via the N terminus. To determine the domain where ARNT interacts with NPAS1, FLAG_fl ARNT, FLAG_ARNT(N), and FLAG_ARNT(C) were individually overexpressed with HA_fl NPAS1 in HEK293 cells, and agarose conjugated to FLAG Ab was used for the pulldown assay. As shown in Fig. 5B, only fl ARNT and ARNT(N), but not ARNT(C), were able to pull down fl NPAS1. This shows that ARNT also interacts with NPAS1 via its N terminus.


Figure 5
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  		FIGURE 5.
Interaction between NPAS1 and ARNT is via their N termini. A, protein lysates from HEK293 cells overexpressing either FLAG_fl NPAS1, FLAG_NPAS1(N), or FLAG_NPAS1(C) with HA_fl ARNT was used for immunoprecipitation (IP) with FLAG Ab-conjugated agarose as described under in under "Experimental Procedures." Bound proteins were detected with FLAG and HA antibodies. Only fl NPAS1 and NPAS1(N) were able to pull down fl ARNT. Western blots (WB) on whole cell lysates (WCL) were carried out to show proper expression of the fusion proteins. Lanes 1, FLAG_fl NPAS1 + HA_fl ARNT; 2, FLAG_NPAS1(N) + HA_fl ARNT; 3, FLAG_NPAS1(C) + HA_fl ARNT. B, protein lysates from HEK293 cells overexpressing either FLAG_fl ARNT, FLAG_ARNT(N), or FLAG_ARNT(C) with HA_fl NPAS1 as used for immunoprecipitation with FLAG antibody-conjugated agarose. Only fl ARNT and ARNT(N) were able to pull down fl NPAS1. A Western blot on WCL was carried out with FLAG and HA to show proper expression of the fusion proteins. Lanes 1, FLAG_fl ARNT + HA_fl NPAS1; 2, FLAG_ARNT(N) + HA_fl NPAS1; 3, FLAG_ARNT(C) + HA_fl NPAS1.

 
There are two ways in which ARNT can facilitate the nuclear translocation of NPAS1. ARNT can either directly interact with NPAS1, thereby guiding it into the nucleus via its own nuclear localization signal (NLS), or it can activate the transcription of some other genes, which in turn bring NPAS1 into the nucleus. To determine whether the latter mechanism is at work, we eliminated the transcriptional activation function of ARNT by deleting the basic region that binds DNA. This mutant ARNT, without the basic region, was designated ARNT{Delta}b and found to have no transcriptional activation function but was still able to interact with NPAS1 (results not shown). From Fig. 6, it was shown that upon co-expression with FLAG_ARNT{Delta}b, NPAS1_GFP was still able to localize to the nucleus, suggesting that the transcriptional regulation of ARNT is not essential for localizing NPAS1 to the nucleus.

Many of the members of the bHLH-PAS domain protein family are known to contain at least one transactivation domain, which is responsible for transcriptional activation (10, 32, 33). We decided to investigate if NPAS1 contains any autonomous transactivation domain using a yeast one-hybrid system. In this case, a heterologous system involving the LexA and GAL4 DNA binding domain was used. Full-length NPAS1 cDNA as well as the N and C terminus of NPAS1 were individually fused downstream of the LexA DBD of the pLexA plasmid to generate NPAS1 fusion proteins that are able to bind to LexA operators. These constructs (Fig. 7A) were transformed into EGY48 yeast cells, which had previously been transformed with the lacZ reporter gene under the control of LexA operators. As shown in Fig. 7, B and C, there was no activation of lacZ by LexA_NPAS1 or LexA fused to different domains of NPAS1. In contrast, the LexA_ARNT and LexA_ARNT(C) fusion proteins were able to activate the lacZ reporter gene, confirming the presence of an activation domain in ARNT, which lies in the C terminus that has already been reported previously (34).

It is known that the yeast cell does not contain all the post-translational modifications found in mammalian cell. To rule out that the lack of reporter activation by NPAS1 (Fig. 7, B and C) was due to certain post-translational modifications missing in the yeast system and rendering NPAS1 non-functional, a similar experiment was carried out in HEK293 cells and MN9D cells. This time round, full-length, or different deletion mutants of NPAS1 were fused downstream to the GAL4 DNA binding domain (Fig. 7D). The profile of reporter gene activation in HEK293 cells (Fig. 7E) was the same as that observed in the yeast system, further supporting the absence of any TAD in NPAS1. However, in MN9D cells, no activation of reporter gene was observed for full-length ARNT, although ARNT(C) induced reporter gene activation. To ensure that the lack of reporter gene activation was not due to the different fusion proteins not being properly expressed, a Western blot was carried out on total protein lysates isolated from the different transfected cells. The blot showed that all the GAL4 fusion proteins were properly expressed (Fig. 7F).


Figure 6
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  		FIGURE 6.
Transcriptional property of ARNT is not required for nuclear translocation of NPAS1. A, internal deletion mutant of ARNT lacking the basic region (aa 59-85) but with its NLS intact. Hatched areas represent the PAS A and PAS B regions. B, MN9D or HEK293 cells were co-transfected with plasmid overexpressing NPAS1_GFP and FLAG_ARNT{Delta}b. After 2 days cells were fixed and stained with Hoechst stain to show position of the nuclei. NPAS1_GFP was localized to both the nucleus and cytoplasm. The cells were stained with FLAG Ab and detected with rhodamine-conjugated secondary Ab to show expression of FLAG_ARNT{Delta}b. The results are representative of three independent experiments. Each scale bar represents 50 µm.

 
Because we could not detect any TAD in NPAS1, we decided to test if NPAS1 contains any repression domain instead. This time around a reporter plasmid consisting of luciferase gene under the control of a strong thymidine kinase promoter and GAL4 binding sites was used. When GAL4 DBD protein was co-expressed with this reporter plasmid, there was a very high level of luciferase (Luc) activity. In contrast, overexpression of GAL4_fl NPAS1, GAL4_NPAS1(N), or GAL4_NPAS1(C) decreased the reporter activity significantly (Fig. 8A), indicating the presence of repression domains.

We went on to further map out the repression domains within the N and C termini of NPAS1 by generating a series of truncation clones as shown in Fig. 8Bi. When these truncation clones were tested for their effect on the reporter system, it was found that three separate deletion mutants were able to suppress the expression of the reporter gene. These results indicate that there are three separate repression domains within NPAS1.

Because it has previously been reported that SIM2 was able to suppress the TAD of ARNT (35), we asked if NPAS1 behaves the same way with ARNT and ARNT2. A heterologous system employing GAL4 DNA binding sites upstream of a CAT reporter gene was used. GAL4_fl ARNT, GAL4_fl ARNT2, or GAL4_ARNT(C) was individually co-expressed with full-length NPAS1 (tagged with GFP) in HEK293 and MN9D cells, and the level of chloramphenicol (CAT) activity was determined (Fig. 9). When fl NPAS1_GFP was co-expressed with GAL4_fl ARNT or GAL4_fl ARNT2, the reporter CAT activity was reduced significantly in both cell lines.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
As transcription factors, the bHLH-PAS domain proteins need to be localized to the nucleus to carry out their role as transcriptional regulators. To date, many different mechanisms for nuclear localization of the different bHLH-PAS domain proteins have been reported. For example, both mammalian ARNT and SIM2 are constitutively localized to the nucleus. A bipartite nuclear localization signal within ARNT is found to be responsible for this nuclear localization (36). On the other hand, both NLS and NES (37) were identified in AhR. In this case, AhR is able to shuttle between the cytoplasm and nucleus but requires the binding of its ligand, TCDD, to be retained in the nucleus (38). Hypoxia-inducible factor is also localized to the nucleus upon exposure to hypoxia (39). In addition, Drosophila Trachealess and SIM2 require the presence of Tango, the ARNT homolog to be localized in the nucleus (29).

Immunohistochemical studies have shown that NPAS1 is localized to the nucleus in interneurons (21) and cortical neurons (22). However, it is not known whether NPAS1 is constitutively localized in the nucleus on its own or it requires other proteins for its nuclear localization. Using NLS prediction software, no NLS was identified in NPAS1. In our studies we showed that NPAS1 was excluded from the nucleus when it was expressed alone but translocated into the nucleus when co-expressed with ARNT or ARNT2 (Fig. 1). We also found that NPAS1 is still excluded from the nucleus upon treatment with leptomycin B (Fig. 2), suggesting that NPAS1 does not shuttle between the cytoplasm and nucleus. This is consistent with the fact that no NLS was predicted to exist within NPAS1. Using deletion studies, we discovered that the N terminus is responsible for this nuclear exclusion (Fig. 3), and the mutation of a predicted NES within this region successfully localized NPAS1 to the nucleus (Fig. 4). In addition, we found that deletion of the basic region of ARNT (which abolishes its transcriptional property) has no effect on its ability to localize NPAS1 to the nucleus (Fig. 5).


Figure 7
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  		FIGURE 7.
Absence of autonomous transactivation domain in NPAS1. A, schematic representations of the different constructs used for transformation into EGY48 yeast strain to test for transactivation domain within NPAS1. The boundary defined by deleted amino acids is indicated by parentheses as subscripts to the name of each construct. Dotted boxes represent the LexA DNA binding domain. Solid boxes represent the bHLH region, and hatched areas represent the PAS A and PAS B regions. B, EGY48 yeast strains were transformed with the respective constructs shown in A and plated out on synthetic dropout plate lacking uracil and histidine, with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) added. All three constructs of NPAS1 (ii, iii, and iv)- and ARNT(N) (vi)-LexA fusion proteins did not activate the reporter gene. Both full-length ARNT (v)- and ARNT(C) (vii)-LexA fusion proteins activate the lacZ reporter gene, as indicated by the dark color. C, graph showing the quantitative differences in the beta-galactosidase activity by the different constructs. The results are from one representative experiment (n = 3) and are presented as the mean and S.E. (n = 5). D, schematic diagram of the different plasmids used for mammalian cells transfection in this study. The boundary defined by deleted amino acids is indicated by parentheses as subscripts to the name of each construct. Dotted boxes represent the GAL4 DNA binding domain. Solid boxes represent the bHLH region, and hatched areas represent the PAS A and PAS B regions. E, HEK293 cells were co-transfected with a CAT reporter plasmid together with the respective plasmids shown in panel C and assayed for CAT activity as described under "Experimental Procedures." All three constructs of NPAS1 (GAL4_fl NPAS1, GAL4_NPAS1(N), and GAL4_NPAS1(C)) and GAL4_ARNT(N) did not activate the reporter gene. Both full-length ARNT (v)- and ARNT(C) (vii)-GAL4 fusion proteins activate the CAT reporter gene. The results are from one representative experiment (n = 3) and are presented as the mean and S.E. (n = 3). F, analysis of GAL4 fusion proteins. Equal amount of transfected cell lysates from HEK293 cells were run on SDS-PAGE gel, and a Western blot was carried out with GAL4 antibodies. All the fusion proteins are expressed properly with their expected molecular weight. Results are representative of three separate experiments.

 


Figure 8
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  		FIGURE 8.
Mapping of repression domains in NPAS1. A, HEK293 cells were transfected with plasmids expressing GAL4, GAL4_fl NPAS1, GAL4_NPAS1(N), or GAL4_NPAS1(C) together with reporter plasmid pGAL4_TK_Luc and internal control plasmid pRL_SV40. Protein lysates were collected from these transfected cells for dual luciferase assay as described under "Experimental Procedures." All GAL4_fl NPAS1, GAL4_NPAS1(N), and GAL4_NPAS1(C) repressed the level of reporter gene. RE, response element. B, HEK293 cells were transfected with a series of GAL4 plasmids expressing NPAS1 deletion mutants together with reporter plasmid pGAL4_TK_Luc and internal control plasmid pSV40_RL. Protein lysates were collected from these transfected cells for the dual luciferase assay as described under "Experimental Procedures." Three deletion mutants, NPAS1(71-165), NPAS1(206-290), and NPAS1(501-594) were able to repress the level of reporter gene. Equal amounts of transfected cell lysates from HEK293 cells were run on SDS-PAGE gel, and a Western blot (WB) was carried out with GAL4 antibodies. All the fusion proteins are expressed properly with their expected molecular weight. All results are from one representative experiment(n = 3) and are presented as the mean and S.E. (n = 3), and shown statistically significant differences according to two-tailed Student t test: *, p < 0.01.

 


Figure 9
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  		FIGURE 9.
Partial trans-repression of NPAS1 on ARNT and ARNT2 transactivation functions. A, there is only partial repression by NPAS1 on ARNT and ARNT2 transactivation functions. HEK293 cells were transfected with increasing amounts of plasmid overexpressing NPAS1_GFP or GFP together with reporter plasmid pGSCAT and internal control plasmid pSV40_beta-gal with either plasmid overexpressing GAL4_ARNT or GAL4_ARNT2. CAT and beta-galactosidase activities were determined according to that described under "Experimental Procedures." The data presented for NPAS1_GFP had been normalized to that of the GFP control. Maximum repression was observed to be about 50% at 1.5 µg of plasmid overexpressing NPAS1_GFP. Bi, NPAS1 represses the transactivation of ARNT and ARNT2 but not ARNT(C) in HEK293 cells. HEK293 cells were transfected with plasmid overexpressing NPAS1_GFP together with reporter plasmid pGSCAT and internal control plasmid pSV40_beta-gal with either plasmid overexpressing GAL4_ARNT, GAL4_ARNT2, or GAL4_ARNT(C). CAT and beta-galactosidase activities were determined according to that described under "Experimental Procedures." Repression by NPAS1 was observed for ARNT and ARNT2 but not ARNT(C). ii, NPAS1 represses the transactivation of ARNT, ARNT2, and ARNT(C) in MN9D cells. MN9D cells were transfected with plasmid overexpressing NPAS1_GFP together with reporter plasmid pGSCAT and internal control plasmid pSV40_beta-gal with either plasmid overexpressing GAL4_ARNT, GAL4_ARNT2, or GAL4_ARNT(C). CAT and beta-galactosidase activities were determined according to that described under "Experimental Procedures." Repression by NPAS1 was observed for ARNT and ARNT2 and ARNT(C). All results are from one representative experiment (n = 3) and are presented as the mean and S.E. (n = 3).

 
Some of the bHLH-PAS domain proteins are activated by the binding of a ligand to the C terminus. One example is that of AhR, upon binding to xenobiotics, translocates into the nucleus and heterodimerizes with ARNT to activate transcription (38, 39). However, it is unlikely that NPAS1 requires a ligand because by simply co-expressing with ARNT or ARNT2, NPAS1 is able to enter the nucleus (Fig. 1). This is in contrast to that of AhR, where the presence of ARNT did not result in AhR entering the nucleus. Moreover, when NPAS1 and ARNT or ARNT2 were co-expressed in other non-neuronal cell lines (Fig. 1B), the same phenomenon was observed, supporting the notion that NPAS1 nuclear translocation is not cell type-specific. This further reinforces the notion that NPAS1 may not require a ligand for nuclear translocation. Moreover, this mechanism of nuclear translocation was also evident in Drosophila (29), suggesting that this mechanism may be conserved throughout the animal kingdom.

Many of the bHLH-PAS domain proteins are known to bind ARNT as a common heterodimeric partner (40) and that such interaction is necessary for binding DNA and regulating transcription (14). A previous study showed that NPAS1 is able to interact with ARNT both in vivo and in vitro and that this interaction is important for NPAS1 binding to erythropoietin enhancer (22). In our study we showed that NPAS1 and ARNT interact via their N termini, respectively. We propose that NPAS1 is initially localized in the cytoplasm. However, the interaction of ARNT with NPAS1 guides NPAS1 into the nucleus, probably via the NLS of ARNT. We postulate that upon heterodimerization with ARNT, the NES within the N terminus of NPAS1 is masked, thereby sequestering NPAS1 in the nucleus.

Many of the bHLH-PAS proteins harbor at least one TAD (32, 33) and upon dimerization with ARNT bind to the respective response element and activate gene transcription. However, our experiments did not identify any TAD in NPAS1 (Fig. 7), suggesting that NPAS1 may not play a positive transcriptional role. A number of bHLH-PAS domain proteins are known to negatively regulate transcription, which include inhibitory PAS domain protein (41) and SIM2 (35). In the case of SIM2, such negative regulation is due to the presence of two repression domains within the C terminus of murine SIM2. Upon heterodimerization with ARNT, the effect of these repression domains overrides the ARNT activation domain.

Although it has been reported that NPAS1 suppressed the expression of erythropoietin (22), there are no published data supporting the presence of repression domains within NPAS1. Using yeast one-hybrid assays, we have identified at least three non-overlapping repression domains within NPAS1 (Fig. 8). Using a heterologous system, when full-length NPAS1 was co-expressed with ARNT or ARNT2, the transcriptional activation functions of ARNT and ARNT2 were reduced, suggesting that NPAS1 negatively regulate the activity of ARNT and ARNT2 (Fig. 9B). However, unlike SIM2, which totally abolished the transactivation function of ARNT (33), NPAS1 only partially suppressed ARNT and ARNT2 (Fig. 9A). We postulate that some other factors may cooperate synergistically with NPAS1 to totally abolish the transactivation of ARNT and ARNT2. In this experiment an exogenous GAL4 promoter was used (Fig. 9), indicating that it is not likely that NPAS1 inhibits ARNT and ARNT (2) transactivation function through competitive binding with other factors to the promoter. Because we have shown that NPAS1 is able to interact with ARNT (Fig. 5) and that deleting the N-terminal domain of NPAS1 (which is required for interaction with ARNT) abolishes the ability of NPAS1 to negatively regulate the activity of ARNT,3 we propose that NPAS1 may inhibit transcription by overriding the transactivation function of adjacent transcription factors, a mechanism similar to the one used by SIM2 to repress transcription (35).


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 65-65163334; Fax: 65-67792486; E-mail: dbsltm{at}nus.edu.sg.

2 The abbreviations used are: bHLH, basic helix-loop-helix; SIM, Single-minded; PAS, PER, ARNT, AHR, SIM homology region; AhR, arylhydrocarbon receptor; ARNT, arylhydrocarbon nuclear translocator; ARNT2, arylhydrocarbon nuclear translocator 2; NPAS1, neuronal PAS domain protein 1; NPAS3, neuronal PAS domain protein 3; TAD, transactivation domain; GFP, green fluorescence protein; NES, nuclear export signal; NLS, nuclear localization signal; DBD, DNA binding domain; TK, thymidine kinase; Luc, luciferase; CAT, chloramphenicol acetyltransferase; aa, amino acids; HA, hemagglutinin; HEK cells, human embryonic kidney cells; PBS, phosphate-buffered saline; Ab, antibody; beta-gal, beta-galactosidase. Back

3 C. H. L. Teh, C. C. Loh, K. K. Y. Lam, T. Yan, and T. M. Lim, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank the Biomedical Research Council, Agency for Science Technology and Research, Singapore, for supporting this project.



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 RESULTS
 DISCUSSION
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