 |
INTRODUCTION |
The differentiation of mammalian neurons during development is a
highly complex process involving regulation and coordination of gene
expression at multiple steps. To understand the basic mechanisms
underlying this complex pathway, identification of genes that are
differentially expressed during neural differentiation is an important
approach. P19 embryonal carcinoma
(EC)1 cells derived from a
mouse embryo have been used extensively as a model system for in
vitro neural differentiation (1, 2). Exposure of aggregated P19
cells with retinoic acid (RA) results in the differentiation of cells
with many fundamental phenotypes of mammalian nervous system (3).
During the early stages of neuronal differentiation of P19 cells, the
mammalian homologues of several Drosophila gene products
such as Motch, Mash-1, Wnt-1, and transduction-like enhancers of
split are expressed (4-7). Moreover, a number of proteins including
retinoic acid receptors, retinoid X receptors, epidermal growth factor
receptor, and transcription factors such as Oct-3/4, Brn-2, and Bdm-1
have been identified (8-10). Perhaps a limited number of RA- and
aggregation-responsive genes trigger the neuronal differentiation
pathway of P19 cells.
Recently we found that a transcriptional corepressor, a component of
COP9 signalosome, Trip15/CSN2, was highly expressed at the early stage
of neural differentiation of RA-treated P19 cells. The deduced amino
acid sequence of rat Trip15/CSN2 gene is completely identical with
those of mouse and human homologues (DDBJ/EMBL/GenBankTM
accession no. AB081072; Ref. 11). Enforced expression of sense
rat Trip15/CSN2 RNA was sufficient to convert P19 cells into neurons,
but not glial cells in the absence of RA, only after the aggregation
treatment, accompanying the down-regulation of Oct-3/4 transcript,
which maintains the undifferentiated state of P19 cells. Thus, the
induction of Trip15/CSN2 prior to down-regulation of Oct-3/4 gene
expression is required for the commitment of P19 cells to neuronal lineage.
Trip15/CSN2 was originally identified as a thyroid hormone receptor
(TR)-interacting protein and acts as a transcriptional corepressor
(12). Trip15/CSN2 interacts with a subset of nuclear hormone receptors
such as DAX-1, ecdysone receptor, chicken ovalbumin promoter
transcription factor 1 (COUP-TF1), its Drosophila homologue Seven-up, Fushi-tarazu-F1 (Ftz-F1), and TRs, but not with retinoic acid
receptors and retinoid X receptors (13, 14). Trip15/CSN2 is conserved
in a wide range of organisms and was also identified as a component of
a 26 S proteasome lid-like complex termed COP9 signalosome (CSN; Refs.
15 and 16). The CSN complex was originally identified as a repressor of
light-controlled development in Arabidopsis thaliana (17).
In animals, the CSN complex is localized in the nucleus and possesses a
kinase activity that specifically phosphorylates transcriptional
regulators such as p105, I
B
, c-Jun, and p53 (18-20). Although
the mutual interaction between Trip15-nuclear receptor complex and CSN
complex is still unknown, these facts indicate that the CSN complex
participates in various signal transduction pathways. The
proteasome-COP9 complex-initiation factor 3 domain in the C-terminal
region of Trip15/CSN2 stabilizes protein-protein interaction within the
CSN complex (21-24). The N-terminal region of Trip15/CSN2 has been
reported to be sufficient for the effector functions of Trip15/CSN2. In
fact, Trip15/CSN2 associates with its binding partners, such as TR and
DAX-1, through its N-terminal region (12-14).
Considering the importance of Trip15/CSN2 in neuronal differentiation,
we tried to find out novel molecules that bind to Trip15/CSN2 and
possibly act as new downstream targets or regulators of Trip15/CSN2. We
used the N-terminal region of rat Trip15/CSN2 as a bait for the yeast
two-hybrid screening of RA-treated P19 cell cDNA library. Five of
17 positive cDNA clones have been turned out to be mouse Nif3l1 (NGG1-interacting factor 3-like 1) gene.
Nif3l1 cDNA was isolated through a suppression
subtractive hybridization between the spermatogonia- and
spermatocyto-derived cell lines and possesses a high homology to yeast
Nif3 (Ngg1-interacting factor 3) gene (25). The mouse Nif3l1
gene encodes a cytoplasmic protein consisted of 376 amino acids and is
highly conserved from bacteria to mammals. Expression of Nif3l1
transcript is detected throughout mouse embryonic development (25);
however, the real biological function of Nif3l1 is still unknown.
Yeast Nif3 was originally identified in yeast two-hybrid screening as a
NGG1-interacting protein (26). NGG1 was isolated based on its
requirement for the full inhibition of transcriptional activation by
GAL4 protein in glucose media (27). Independently, ADA3/NGG1 was
isolated based on the ability of mutations to suppress the toxic
effects of overexpression of the viral activator VP16 in yeast (28).
Therefore, ADA3/NGG1 was involved in transcriptional activation and
repression (26, 29, 30). Alternation deficiency in activation (ADA)
proteins have been found to be required for transcriptional activation
by a number of yeast activators (28, 31, 32). In yeast, ADA3/NGG1 is
found as multisubunit complexes containing three to four additional ADA
proteins and different TAFs and Spt (30, 31, 33-36). In mammalian
cells, the majority of ADA3/NGG1 protein also seems to be complexed
with Spt and TAF or TAF-like factors, making up several types of
complexes (37-41). These complexes are thought to be functional
homologs of the yeast ADA complexes (37, 39, 41). Recently, genetic
studies in yeast have demonstrated a crucial role of ADA complex in the
transactivation function of mammalian nuclear hormone receptors
(42-45). Nonetheless, there is no report concerning about the function
of Nif3l1 in transcriptional regulation.
In this study, we show that the cytoplasmic Nif3l1 protein could be
translocated into the nuclei by the association with Trip15/CSN2 and
that it synergized the transcriptional repression activity of
Trip15/CSN2. In addition, Nif3l1 implicates in neural differentiation of P19 cells, perhaps through the down-regulation of Oct-3/4
transcript, which suppresses neurogenic genes including Mash-1 to
maintain the undifferentiated state of P19 cells. Considering these
results and the expression of Nif3l1 in early developing
brain, it seems likely that both Nif3l1 and Trip15/CSN2 play an
important role in neural differentiation.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Experimental Animals--
Mouse P19 embryonal
carcinoma cells were obtained from American Type Culture Collection
(Bethesda, MD). Cells were maintained in
-minimal essential medium
(Invitrogen) containing 10% fetal calf serum (FCS) at 37 °C
in a humidified atmosphere of 5% CO2 in air. To induce
neural differentiation, 1 × 106 P19 cells aggregated
in 10-cm bacteriological grade dishes were cultivated in 10 ml of
-minimal essential medium containing 10% FCS and 5 × 10
7 M all-trans-retinoic acid (RA)
(Sigma) for 4 days. Cell aggregates were then suspended with mild
pipetting and transferred to tissue culture dishes. The cells were
cultivated in RA-free
-minimal essential medium containing 10% FCS
for additional 3 days to induce
-tubulin type III-positive neurons
and for 7 days to induce glial fibrially acidic protein (GFAP)-positive
glial cells. COS-7 cells were obtained from Japanese Cancer Resources
Bank (Tokyo, Japan) and maintained in Dulbecco's modified Eagle's
medium containing 5% FCS.
Male and female ICR mice were purchased from Charles River Japan
(Kanagawa, Japan) and allowed to mate to produce offspring at the
experimental animal facility (Tokyo University of Science). All mouse
were kept under a 12-h light/12-h dark cycle at 22-24 °C. Standard
laboratory feed (MR standard, Nousan Ltd, Kanagawa, Japan) and tap
water were given ad libitum. Mouse care and handling conformed to the National Institutes of Health guidelines for animal
research. The experimental protocols were approved by the Institutional
Animal Care and Use Committee.
Yeast Two-hybrid Screening--
Rat Trip15/CSN2 full-length
cDNA (amino acids 1-443) and its fragment covering amino acids
1-275 were inserted into pAS2-1 (Clontech, Palo
Alto, CA) in frame with the coding sequence for GAL4 DNA binding domain
(DBD) and used for yeast two-hybrid screening as bait vectors. A
RA-treated P19 cell cDNA library was constructed using plasmid
pACT-2 (Clontech). Poly(A)+ RNA was
prepared from the P19 cells treated with 5 × 10
7
M RA for 12 h, and cDNA was prepared by a cDNA
Synthesis Kit (Stratagene, Toyobo Co. Ltd., Tokyo, Japan). cDNA
fragments digested with XhoI and EcoRI were
inserted into pACT-2 in frame with the coding sequence for the GAL4
activation domain. Yeast two-hybrid screening was performed as
described for the Matchmaker Two-Hybrid System 2 Protocol
(Clontech). Briefly, competent Y153 yeast tester strain (His
, Leu
, Trp
)
containing the His3 and LacZ genes linked to the
GAL4 promoter were cotransformed with the bait and library plasmids,
and colonies were selected on His
, Leu
, and
Trp
plate with 25 mM 3-aminotriazole. The
colonies were confirmed to be truly positive for His and
-galactosidase (
-gal) production.
Full-length mouse Nif3l1 cDNA was amplified by reverse
transcriptase-polymerase chain reaction (RT-PCR) using total P19 cell RNA and the primers corresponding to mouse Nif3l1 cDNA
(24). The primers used are as follows: 5'-primer, 5'-GAA AGA GCT TCT GCG ACT GG-3'; 3'-primer, 5'-CAC AGC CGT TTC AAT CCA GG-3'. The PCR
product was inserted into the EcoRV site of pGEM-5zf(+)
(Promega, Madison, WI). The plasmid was designated as
pGEM-5zf(+)-Nif3l1.
Glutathione S-Transferase (GST) Pull-down Assay--
To prepare
GST-Trip15/CSN2 fusion proteins, Trip15/CSN2 cDNA fragment was
inserted into the expression vector pGEX-2T (Amersham Biosciences,
Buckinghamshire, UK) in frame with the coding sequence for GST. The
plasmid was designated as a pGEX-Trip15/CSN2. pGEX-Trip15/CSN2 was
introduced into Escherichia coli JM109, and the fusion
protein synthesized was purified on glutathione-Sepharose beads
(Amersham Biosciences) as described by Kaelin et al. (46).
Glutathione-Sepharose beads were washed three times with the binding
buffer (50 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 150 mM NaCl, 10% glycerol, 0.5 mg/ml
bovine serum albumin, 5 mM 2-mercaptoethanol, 0.5% Nonidet
P-40), and subsequently 5 µg of the purified fusion protein were
incubated with 2% glutathione-Sepharose beads in a final volume of 500 µl for 1 h at 4 °C with gentle rotation. GST encoded in the
vector was also prepared under the same conditions and immobilized as a
control. The beads preloaded with GST-Trip15/CSN2 fusion proteins were
washed three times with the binding buffer, and 35S-labeled
Nif3l1 proteins synthesized by the RiboMaxTM Large Scale
RNA production system (Promega) were then added. The beads were rocked
continuously for 1 h at 4 °C. The beads were washed five times
with 500 µl of the binding buffer, and the bound proteins were eluted
with 15 µl of the SDS sample buffer (62.5 mM Tris-HCl, pH
6.8, 2% SDS, 10% glycerol) and electrophoresed on 12% SDS-PAGE. The
gel was fixed, dried up, and exposed to the x-ray film.
Immunoprecipitation Assay--
Flag-tagged Nif3l1 expression
vector was constructed by the insertion of blunt-ended
NcoI-SalI ORF fragment of pGEM-5zf(+)-Nif3l1 into
the blunt-ended EcoRI-SalI site of pFLAG-CMV2
(Sigma). Full-length rat Trip15/CSN2 cDNA clone was transferred
from pGEM-7zf(+) (Promega) to pACT2 after cleavage with
EcoRI and XhoI. HA-tagged Trip15/CSN2 expression
vector was constructed by the insertion of blunt-ended XhoI-BglII ORF fragment of pACT2-Trip15/CSN2 into
the blunt-ended BamHI-EcoRV site of pcDNA3
(Invitrogen). For immunoprecipitation studies, COS-7 cells (2 × 106) were transfected with pcDNA3-HA-Trip15/CSN2 and/or
pFlag-CMV2-Nif3l1 by lipofection with DOTAP (Roche Molecular
Biochemicals). Forty-eight hours after the transfection, the cells were
lysed in 0.5 ml of TNM buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride). The cell
lysate was incubated with 2% anti-Flag M2-agarose affinity gel (Sigma) at 4 °C overnight. The affinity gels were washed three times with 1 ml of TNM buffer and suspended in 15 µl of the SDS sample buffer containing 5% 2-mercaptoethanol. The affinity gels were heated at
95 °C for 5 min and subjected to 12% SDS-PAGE. The proteins were
transferred to a Clear Blot Membrane (Atto, Tokyo, Japan). The membrane
were blocked in TBST (20 mM Tris-HCl, pH 7.4-buffered saline, 0.02% Tween 20) containing 1% nonfat dry milk for 1 h at
room temperature and incubated overnight at 4 °C with anti-HA antibody (1:1000 dilution, Sigma), anti-Flag antibody (1:400 dilution, Sigma), or anti-rat Trip15/CSN2 antibody (1:1000 dilution; Ref. 11).
After washing three times with TBST, the membrane was incubated with
anti-mouse IgG conjugated with horseradish peroxidase (1:5000 dilution,
Sigma). Signals were visualized with the ECL system (Amersham
Biosciences) according to the protocol from the manufacturer.
Northern Blot Analysis--
Total RNAs were extracted from P19
cells and various mouse tissues by the acidic guanidine
thiocyanate-phenol-chloroform method (47). Aliquots of 20 µg total
RNA were electrophoresed on 1% agarose, 6% formaldehyde gel,
transferred to Hybond-N+ nylon membrane (Amersham
Biosciences), and hybridized with 32P-labeled 0.6-kb
EcoRI-DraI fragment of pGEM-5zf(+)-Nif3l1, 2.1-kb BamHI-EcoRI fragment of pGEM-rTrip15/CSN2 (11),
and PCR-amplified mouse Oct-3/4 cDNA (5'-primer, 5'-CCT GGC TAA GCT
TCC AAG GGC-3'; 3'-primer, 5'-GTT CTA GCT CCT TCT GCA GGG C-3') (48),
Mash-1 cDNA (5'-primer, 5'-CAC AAG TCA GCG GCC AAG CAG-3';
3'-primer, 5'-GAT CCC TCG TCG GAG GAG TAG-3') (49), and acidic
ribosomal phosphoprotein (PO) cDNA (5'-primer, 5'-CAG CTC TGG AGA
AAC TGC TG-3'; 3'-primer, 5'-GTG TAC TCA GTC TCC ACA GA-3') (50). The PO gene, also called 36B4 (51), was used as an internal control gene,
because its expression level has been shown to be invariant during
RA-induced differentiation of P19 cells (6). Developed x-ray films were
scanned in a Macintosh Performa 6410, and the expression levels of RNAs
were quantified using Image 1.62 ppc program (National Institutes of
Health, Bethesda, MD).
Construction of Tetracycline (Tet)-controlled Nif3l1 Expression
System--
Tet-controlled Nif3l1 expression in P19 cells was
performed using the Tet-OffTM gene expression
system (Clontech). At the first step, P19 cells were transfected with a pTet-Off vector using DOTAP and cultivated in
the presence of 400 µg/ml G418 (Wako, Tokyo, Japan) for selection. Resulting G418-resistant colonies were then screened by the transient transfection with a pTRE2-Luc vector (Clontech) to
isolate the best P19 Tet-Off cells exhibiting a low background and high
Tet-dependent induction of luciferase (Luc) by the
withdrawal of Tet. The selected P19 Tet-Off cell line was designated as
R13. pTRE2-sense and -antisense Nif3l1 expression vectors were
constructed by the insertion of blunt-ended
NcoI-SalI ORF fragment of pGEM5zf(+)-Nif3l1 into
the blunt-ended HindIII-SalI and
SalI-EcoRV sites of pTRE2
(Clontech), respectively. At the second step, R13
cells were transfected with pTRE2-sense Nif3l1 or pTRE2-antisense
Nif3l1 together with pTK-Hyg (Clontech) and
cultivated in the presence of 400 µg/ml hygromycin (Wako) and 2 µg/ml Tet (Sigma) for selection. Hygromycin-resistant colonies were
then screened for the induction of sense and antisense Nif3l1 RNAs in the absence of Tet by Northern blot and
RT-PCR. The cell lines that express sense and antisense Nif3l1
RNA after Tet removal thus obtained were designated as R13NifS and
R13NifA, respectively.
Immunocytochemistry--
P19 cells were cultivated in a Lab-Tek
II Chamber slide (Nalge Nunc International, Naperville, IL) and fixed
with 4% paraformaldehyde. The cell samples were incubated in
Ca2+,Mg2+-free phosphate-buffered saline
(PBS(
)) containing 10% normal rabbit serum for 30 min at 37 °C.
The samples were then incubated for 2 h at 37 °C with
antibodies against
-tubulin III (1:1000 dilution, Sigma) or GFAP
(1:400 dilution, Sigma) in the same solution described above. After
washing with PBS(
), the samples were incubated with a
biotin-conjugated rabbit anti-mouse IgG+IgA+IgM (Nichirei, Tokyo,
Japan) as a secondary antibody and followed by the incubation with a
peroxidase-conjugated streptavidin (Nichirei). Visualization of the
signal was carried out using 3,3'-diaminobenzidine. Nuclei were
counterstained with hematoxylin.
Western Blot Analysis--
For Western blot analysis, cells were
collected, lysed in the SDS sample buffer without bromphenol blue and
2-mercaptoethanol, and sonicated for 4 s. The resulting lysates
were cleared by centrifugation at 15,000 rpm for 10 min. After protein
concentration was determined by BCA kit (Pierce), 5% 2-mercaptoethanol
(final concentration) was added. Aliquots of 20 µg of cell lysate
were heated at 95 °C for 5 min and subjected to 12% SDS-PAGE. The
proteins were transferred to a Clear Blot Membrane (Atto). The membrane
were blocked in TBST containing 1% nonfat dry milk for 1 h at
room temperature and incubated overnight at 4 °C with
anti-
-tubulin type III antibody (1:1000 dilution, Sigma) or
anti-GFAP antibody (1:400 dilution, Sigma). After washing three times
with TBST, the membrane was incubated with anti-mouse IgG conjugated
with horseradish peroxidase (1:5000 dilution, Sigma). Signals were visualized with the ECL system (Amersham Biosciences) according to the
protocol from the manufacturer. Densitometric analysis was performed
using Image 1.62 ppc program as described above.
Luciferase Reporter Assay--
A luciferase reporter construct
was generated by the insertion of the thymidine kinase (TK) basal
promoter derived from pMC1neoPolyA (Stratagene) into
XhoI-BamHI site of pTRE2-Luc
(Clontech) containing luciferase gene. In addition,
blunt-ended fragment containing five copies of GAL4 binding sequence
was inserted into blunt-ended XhoI sites of pTRE2-Luc. The
plasmid was designated as pGAL4-TK-Luc. The GAL4DBD expression
vector was constructed by the insertion of the GAL4DBD sequence derived
from pAS2-1 into blunt-ended EcoRI sites of pcDNA3. The
plasmid was designated as pcDNA3GAL4DBD. The GAL4DBD-fused Nif3l1
and Trip15/CSN2 expression vectors were constructed by the insertions
of entire coding region of Nif3l1 and Trip15/CSN2 in frame into the
downstream of GAL4DBD-coding sequence in pcDNA3GAL4DBD and
designated as pGAL4DBD-Nif3l1 and pGAL4DBD-Trip15/CSN2, respectively.
Trip15/CSN2 expression vector was constructed by insertion of the
BamHI-EcoRI fragment containing entire ORF of
pGEM7zf(+)-Trip15/CSN2 into the BamHI-EcoRI sites of pcDNA3 and designated as pcDNA3-Trip15/CSN2 (11). For each transfection, 2 × 105 P19 cells/35-mm diameter dish
was transfected with 1 µg of pGAL4-TK-Luc, 0.5 µg of
-gal
expression vector pcDNA3.1/Myc-His/lacZ (Invitrogen), 0.5 µg of
GAL4DBD-fused Nif3l1 or Trip15/CSN2 expression vector or empty
expression vector (pcDNA3) which indicated, 0.5 µg of Trip15/CSN2
expression vector was also cotransfected. At 48 h after
transfection, cells were lysed with the lysis buffer of luciferase
assay kit (Promega), and luciferase activities were determined
according to the instructions from the manufacturer using a Luminous
CT9000D (Dia-Iatron, Tokyo, Japan). Reporter gene activities were
normalized using
-gal activity as an internal control.
Cytochemical Analysis--
Enhanced green fluorescent protein
(EGFP)-fused Nif3l1 and Trip15/CSN2 expression vectors were constructed
by the insertions of entire cording region of Nif3l1 and Trip15/CSN2
into the downstream of EGFP-coding sequence in frame in pEGFP-C1
expression vector (Clontech) and designated as
pEGFP-Nif3l1 and pEGFP-Trip15/CSN2, respectively. COS-7 cells (5 × 105 cells/6-cm dish) were transfected with these vectors
in various combinations using DOTAP. pEF/Myc/ER/GFP (Invitrogen) having
a targeting signal of endoplasmic reticulum (ER) was also transfected as a marker of ER. After 36 h, the cells were fixed with 4%
paraformaldehyde and washed with PBS(
). To see the cellular
localization of Nif3l1, P19 cells (1 × 106
cells/10-cm dish) were transfected with pEGFP-Nif3l1 expression vector
using LipofectAMINE PLUSTM (Invitrogen). After 12 h,
the cells were treated with 5 × 10
7 M
RA and cultivated for various times. The cells were then fixed with 4%
paraformaldehyde and subsequently washed with PBS(
). The nuclei were
stained with 1 µg/ml Hoechst 33258. The cells were observed under a
fluorescent microscope (Axioplan2, Carl Zeiss, Oberkochen, Germany).
 |
RESULTS |
Identification of Trip15/CSN2-binding Proteins--
The
N-terminal region of Trip15/CSN2 has been shown to be sufficient for
its effective function and for association with its binding partners
including TR and DAX-1 via its N-terminal region (12-15). To analyze
the role of Trip15/CSN2 in the process of neuronal differentiation,
cDNA clones encoding novel Trip15/CSN2-binding proteins were
searched for by using a yeast two-hybrid screening system. A yeast
strain Y153 (His
, Trp
, Leu
)
was cotransfected with the bait vector, which expresses a fusion protein composed of GAL4DBD and the N-terminal region (amino acids 1-275) of rat Trip15/CSN2, and a RA-treated P19 cell cDNA library, which direct the synthesis of fusion proteins composed of
cDNA-encoded proteins and the GAL4 transcriptional activation
domain. Y153 strain contains the His3 and LacZ
genes linked to the GAL4 promoter. By the transfection of 3 × 106 Y153 cells, 52 His+ colonies were
developed and 17 of 52 colonies were
-gal-positive. Sequence
analysis of these clones showed that 5 of 17 clones encode the
C-terminal region (amino acids 243-376) of mouse Nif3l1
(Ngg1-interacting factor 3 like-1), which possesses a high homology to
yeast Ngg1-interacting factor 3 homolog (25). Based on this finding, we
focused on the functional analysis of Nif3l1 in neural differentiation.
Full-length Nif3l1 cDNA was amplified by RT-PCR using
total RNA from mouse P19 cells and Nif3l1-specific primers (25).
Interaction of Nif3l1 with Trip15/CSN2 in Vitro and in
Vivo--
We found that Nif3l1 possesses two putative leucine zipper
(Leu Zip) motifs (210-224 and 264-278) mediating a protein-protein interaction (Fig. 1B) (52). To
determine the binding region of Trip15/CSN2 to the C-terminal region
(243-376) of Nif3l1, various regions of Trip15/CSN2 were inserted into
pAS2-1 and obtained the bait constructs that express fusion proteins
of GAL4DBD and Trip15/CSN2 fragments. These constructs, Full (amino
acids 1-443), N1 (1-127), N2 (1-275), M (128-275), C1 (128-443),
and C2 (276-443), are shown in Fig. 1A. These bait
constructs were transfected into yeast strain Y153 cells, which allow
selection for two different markers: His and
-gal. Cotransformation
with the vector for GAL4 activation domain-fused C-terminal region of
Nif3l1 (243-376) showed that the N2 region selectively bound to the
C-terminal region of Nif3l1 (Fig. 1A). The full-length
Trip15/CSN2 bound to Nif3l1 weakly. The result suggests that the N2
region of Trip15/CSN2 containing a putative Leu Zip, nuclear
localization signal (NLS), and corepressor motif
((I/L)XX(I/V)I; Ref. 53), which is required for binding to a
nuclear hormone receptor DAX-1 (14), is necessary and sufficient for
interaction with the C-terminal region of Nif3l1 containing a putative
Leu Zip (Fig. 1C). It seems likely that these putative
motifs participate in the interaction between Trip15/CSN2 and Nif3l1
and the weak interaction of full-length Trip15/CSN2 with Nif3l1 is
related to the transcriptional corepressor activity of Trip15/CSN2
(12-14).

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Fig. 1.
Characterization of binding region of
Trip15/CSN2 to Nif3l1. A, yeast two-hybrid analysis of
the Trip15/CSN2 domain required for binding to Nif3l1. Various
fragments of Trip15/CSN2 cDNA were ligated in frame with the
GAL4DBD in pAS2-1 and used as baits. Nif3l1 cDNA
corresponding to amino acid residues 243-376 was ligated with the GAL4
activation domain in pACT-2. These Nif3l1 and Trip15/CSN2 expression
constructs were simultaneously introduced into yeast strain Y153 cells.
The cells were streaked on both selection (Leu ,
Trp , His , and 3-aminotriazole+)
and non-selection (Leu , Trp ) media.
-Galactosidase assay was carried out with the cells grown on a
non-selection medium. B, amino acid sequence of mouse Nif3l1
and its putative functional domains. Amino acid sequence of mouse
Nif3l1 has been reported by Tascou et al. (25). We found a
putative Leu Zip (open box) sequence, which is
commonly observed in a protein-protein interaction site (52).
Underline indicates the region of Nif3l1 used as a prey.
C, schematic view of protein binding regions of Nif3l1 and
Trip15/CSN2. The regions of Trip15/CSN2 required for interaction with
DAX-1, TR (14), and COP9 signalosome (21-24) have been previously
reported.
|
|
To further confirm the interaction between the full-length Nif3l1 and
Trip15/CSN2 in vitro, GST pull-down assay was performed. GST-fused Trip15/CSN2 protein was bound to glutathione-Sepharose beads,
and then 35S-labeled Nif3l1 synthesized by a in
vitro translation was added to the beads. After washing with the
binding buffer, 35S-labeled Nif3l1 retained in the beads
was eluted with the SDS sample buffer and analyzed by 12% SDS-PAGE. As
shown in Fig. 2A, the 42-kDa
Nif3l1 protein specifically bound to GST-Trip15/CSN2 fusion protein.

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Fig. 2.
Interaction between Nif3l1 and Trip15/CSN2
in vivo and in vitro.
A, binding of Trip15/CSN2 with Nif3l1 analyzed by GST
pull-down assay. Bacterially expressed GST-Trip15/CSN2 or GST were
preloaded to glutathione-Sepharose beads. The beads were incubated with
[35S]methionine-labeled Nif3l1 protein. The retained
proteins were analyzed by 12% SDS-PAGE. B, interaction
between Trip15/CSN2 and Nif3l1 in COS-7 cells. COS-7 cells were
transfected with pFlag-CMV2-Nif3l1, pcDNA3-HA-Trip15/CSN2, or both,
and the cell lysates prepared after 48 h were subjected to
immunoprecipitation with a monoclonal anti-Flag antibody. The
immunoprecipitates were analyzed by Western blot with anti-HA antibody
(upper panel). Expression of Flag-Nif3l1 and HA-Trip15 was
confirmed in the cells transfected with the corresponding vector alone
by Western blot (middle and lower panels,
respectively). C, interaction of Nif3l1 with endogenous
Trip15/CSN2. P19 cells were transfected with pFlag-CMV2 or
pFlag-CMV2-Nif3l1 vector and then treated with RA for 12 h to
induce the Trip15/CSN2 gene expression. The cell lysates were prepared,
immunoprecipitated with anti-Flag antibody, and subjected to
Western blot with anti-Trip15/CSN2 antibody (upper panel;
Ref. 11) and anti-Flag antibody (lower panel). The cell
lysate from parental P19 cells treated with RA for 12 h was also
analyzed to confirm the induction level of Trip15/CSN2.
|
|
To demonstrate that the interaction between these two proteins also
occurs in vivo, COS7 cells were transfected with expression vectors for pcDNA3-HA-Trip15/CSN2, pFlag-CMV2-Nif3l1, or both vectors to express HA-tagged Trip15/CSN2 and Flag-tagged Nif3l1 proteins. After 48 h, the cell lysates were prepared and subjected to Western blot analysis. The transient expression detected two proteins of 43 and 53 kDa that correspond to Flag-tagged Nif3l1 and
HA-tagged Trip15/CSN2, respectively, judging from the estimated molecular mass of these constructs (Fig. 2B,
middle and lower panels, respectively).
Immunoprecipitation of the extracts prepared from the cells transfected
with both vectors with anti-Flag antibody revealed coprecipitation of
Trip15/CSN2 (Fig. 2B, upper panel). Coprecipitation was not observed in the cells transfected with either
one of expression vectors. No effect on the expression level of either
Trip15/CSN2 or Nif3l1 was observed in the cotransfected cells as
determined by Western blot analysis. To examine further the interaction
between Nif3l1 and endogenous Trip15/CSN2, P19 cells were transfected
with pFlag-CMV2-Nif3l1 vector and then treated with RA to induce the
Trip15/CSN2 gene expression. After 12 h when the maximal induction
of Trip15/CSN2 protein was observed (11), the cell lysate was prepared,
immunoprecipitated with anti-Flag antibody and subjected to Western
blot with polyclonal anti-Trip15/CSN2 antibody (11). As shown in Fig.
2C, endogenous Trip15/CSN2 was coimmunoprecipitated with
Nif3l1. These results indicate that the interaction between Nif3l1 and
Trip15/CSN2 occurs in vivo.
Expression Pattern of the Nif3l1 Gene during Neural
Differentiation--
To examine the expression level of
Nif3l1 mRNA during the neural differentiation of P19
cells, total RNAs were extracted from the aggregated P19 cells treated
with 5 × 10
7 M RA for various times and
analyzed by Northern blotting with the 0.6-kb
EcoRI-DraI fragment of pGEM-5zf(+)-Nif3l1 as a
probe. As shown in Fig. 3A,
the Nif3l1 gene was mainly expressed as 1.85-kb mRNA and
the levels of two additional transcripts of 2.4 and 3.4 kb were very
low. The expression pattern of the Nif3l1 gene in P19 cells
resembles those of the spermatogonia-derived GC-1 spg cells and the
mouse teratocarcinoma F9 cells (25). Three species of mRNAs might
be generated by alternative splicing, because the Nif3l1
gene has been shown to be a single-copy gene (25). During neural
differentiation of RA-treated P19 cells, the level of Nif3l1 gene expression was not changed. On the other hand, the expression of
Trip15/CSN2 mRNA was induced shortly after the addition of RA
reaching a maximal level at 3 h after the treatment. The level then decreased and became barely detectable after 12 h. However, Trip15/CSN2 seemed to be accumulated during this period, and its ability to translocate Nif3l1 into the nuclei was augmented along with
the progression of neural differentiation as shown below (see Fig.
10B). The result suggested that Nif3l1 interacts with Trip15/CSN2 for only the limited period during the early stage of
neuronal differentiation. To analyze the tissue-specific expression of
the Nif3l1 gene, total RNAs were extracted from various
adult mouse tissues and analyzed by Northern blot. The
Nif3l1 gene was expressed as 1.85-, 2.4-, and 3.4-kb
mRNAs in all the tissues so far examined, but the expression levels
vary among these tissues (Fig. 3B). Higher levels of
expression were observed in cerebellum, heart, and kidney and low
levels of expression in spleen and muscle. The levels of expression in
cerebrum, lung, and liver were intermediate. In cerebellum and kidney,
the levels of 2.4-kb mRNA were higher than those of other tissues.
We also analyzed the Nif3l1 gene expression during the
development of mouse brain. A high level of Nif3l1
expression as 2.4- and 1.85-kb mRNAs was already detectable in the
brain at E10.5 and continued until E14.5 (Fig. 3C). The levels decreased steeply after E16.5. The second up-regulation was
observed at postnatal day (P5), and the levels were maintained relatively constant thereafter. The level of 3.4-kb mRNA became visible as a clear band as did 2.4- and 1.85-kb mRNAs (Fig.
3D). Although Tascou et al. (25) analyzed the
Nif3l1 gene expression in the mouse tissues and whole embryo
by Northern blot, they did not detect the changes of expression among
these splicing variants. The reason is not clear at present. These
results support the idea that the Nif3l1 gene plays an
important role in neural differentiation and development, and in
maintenance of neural function.

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Fig. 3.
Expression levels of Nif3l1
mRNA in RA-treated P19 cells and in various mouse
tissues. A, expression patterns of Nif3l1
and Trip15/CSN2 mRNAs during RA-primed P19 cell neural
differentiation. Total RNAs were extracted from the P19 cells treated
with 5 × 10 7 M RA for various times and
analyzed by Northern blot using a 0.6-kb
EcoRI-DraI fragment of mouse Nif3l1
cDNA (upper panel) and a 2.1-kb EcoRI
fragment of rat Trip15/CSN2 cDNA (middle panel) as
probes. Expression levels of Nif3l1 mRNA in various
adult mouse tissues (B), embryonic mouse brain
(C), and postnatal mouse brain (D) were similarly
analyzed. Pictures of ethidium bromide-stained 28 S ribosomal RNA are
included for comparison of total amount of RNA employed.
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Implication of Nif3l1 in Neural Differentiation--
To
demonstrate the implication of Nif3l1 in neural differentiation, we
established P19 derivative cell lines in which the exogenous
expressions of sense and antisense mouse Nif3l1 RNAs could
be initiated by the withdrawal of Tet. Briefly, pTRE2-sense Nif3l1 and
pTRE2-antisense Nif3l1 vectors were transfected into the pTet-Off
vector-introduced P19 (termed as R13) cells as described under
"Experimental Procedures." The resulting stable transformants introduced with sense and antisense Nif3l1 vectors were designated as
R13NifS and R13NifA, respectively. To examine whether the expression of
exogenous sense and antisense Nif3l1 RNA could be induced by the removal of Tet, we analyzed the expression levels of
Nif3l1 mRNA in R13, R13NifS, and R13NifA cells in the
presence and absence of Tet (Fig. 4). The
endogenous expression level of Nif3l1 mRNA in R13 cells
was not changed in the Tet-free medium (Fig. 4A). In R13NifS
cells, the expression of Nif3l1 1.85-kb mRNA analyzed by
Northern blot was substantially induced by the withdrawal of Tet and
after 12 h, the level increased to 6.5-fold higher than that
expressed in the presence of Tet (0 h) (Fig. 4B). In
contrast, the expression level of endogenous Nif3l1 mRNA
in R13NifA cells detected by RT-PCR was decreased after the removal of
Tet and lowered to 20% of the original level after 36-48 h (Fig.
4C). The expression level of PO mRNA examined as an
internal control in R13NifA cells was not changed by the removal of
Tet, showing that antisense Nif3l1 RNA specifically
interacted with endogenous Nif3l1 mRNA. These results
indicate that the exogenous expression of the sense and antisense
Nif3l1 RNAs could really be controlled by Tet.

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Fig. 4.
Induction of sense and antisense Nif3l1 RNAs
by Tet removal. Expression levels of Nif3l1 mRNA in
R13 cells in the presence and absence of Tet (A) and in
R13NifS cells after removal of Tet (B). The levels were
analyzed by Northern blot using a 0.6-kb
EcoRI-DraI fragment of Nif3l1 cDNA
as a probe. C, reduction of endogenous Nif3l1
mRNA expression in R13NifA cells after removal of Tet. Expression
levels of endogenous Nif3l1 mRNA were analyzed by
RT-PCR. The primers annealed to mouse Nif3l1 cDNA (25)
are as follows: 5'-primer, 5'-CAG CGG CCT GGA GTG GGA AGC AG-3';
3'-primer, 5'-CTC CTC CAG TAC CTG CTC CGA G-3'. Expression levels of PO
mRNA were also analyzed by RT-PCR as an internal control using the
following primers. 5'-primer, 5'-CAG CTC TGG AGA AAC TGC TG-3';
3'-primer, 5'-GTG TAC TCA GTC TCC ACA GA-3' (50).
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Using these stable transformants, we analyzed the effect of exogenous
expression of Nif3l1 on the neural differentiation of P19 cells
immunocytochemically (Fig. 5). R13,
R13NifS, and R13NifA cells could not differentiate into neurons after
the simple aggregation culture without the addition of RA, irrespective
of the absence of Tet (Fig. 5A (a, d,
and g)). On the other hand, in the presence of RA all
transformants were differentiated into
-tubulin III-positive neurons
even if Tet was added (Fig. 5A (b, e,
and h)). Nonetheless, by the removal of Tet, the neuronal
differentiation of R13NifS cells was significantly stimulated (Fig. 5,
A (f) and B). In contrast, the
neuronal differentiation of R13NifA cells was slightly decreased (Fig.
5, A (i) and B). In R13 cells, the
effect of Tet removal on RA-primed neuronal differentiation was not
observed (Fig. 5, A (c) and B),
showing that Tet itself did not affect the RA-primed neuronal
differentiation.

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Fig. 5.
Effects of enforced expression of sense and
antisense mouse Nif3l1 RNAs on RA-primed P19 cell
neural differentiation. A, effects of
Tet-controlled expression of sense and antisense Nif3l1 RNAs
on differentiation to -tubulin III-positive neurons. R13
(a-c), R13NifS (d-f), and R13NifA
(g-i) cells were treated without RA (a,
d, and g) and with RA (b,
c, e, f, h, and
i) in the presence (b, e, and
h) and absence (a, c, d,
f, g, and i) of Tet for 4 days.
Immunocytochemical analysis was performed after 3 days of replating.
Scale bar presents 100 µm. B,
quantification of effects of sense and antisense Nif3l1 RNAs on
differentiation to -tubulin III-positive neurons. -Tubulin
III-positive neurons were counted at least 5 fields/slide under a
microscope (original magnification, ×100) and estimated the
Tet( )/Tet(+) ratios. Each value is the average ± S.E. of
triplicate chamber slides. *, p < 0.001; **,
p < 0.05 compared with the control R13 cells.
C, effects of Tet-controlled expression of sense and
antisense Nif3l1 RNAs on differentiation to GFAP-positive
glial cells. R13 (j and k), R13NifS (l
and m), and R13NifA (n and o) cells
were treated with RA in the presence (j, l, and
n) and absence (k, m, and
o) of Tet for 4 days. Immunocytochemical analysis was
carried out at 7 days after replating. Scale bar
presents 500 µm. D, quantification of effects of sense and
antisense Nif3l1 RNAs on differentiation to GFAP-positive glial cells.
GFAP-positive areas were measured at least 6 fields/slide using a Image
Gauge software (Fujifilm) and estimated the Tet( )/Tet(+) ratios. Each
value is the average ± S.E. of triplicate chamber slides. *,
p < 0.001; **, p < 0.001 compared
with the control R13 cells.
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The effect of Nif3l1 on the GFAP-positive glial cell differentiation
was also examined. Although differentiation of all transformants to
GFAP-positive glial cells was not observed without the addition of RA,
as was the case in neural differentiation (data not shown), differentiation of R13NifS cells to glial cells induced by RA was
stimulated by the removal of Tet (Fig. 5C, compare
m with l). In contrast, the glial differentiation
of R13NifA cells induced by RA was significantly suppressed after Tet
removal (Fig. 5C, compare o with n),
whereas in R13 cells, the effects of Tet removal was not evident (Fig.
5C (k)). These results were supported by the
quantitative analysis of GFAP-positive area using an Image Gauge
software (Fujifilm, Tokyo, Japan) as shown in Fig. 5D.
We further analyzed the effects of exogenous expression of Nif3l1 on
the expression of neuron and glial cell markers by Western blotting
with anti-
-tubulin III and anti-GFAP antibodies. In aggregated
R13NifS cells, the expression levels of
-tubulin III and GFAP were
not induced by the withdrawal of Tet in the absence of RA (data not
shown). In the presence of RA, the additive effect of the exogenous
expression of Nif3l1 was observed in aggregated R13NifS
cells on the expression of
-tubulin III (Fig.
6, A and C) and
GFAP (Fig. 6, B and D). On the other hand, the
expression of
-tubulin III and GFAP in R13NifA cells was
conspicuously reduced by the expression of antisense Nif3l1
RNA in the presence of RA. These results were consistent with the data
obtained from immunocytochemical analysis as shown in Fig. 5. Taken
together, it seems likely that the Nif3l1 gene plays a
crucial role in the RA-primed neural differentiation signal
pathway.

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Fig. 6.
Effects of enforced expression of sense and
antisense Nif3l1 RNAs on expression of neuron and
glial cell marker proteins. R13, R13NifS, and R13NifA cells were
treated with RA for 4 days in the presence or absence of Tet.
Expression levels of -tubulin III (A) and GFAP
(B) were analyzed by Western blot with corresponding
antibodies at 3 and 7 days after plating, respectively. C,
the graph shows the Tet( )/Tet(+) ratio of -tubulin III expression
levels. Values are shown as the mean ± S.E. of four experiments.
*, p < 0.004; **, p < 0.002 compared
with the control R13 cells. D, the graph shows the
Tet( )/Tet(+) ratio of GFAP expression levels. Values are shown as the
mean ± S.E. of four experiments. *, p < 0.001;
**, p < 0.006 compared with the control R13
cells.
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Implication of Nif3l1 Gene in Early Commitment of P19 Cells to
Neural Lineage--
The enforced expression of sense Nif3l1
RNA in R13NifS cells by the removal of Tet enhanced differentiation
into
-tubulin III-positive neurons in the presence of RA (Fig.
5A). To investigate which periods of sense Nif3l1
RNA expression are effective for RA-primed neuronal differentiation, we
removed Tet for various time periods and the extents of cell
differentiation and the levels of
-tubulin III were analyzed by
immunocytochemistry and Western blot, respectively. We confirmed that
sense Nif3l1 RNA expression was induced just after 30 min of
the Tet removal (Fig. 7H).
Immunocytochemical analysis revealed that neuronal differentiation of
R13NifS cells was significantly stimulated by Tet removal, only for
3 h (Fig. 7, B and E). Longer removal of
12 h stimulated the differentiation further, but only to a small
extent, and the extent was even slightly decreased by removal for
24 h (Fig. 7, C, D, and E).
Western blot analysis also showed that the expression of
-tubulin
III was induced to near maximal level during 3 h of Tet removal
(Fig. 7, F and G). These results indicate that
Nif3l1 acts on the very early stage of RA-primed neuronal
differentiation.

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Fig. 7.
Effect of Tet-controlled expression periods
of sense Nif3l1 RNA on neuronal differentiation.
R13NifS cells were treated with RA in the presence of Tet
(A) or absence of Tet for 3 h (B), 12 h
(C), and 24 h (D) and then cultivated in the
medium containing RA and 2 µg/ml Tet up to 4 days. Immunocytochemical
analysis was performed using antibody against -tubulin III at 3 days
after replating. Scale bar presents 500 µm.
E, effect of enforced expression periods of sense
Nif3l1 RNA on the fraction of R13NifS cells differentiated
to -tubulin III-positive neurons. The differentiated neurons were
counted at least 5 fields/slide. Values are the average ± S.E. of
three independent chamber slides. *, p < 0.05 compared
with the Tet(+) control. F, effect of enforced expression
periods of sense Nif3l1 RNA on -tubulin III expression.
Cell lysates were prepared at 3 days after replating and analyzed by
Western blot. G, the result obtained in F was
quantitatively shown as relative expression levels. The -tubulin III
level expressed at time 0 was taken as 1. Values are the average ± S.E. of three experiments. *, p < 0.001 compared
with the Tet(+) control. H, rapid induction of sense
Nif3l1 RNA in R13NifS cells by Tet removal. R13NifS cells
were cultivated in the absence of Tet for various times, and expression
levels of Nif3l1 RNA were analyzed by Northern blot.
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Effect of Nif3l1 on Oct-3/4 and Neurogenic Gene
Expressions--
Transcription factors regulate expression of specific
genes to control cellular phenotype. Oct-3/4 is a transcription factor and acts to maintain the undifferentiated state of P19 cells as well as
embryonic stem cells. Its mRNA expression is dramatically diminished within 24 h of the RA treatment (3). Thus, the
down-regulation of Oct-3/4 is required for the onset of neural
differentiation. In addition, Nif3l1 and Trip15/CSN2 are known as
transcriptional regulator and transcriptional corepressor, respectively
(12-14, 25, 26). On the basis of these facts, whether Nif3l1
participates in neural differentiation through the down-regulation of
Oct-3/4 gene expression was analyzed in aggregated R13NifS cells after the removal of Tet in the presence and absence of RA by Northern blot
(Fig. 8A). When the cells were
treated with RA in the absence of Tet, the level of Oct-3/4 mRNA
began to decrease after 36 h and lowered to an almost undetectable
level after 60 h (Fig. 8A). The reduction of Oct-3/4
mRNA level after 60 h, however, was not observed in the
presence of Tet, suggesting that Nif3l1 is involved in the repression
of the Oct-3/4 gene and the effect became visible after 60 h. This
residual effect of Nif3l1 was also seen in the absence of RA, and the
level was reduced to 75% of the original level at 96 h after
removal of Tet (Fig. 8A).

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Fig. 8.
Effect of enforced expression of
Nif3l1 mRNA on the levels of Oct-3/4 and Mash-1
mRNAs. R13NifS cells were treated or untreated with RA in the
presence or absence of Tet for various times. Expression levels of
Oct-3/4 (A) and Mash-1 (B) mRNAs were
analyzed by Northern blot. PO mRNA expression (C) was
analyzed simultaneously as an internal control.
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We further analyzed the expression level of neurogenic Mash-1 gene
during neural differentiation, because it plays a key regulatory role
in the downstream of Oct-3/4 pathway, which leads to RA-primed neuronal
differentiation of P19 cells (6). As shown in Fig. 8B, the
expression of Mash-1 mRNAs was hardly detected in R13NifS cells not
treated with RA. By the addition of RA, expression of Mash-1 mRNA
became detectable after 60 h when the down-regulation of Oct-3/4
mRNA was enhanced. This induction of Mash-1 gene expression seemed
to be influenced by exogenously expressed Nif3l1, although the effects
was not so evident. Under these conditions, the level of PO mRNA,
analyzed as an internal control, was not significantly altered,
although some bias was observed (Fig. 8C).
Nif3l1 Harbors an Autonomous Silencing Function and Cooperates with
Trip15/CSN2--
Trip15/CSN2 interacts with a subset of
nuclear hormone receptors such as DAX-1, ecdysone receptor, COUP-TF1,
Ftz-F1, and TRs, but not with retinoic acid receptors and retinoid X
receptors (12-14). Trip15/CSN2 interacts with TR
and harbors an
autonomous silencing function. This interaction is inhibited by
increasing amounts of thyroid hormone (13). Thus, Trip15/CSN2
represents a member of novel class of corepressors specific for a
selected member of the nuclear hormone receptor family (13, 14). In addition, Nif3l1 interacts with transcriptional activator/repressor protein ADA3/NGG1 (26, 29, 30). Therefore, if Nif3l1 is involved in a
transcriptional silencing, we expected that Nif3l1 should harbor an
autonomous silencing function and cooperate with Trip15/CSN2 in a
transcriptional silencing.
To localize Nif3l1 and Trip15/CSN2 effectively in nuclei, expression
vectors containing the full-length Nif3l1 and Trip15/CSN2 cDNAs fused to the GAL4DBD coding sequence downstream of the CMV promoter were constructed, because GAL4DBD possesses PKTKRSP
sequence as a NLS. GAL4DBD itself and GAL4DBD-fused Nif3l1 transiently expressed in P19 cells were actually localized in nuclei (Fig. 9A). The effect of Nif3l1 and
Trip15/CSN2 on the suppression of TK promoter activity in P19 cells was
studied using these vectors and a luciferase reporter construct,
pGAL4-TK-Luc which contains GAL4 binding sequence upstream of the TK
basal promoter. P19 cells were transfected with pGAL4-TK-Luc together
with expression vectors of pGAL4DBD, pGAL4DBD-Nif3l1,
pGAL4DBD-Trip15/CSN2, pcDNA3-Trip15/CSN2, or a combination of these
expression vectors. As shown in Fig. 9B, Nif3l1
significantly suppressed the TK promoter activity to one eighth of the
activity expressed by cotransfection with pGAL4DBD, which expresses
GAL4DBD alone. Trip15/CSN2 also suppressed the TK promoter activity but
to a much lesser extent. When Nif3l1 was coexpressed with Trip15/CSN2
by cotransfection with pcDNA3-Trip15/CSN2, additive effect was
observed on the suppression of TK promoter activity. These results
support the idea that a transcriptional silencer Nif3l1 cooperates with
Trip15/CSN2 for the suppression of the TK promoter activity and perhaps
implicates in the down-regulation of Oct-3/4 gene.

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Fig. 9.
Nif3l1 harbors an autonomous silencing
function. A, nuclear localization of GAL4DBD and
GAL4DBD-fused Nif3l1 proteins. P19 cells were transfected with either
pGAL4DBD (upper panel) or pGAL4DBD-Nif3l1 (middle
panel) and stained with anti-GAL4 antibody after 48 h (Santa
Cruz Biotechnology, Santa Cruz, CA). Nuclei were stained with
hematoxylin (lower panel). Scale bar
presents 20 µm. B, both luciferase reporter plasmid
pGAL4-TK-Luc (1 µg) and -gal expression plasmid
pcDNA3.1/Myc-His/lacZ (0.5 µg) were transfected into P19 cells
together with 0.5 µg of pGAL4DBD, pGAL4DBD-Nif3l1, or
pGAL4DBD-Trip15/CSN2 in combination with 0.5 µg of
pcDNA3-Trip15/CSN2 or empty pcDNA3. At 48 h after
transfection, the cells were lysed and assayed for luciferase activity.
Luciferase activities were normalized using -gal activity as an
internal control, and the activity expressed by cotransfection with
pGAL4DBD as a control was taken as 1. Each value is shown as
average ± S.E. of triplicate culture dishes. *,
p < 0.002; **, p < 0.001;
***, p < 0.001 compared with the pGAL4DBD
control.
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Cellular Localization of Nif3l1--
To investigate the
subcellular localization of Nif3l1, we constructed a EGFP-fused Nif3l1
expression vector by the insertion of full-length Nif3l1
cDNA into pEGFP-C1 downstream of EGFP coding sequence in frame. The
EGFP-fused Nif3l1 expression vector was introduced into COS-7 cells,
and subcellular distribution of the fused protein was observed after
48 h. As shown in Fig.
10A (a), EGFP-Nif3l1 fusion protein was localized predominantly in the cytoplasm, although fluorescence was homogenously detected both in the
nuclei and cytoplasm when only EGFP protein was expressed (data not
shown). The localization of EGFP-Nif3l1 was different from that of ER
retention signal-tagged GFP, which was concentrated around the nuclei
(Fig. 10A (e)). On the other hand, EGFP-fused Trip15/CSN2 was localized in the nuclei predominantly (Fig.
10A (c)) and showed an image similar to the
nuclei that are stained with Hoechst 33258, which intercalate to DNA
(Fig. 10A (c and f)). For the
expression of transcriptional silencing activity, however, the
translocation of Nif3l1 from the cytoplasm into the nuclei is
absolutely required. As described above, Nif3l1 bound to Trip15/CSN2 in vivo and in vitro, and cooperated with
Trip15/CSN2 for the suppression of the TK promoter activity, suggesting
that Trip15/CSN2 participates in the translocation of Nif3l1 from the
cytoplasm to the nuclei. To confirm this assumption, the effect of
Trip15/CSN2 on the intracellular localization of Nif3l1 was examined by
cotransfection of COS-7 cells with pEGFP-Nif3l1 and
pcDNA3-Trip15/CSN2 and the cells exhibiting distinct fluorescence
in the nuclei or cytoplasm were counted. The fraction of EGFP-fused
Nif3l1 localized predominantly in the nuclei was increased depending on
the increase in the amount of the Trip15/CSN2 vector transfected, and
in the ratio of 1: 9, the fraction of cells exhibiting EGFP-fused
Nif3l1 in the nuclei (Fig. 10A (b)) was ~4-fold
higher than that observed in the absence of Trip15/CSN2 (Fig.
10A (g)). In contrast, the intercellular
localization of EGFP-fused Trip15/CSN2 was not affected by the
coexpression of Nif3l1 (Fig. 10A (d and
h)).

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Fig. 10.
Nuclear localization of Nif3l1 controlled by
Trip15/CSN2. A, implication of Trip15/CSN2 in
translocation of Nif3l1. COS-7 cells was transfected with pEGFP-Nif3l1
alone (a) or together with pcDNA3-Trip15/CSN2 in the
ratio of 1:9 (b). COS-7 cells were also transfected with
pEGFP-Trip15/CSN2 alone (c) or together with
pcDNA3-Nif3l1 in the ratio of 1:9 (d). Transfection with
pEF/Myc/ER/GFP (Invitrogen) was performed as a marker of ER
(e). Nuclei were stained with Hoechst 33258 (f).
At 48 h after transfection, the cells were observed under a
fluorescent optics (Axioplan2; Carl Zeiss). Scale
bar presents 40 µm. g, quantitative analysis of
the effect of Trip15/CSN2 on intracellular localization of EGFP-fused
Nif3l1. The cells exhibiting distinct fluorescence in the cytoplasm and
nuclei as shown in a and b, respectively, were
counted. Each value is the average ± S.E. of triplicate culture
dishes. *, p < 0.004 compared with the cells
transfected with pEGFP-Nif3l1 alone. h, quantitative
analysis of the effect of coexpression of Nif3l1 on intracellular
localization of EGFP-fused Trip15/CSN2. Each value is the average ± S.E. of triplicate culture dishes. B, translocation of
Nif3l1 during neural differentiation. The pEGFP-Nif3l1-transfected P19
cells were cultivated in the absence of RA (a-c) or in the
presence of RA (d-f) for 12 h. Intracellular
localization of EGFP-Nif3l1 (a and d) and the
nuclei stained with Hoechst 33258 (b and e) were
observed under a confocal microscope (Radiance2100; Bio-Rad).
c and f present merges of a and
b, and d and e, respectively.
Scale bar presents 40 µm. g,
time-dependent translocation of EGFP-fused Nif3l1 during
RA-primed P19 cell neural differentiation. Values are presented as the
average ± S.E. of four independent dishes. *, p < 0.03 compared with of RA( ) control.
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Because the Trip15/CSN2 gene was highly expressed in an early stage of
neural differentiation of RA-treated P19 cells (Fig. 3A), we
assumed that the translocation of Ni3fl1 from the cytoplasm into the
nuclei might be enhanced at this early stage of neuronal differentiation. To confirm this assumption, P19 cells were transfected with pEGFP-Nif31l and cultivated with and without RA for various times
and the intracellular localization of EGFP-fused Nif3l1 was examined.
The localization in the nuclei was confirmed by staining the cells with
Hoechst 33258. Although in the absence of RA, EGFP-fused Nif3l1 was
mainly detected in the cytoplasm (Fig. 10B
(a-c)), the fraction of cells exhibiting EGFP-fused Nif3l1 predominantly in the nuclei was augmented by the treatment with RA in a
time-dependent manner and, after 24 h, increased to
~3.5-fold of that observed in the absence of RA (Fig. 10B
(d-g)). The merge of two images obtained by the EGFP
fluorescence and by staining with Hoechst 33258 showed that Nif3l1
localizes as nuclear patches (Fig. 10B (f)).
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DISCUSSION |
The data presented here provide the first insight into the
molecular mechanism underlying the involvement of Nif3l1 in neural differentiation through the association with Trip15/CSN2.
Using a yeast two-hybrid method, we found that the N-terminal region
(amino acids 1-275) of Trip15/CSN2 bound to the C-terminal region
(amino acids 243-376) of Nif3l1 (Fig. 1). The interaction was
confirmed by a pull-down assay and an epitope-tagged
coimmunoprecipitation (Fig. 2, A and B). In
addition, the interaction between Nif3l1 and endogenous Trip15/CSN2 in
RA-primed P19 cells was also confirmed (Fig. 2C).
Trip15/CSN2 possesses a putative NLS and Leu Zip sequence and is
functioning in the nucleus (54), whereas its binding partner Nif3l1
does not contain a NLS (25). One explanation could be that Nif3l1 binds
to Trip15/CSN2 in the cytoplasm and subsequently enters the nucleus via
cotransport as reported for interleukin-5 and its receptor subunit
(55). In fact, this assumption was demonstrated based on the finding
that the translocation of Nif3l1 from the cytoplasm to the nucleus was
dependent on the expression level of Trip15/CSN2 protein, whereas the
nuclear localization of Trip15/CSN2 was not affected by the
coexpression of Nif3l1 protein (Fig. 10A). Furthermore, the
nuclear localization of Nif3l1 was essentially required for its
functional expression, because Nif3l1 acted as a transcriptional
silencer and synergized with Trip15/CSN2 for repression of TK the
promoter activity of a reporter construct, pGAL4-TK-Luc (Fig. 9).
Yeast Nif3 was originally identified as a NGG1-interacting protein by a
yeast two-hybrid screening (26). NGG1 was isolated based on its
requirement for the inhibition of transcriptional activation of the
genes involved in the utilization of galactose by GAL4 protein when
yeast was grown in glucose medium (27). ADA3/NGG1 was also isolated
based on its ability to suppress mutations and the toxic effects of
overexpression of the viral activator protein, VP16 in yeast (28),
suggesting that ADA3/NGG1 is involved in transcriptional activation and
repression by Nif3l1 (26, 29, 30). Genetic studies on the
transcriptional activation in yeast have identified ADA3/NGG1 as a
critical component of coactivator complexes that link to
transcriptional activators (56). ADA3/NGG1 and its associated adapter
ADA2 form a complex that recruit a histone acetyltransferase GCN5 to
promoters (35, 37, 57, 58). Through a similar mechanism, coactivator
complexes could also implicate in transcriptional repression. It is
well known that Trip15/CSN2 is a member of corepressors specific for the nuclear hormone receptor superfamily (12-14). Using the luciferase reporter assay, we revealed that a Trip15/CSN2 binding partner, Nif3l1,
possesses a transcriptional silencing function (Fig. 9). Therefore, we
propose that Nif3l1 and Trip15/CSN2 form a novel corepressor complex
that might recruits an inhibitor of histone acetyltransferase or a
histone deacetylase to target promoter(s) during neural
differentiation, although direct evidence is needed.
Enforced expression of sense Nif3l1 RNA by the Tet
expression system caused a small but significant enhancement of
RA-primed neural differentiation of P19 cells into
-tubulin
III-positive neurons and GFAP-positive glial cells (Figs. 5 and 6).
Interestingly, the enforced expression for only up to 3 h was
nearly sufficient for the enhancement (Fig. 7). This time corresponds
to that when the level of Trip15/CSN2 induced by the treatment with RA
becomes nearly maximal in accordance with the cooperative action of
Nif3l1 and Trip15/CSN2 for induction of neural differentiation.
Moreover, in relation to the intense down-regulation of Oct-3/4, which
maintains an undifferentiated state of P19 cells (3), the expression of
neurogenic Mash-1 gene was rapidly induced by the enforced expression
of Nif3l1 mRNA (Fig. 8). It appeared that
Nif3l1-Trip15/CSN2 complex could be implicated in the commitment of
multipotent P19 cells to neural lineage via the Oct-3/4
down-regulation. In the developing brain, a common cortical progenitor
cell gives rise first to a variety of layer-specific neurons and then
switches to producing astrocytes and ultimately oligodendrocytes (59, 60). As in the case of neurogenesis in vivo, neurons appear earlier than glial cells during RA-primed P19 cell neural
differentiation (61). Therefore, the enhancement of both neurogenesis
and gliogenesis by Nif3l1 might be caused by the increase in the
fraction of cells committed to differentiate to neural lineage,
although the precise estimation of the fraction of neural lineage was
difficult because of the occurrence of the cells that underwent
proliferation and apoptosis during P19 cell neural differentiation
(62).
The mouse Nif3l1 gene has been isolated by a suppression
subtractive hybridization between the spermatogonia-derived cell line
GC-1 spg and spermatocyte-derived cell line GC-4 spc (25). Yeast Nif3
and NGG1 form a complex for effective inhibition of the transcriptional
activation by GAL4 (27). The Nif3l1 gene was expressed
throughout embryonic development and in the various adult mouse tissues
ubiquitously accompanying the putative alternative splicing (Fig. 3).
The widespread expression of Nif3l1 gene suggests that the
gene acts as a common repressor and is unlikely to be restricted to
specify neural identities by itself. The expression of
Nif3l1 gene was also observed in the undifferentiated P19
cells, and the expression level was not changed significantly during RA-primed neural differentiation. On the other hand, a binding partner
Trip15/CSN2 gene expression was markedly induced by the treatment of RA
(Fig. 3A; Ref. 11). These observations suggest that
Nif3l1-Trip15/CSN2 complex is specifically required for the early stage
of neural differentiation and various regulatory factors distinct from
Trip15/CSN2 cooperate with Nif3l1 in the process of the important
cellular events such as spermatogenesis and maintenance of cell
type-specific functions.
P19 cells possess many properties similar to embryonic stem cells
isolated from mice and humans (63). Therefore, it may be possible that
the expression system of Nif3l1-Trip15/CSN2 complex can be utilized for
the production of large amount of neurons from human embryonic stem
cells in combination with the expression of key neurogenic genes.