The Role of Transcriptional Corepressor Nif3l1 in Early Stage ofNeural Differentiation via Cooperation with Trip15/CSN2*

Received for publication, September 25, 2002, and in revised form, November 18, 2002Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M209856200

Hirotada Akiyama, Naoko Fujisawa, Yousuke Tashiro, Natsuko Takanabe, Akinori Sugiyama,and Fumio Tashiro‡

From the Department of Biological Science and Technology, Faculty of Industrial Science and Technology, TokyoUniversity of Science, Yamazaki, Noda-shi, Chiba 278-8510, Japan

Mouse Nif3l1 gene is highly conserved from bacteriato human. Even though this gene is expressed through-out embryonic development, its biological function isstill obscure. Here, we show that Nif3l1 participates inretinoic acid-primed neural differentiation of P19 em-bryonic carcinoma cells through cooperation withTrip15/CSN2, a transcriptional corepressor/componentof COP9 signalosome. We isolated Nif3l1 cDNA from P19cell cDNA library by a yeast two-hybrid screening usingTrip15/CSN2 as a bait. This interaction was confirmedby a pull-down assay and an epitope-tagged coimmuno-precipitation. Although Nif3l1 was mainly detected inthe cytoplasm, the translocation of Nif3l1 into the nucleiwas observed in retinoic acid-primed neural differenti-ation of P19 cells and enhanced by the enforced expres-sion of Trip15/CSN2. Furthermore, enforced expressionof sense Nif3l1 RNA, but not antisense RNA, enhancedthe neural differentiation of P19 cells accompanying theintense down-regulation of Oct-3/4 mRNA expressionand the rapid induction of Mash-1 mRNA expression.Luciferase reporter assay showed that Nif3l1 could actas a transcriptional repressor and synergized the tran-scriptional repression by Trip15/CSN2. These results in-dicate that Nif3l1 implicates in neural differentiationthrough the cooperation with Trip15/CSN2.

The differentiation of mammalian neurons during develop-ment is a highly complex process involving regulation andcoordination of gene expression at multiple steps. To under-stand the basic mechanisms underlying this complex pathway,identification of genes that are differentially expressed duringneural differentiation is an important approach. P19 embryo-nal carcinoma (EC)1 cells derived from a mouse embryo havebeen used extensively as a model system for in vitro neural

differentiation (1, 2). Exposure of aggregated P19 cells withretinoic acid (RA) results in the differentiation of cells withmany fundamental phenotypes of mammalian nervous system(3). During the early stages of neuronal differentiation of P19cells, the mammalian homologues of several Drosophila geneproducts such as Motch, Mash-1, Wnt-1, and transduction-likeenhancers of split are expressed (4–7). Moreover, a number ofproteins including retinoic acid receptors, retinoid X receptors,epidermal growth factor receptor, and transcription factorssuch as Oct-3/4, Brn-2, and Bdm-1 have been identified (8–10).Perhaps a limited number of RA- and aggregation-responsivegenes trigger the neuronal differentiation pathway of P19 cells.

Recently we found that a transcriptional corepressor, a com-ponent of COP9 signalosome, Trip15/CSN2, was highly ex-pressed at the early stage of neural differentiation of RA-treated P19 cells. The deduced amino acid sequence of ratTrip15/CSN2 gene is completely identical with those of mouseand human homologues (DDBJ/EMBL/GenBankTM accessionno. AB081072; Ref. 11). Enforced expression of sense ratTrip15/CSN2 RNA was sufficient to convert P19 cells intoneurons, but not glial cells in the absence of RA, only after theaggregation treatment, accompanying the down-regulation ofOct-3/4 transcript, which maintains the undifferentiated stateof P19 cells. Thus, the induction of Trip15/CSN2 prior to down-regulation of Oct-3/4 gene expression is required for the com-mitment of P19 cells to neuronal lineage.

Trip15/CSN2 was originally identified as a thyroid hormonereceptor (TR)-interacting protein and acts as a transcriptionalcorepressor (12). Trip15/CSN2 interacts with a subset of nu-clear 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 andretinoid X receptors (13, 14). Trip15/CSN2 is conserved in awide range of organisms and was also identified as a compo-nent of a 26 S proteasome lid-like complex termed COP9 sig-nalosome (CSN; Refs. 15 and 16). The CSN complex was orig-inally identified as a repressor of light-controlled developmentin Arabidopsis thaliana (17). In animals, the CSN complex islocalized in the nucleus and possesses a kinase activity thatspecifically phosphorylates transcriptional regulators such asp105, I�B�, c-Jun, and p53 (18–20). Although the mutual in-teraction between Trip15-nuclear receptor complex and CSNcomplex is still unknown, these facts indicate that the CSNcomplex participates in various signal transduction pathways.The proteasome-COP9 complex-initiation factor 3 domain inthe C-terminal region of Trip15/CSN2 stabilizes protein-pro-tein interaction within the CSN complex (21–24). The N-termi-nal region of Trip15/CSN2 has been reported to be sufficient forthe effector functions of Trip15/CSN2. In fact, Trip15/CSN2

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ To whom correspondence should be addressed. Tel.: 81-4-7124-1501; Fax: 81-4-7125-1841; E-mail: ftashir@rs.noda.tus.ac.jp.

1 The abbreviations used are: EC, embryonal carcinoma; RA, retinoicacid; RT, reverse transcription; ORF, open reading frame; E, embryonicday; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; CSN, COP9 signalosome; TR, thyroid hormone receptor;PBS(�), Ca2�,Mg2�-free phosphate-buffered saline; HA, hemaggluti-nin; DBD, DNA binding domain; �-gal, �-galactosidase; Tet, tetracy-cline; Luc, luciferase; GFAP, glial fibrially acidic protein; FCS, fetal calfserum; Leu Zip, leucine zipper; ADA, alternation deficiency in activa-tion; TAF, TATA box-binding protein-associated factor; CMV, cytomeg-alovirus; PO, ribosomal phosphoprotein; ER, endoplasmic reticulum;GFP, green fluorescent protein; TBST, Tris-buffered saline plus Tween20; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulfate; NLS, nuclear localization signal; TK, thymidine kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 12, Issue of March 21, pp. 10752–10762, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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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 dif-ferentiation, we tried to find out novel molecules that bind toTrip15/CSN2 and possibly act as new downstream targets orregulators of Trip15/CSN2. We used the N-terminal region ofrat Trip15/CSN2 as a bait for the yeast two-hybrid screening ofRA-treated P19 cell cDNA library. Five of 17 positive cDNAclones have been turned out to be mouse Nif3l1 (NGG1-inter-acting factor 3-like 1) gene. Nif3l1 cDNA was isolated througha suppression subtractive hybridization between the spermato-gonia- and spermatocyto-derived cell lines and possesses a highhomology to yeast Nif3 (Ngg1-interacting factor 3) gene (25).The mouse Nif3l1 gene encodes a cytoplasmic protein consistedof 376 amino acids and is highly conserved from bacteria tomammals. Expression of Nif3l1 transcript is detected through-out mouse embryonic development (25); however, the real bio-logical function of Nif3l1 is still unknown.

Yeast Nif3 was originally identified in yeast two-hybridscreening as a NGG1-interacting protein (26). NGG1 was iso-lated based on its requirement for the full inhibition of tran-scriptional activation by GAL4 protein in glucose media (27).Independently, ADA3/NGG1 was isolated based on the abilityof mutations to suppress the toxic effects of overexpression ofthe viral activator VP16 in yeast (28). Therefore, ADA3/NGG1was involved in transcriptional activation and repression (26,29, 30). Alternation deficiency in activation (ADA) proteinshave been found to be required for transcriptional activation bya number of yeast activators (28, 31, 32). In yeast, ADA3/NGG1is found as multisubunit complexes containing three to fouradditional ADA proteins and different TAFs and Spt (30, 31,33–36). In mammalian cells, the majority of ADA3/NGG1 pro-tein also seems to be complexed with Spt and TAF or TAF-likefactors, making up several types of complexes (37–41). Thesecomplexes are thought to be functional homologs of the yeastADA complexes (37, 39, 41). Recently, genetic studies in yeasthave demonstrated a crucial role of ADA complex in the trans-activation function of mammalian nuclear hormone receptors(42–45). Nonetheless, there is no report concerning about thefunction of Nif3l1 in transcriptional regulation.

In this study, we show that the cytoplasmic Nif3l1 proteincould be translocated into the nuclei by the association withTrip15/CSN2 and that it synergized the transcriptional repres-sion activity of Trip15/CSN2. In addition, Nif3l1 implicates inneural differentiation of P19 cells, perhaps through the down-regulation of Oct-3/4 transcript, which suppresses neurogenicgenes including Mash-1 to maintain the undifferentiated stateof P19 cells. Considering these results and the expression ofNif3l1 in early developing brain, it seems likely that bothNif3l1 and Trip15/CSN2 play an important role in neuraldifferentiation.

EXPERIMENTAL PROCEDURES

Cell Culture and Experimental Animals—Mouse P19 embryonal car-cinoma 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 humid-ified atmosphere of 5% CO2 in air. To induce neural differentiation, 1 �106 P19 cells aggregated in 10-cm bacteriological grade dishes werecultivated in 10 ml of �-minimal essential medium containing 10% FCSand 5 � 10�7 M all-trans-retinoic acid (RA) (Sigma) for 4 days. Cellaggregates were then suspended with mild pipetting and transferred totissue culture dishes. The cells were cultivated in RA-free �-minimalessential medium containing 10% FCS for additional 3 days to induce�-tubulin type III-positive neurons and for 7 days to induce glial fibri-ally acidic protein (GFAP)-positive glial cells. COS-7 cells were obtainedfrom Japanese Cancer Resources Bank (Tokyo, Japan) and maintainedin 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 theexperimental animal facility (Tokyo University of Science). All mousewere kept under a 12-h light/12-h dark cycle at 22–24 °C. Standardlaboratory feed (MR standard, Nousan Ltd, Kanagawa, Japan) and tapwater were given ad libitum. Mouse care and handling conformed to theNational Institutes of Health guidelines for animal research. The ex-perimental protocols were approved by the Institutional Animal Careand Use Committee.

Yeast Two-hybrid Screening—Rat Trip15/CSN2 full-length cDNA(amino acids 1–443) and its fragment covering amino acids 1–275 wereinserted into pAS2–1 (Clontech, Palo Alto, CA) in frame with the codingsequence for GAL4 DNA binding domain (DBD) and used for yeasttwo-hybrid screening as bait vectors. A RA-treated P19 cell cDNAlibrary was constructed using plasmid pACT-2 (Clontech). Poly(A)�

RNA was prepared from the P19 cells treated with 5 � 10�7 M RA for12 h, and cDNA was prepared by a cDNA Synthesis Kit (Stratagene,Toyobo Co. Ltd., Tokyo, Japan). cDNA fragments digested with XhoIand EcoRI were inserted into pACT-2 in frame with the coding sequencefor the GAL4 activation domain. Yeast two-hybrid screening was per-formed 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 promoterwere cotransformed with the bait and library plasmids, and colonieswere selected on His�, Leu�, and Trp� plate with 25 mM 3-aminotria-zole. The colonies were confirmed to be truly positive for His and�-galactosidase (�-gal) production.

Full-length mouse Nif3l1 cDNA was amplified by reverse tran-scriptase-polymerase chain reaction (RT-PCR) using total P19 cell RNAand the primers corresponding to mouse Nif3l1 cDNA (24). The primersused 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 productwas 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 prepareGST-Trip15/CSN2 fusion proteins, Trip15/CSN2 cDNA fragment wasinserted into the expression vector pGEX-2T (Amersham Biosciences,Buckinghamshire, UK) in frame with the coding sequence for GST. Theplasmid was designated as a pGEX-Trip15/CSN2. pGEX-Trip15/CSN2was introduced into Escherichia coli JM109, and the fusion proteinsynthesized was purified on glutathione-Sepharose beads (AmershamBiosciences) as described by Kaelin et al. (46). Glutathione-Sepharosebeads 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 bovineserum albumin, 5 mM 2-mercaptoethanol, 0.5% Nonidet P-40), andsubsequently 5 �g of the purified fusion protein were incubated with 2%glutathione-Sepharose beads in a final volume of 500 �l for 1 h at 4 °Cwith gentle rotation. GST encoded in the vector was also preparedunder the same conditions and immobilized as a control. The beadspreloaded with GST-Trip15/CSN2 fusion proteins were washed threetimes with the binding buffer, and 35S-labeled Nif3l1 proteins synthe-sized 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 �l of the binding buffer, andthe bound proteins were eluted with 15 �l of the SDS sample buffer(62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol) and electrophoresedon 12% SDS-PAGE. The gel was fixed, dried up, and exposed to thex-ray film.

Immunoprecipitation Assay—Flag-tagged Nif3l1 expression vectorwas constructed by the insertion of blunt-ended NcoI-SalI ORF frag-ment of pGEM-5zf(�)-Nif3l1 into the blunt-ended EcoRI-SalI site ofpFLAG-CMV2 (Sigma). Full-length rat Trip15/CSN2 cDNA clone wastransferred from pGEM-7zf(�) (Promega) to pACT2 after cleavage withEcoRI and XhoI. HA-tagged Trip15/CSN2 expression vector was con-structed by the insertion of blunt-ended XhoI-BglII ORF fragment ofpACT2-Trip15/CSN2 into the blunt-ended BamHI-EcoRV site ofpcDNA3 (Invitrogen). For immunoprecipitation studies, COS-7 cells(2 � 106) were transfected with pcDNA3-HA-Trip15/CSN2 and/orpFlag-CMV2-Nif3l1 by lipofection with DOTAP (Roche Molecular Bio-chemicals). Forty-eight hours after the transfection, the cells were lysedin 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 celllysate was incubated with 2% anti-Flag M2-agarose affinity gel (Sigma)at 4 °C overnight. The affinity gels were washed three times with 1 mlof TNM buffer and suspended in 15 �l of the SDS sample buffercontaining 5% 2-mercaptoethanol. The affinity gels were heated at95 °C for 5 min and subjected to 12% SDS-PAGE. The proteins weretransferred to a Clear Blot Membrane (Atto, Tokyo, Japan). The mem-brane were blocked in TBST (20 mM Tris-HCl, pH 7.4-buffered saline,

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0.02% Tween 20) containing 1% nonfat dry milk for 1 h at roomtemperature and incubated overnight at 4 °C with anti-HA antibody(1:1000 dilution, Sigma), anti-Flag antibody (1:400 dilution, Sigma), oranti-rat Trip15/CSN2 antibody (1:1000 dilution; Ref. 11). After washingthree times with TBST, the membrane was incubated with anti-mouseIgG 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 cellsand various mouse tissues by the acidic guanidine thiocyanate-phenol-chloroform method (47). Aliquots of 20 �g total RNA were electropho-resed 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-kbBamHI-EcoRI fragment of pGEM-rTrip15/CSN2 (11), and PCR-ampli-fied mouse Oct-3/4 cDNA (5�-primer, 5�-CCT GGC TAA GCT TCC AAGGGC-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 acidicribosomal phosphoprotein (PO) cDNA (5�-primer, 5�-CAG CTC TGGAGA AAC TGC TG-3�; 3�-primer, 5�-GTG TAC TCA GTC TCC ACAGA-3�) (50). The PO gene, also called 36B4 (51), was used as an internalcontrol gene, because its expression level has been shown to be invari-ant during RA-induced differentiation of P19 cells (6). Developed x-rayfilms were scanned in a Macintosh Performa 6410, and the expressionlevels of RNAs were quantified using Image 1.62 ppc program (NationalInstitutes of Health, Bethesda, MD).

Construction of Tetracycline (Tet)-controlled Nif3l1 Expression Sys-tem—Tet-controlled Nif3l1 expression in P19 cells was performed usingthe Tet-Off TM gene expression system (Clontech). At the first step, P19cells were transfected with a pTet-Off vector using DOTAP and culti-vated in the presence of 400 �g/ml G418 (Wako, Tokyo, Japan) forselection. Resulting G418-resistant colonies were then screened by thetransient transfection with a pTRE2-Luc vector (Clontech) to isolate the

best P19 Tet-Off cells exhibiting a low background and high Tet-de-pendent induction of luciferase (Luc) by the withdrawal of Tet. Theselected P19 Tet-Off cell line was designated as R13. pTRE2-sense and-antisense Nif3l1 expression vectors were constructed by the insertionof blunt-ended NcoI-SalI ORF fragment of pGEM5zf(�)-Nif3l1 into theblunt-ended HindIII-SalI and SalI-EcoRV sites of pTRE2 (Clontech),respectively. At the second step, R13 cells were transfected withpTRE2-sense Nif3l1 or pTRE2-antisense Nif3l1 together with pTK-Hyg(Clontech) and cultivated in the presence of 400 �g/ml hygromycin(Wako) and 2 �g/ml Tet (Sigma) for selection. Hygromycin-resistantcolonies were then screened for the induction of sense and antisenseNif3l1 RNAs in the absence of Tet by Northern blot and RT-PCR. Thecell lines that express sense and antisense Nif3l1 RNA after Tet re-moval thus obtained were designated as R13NifS and R13NifA,respectively.

Immunocytochemistry—P19 cells were cultivated in a Lab-Tek IIChamber slide (Nalge Nunc International, Naperville, IL) and fixedwith 4% paraformaldehyde. The cell samples were incubated inCa2�,Mg2�-free phosphate-buffered saline (PBS(�)) containing 10%normal rabbit serum for 30 min at 37 °C. The samples were thenincubated for 2 h at 37 °C with antibodies against �-tubulin III (1:1000dilution, Sigma) or GFAP (1:400 dilution, Sigma) in the same solutiondescribed above. After washing with PBS(�), the samples were incu-bated with a biotin-conjugated rabbit anti-mouse IgG�IgA�IgM(Nichirei, Tokyo, Japan) as a secondary antibody and followed by theincubation with a peroxidase-conjugated streptavidin (Nichirei). Visu-alization of the signal was carried out using 3,3�-diaminobenzidine.Nuclei were counterstained with hematoxylin.

Western Blot Analysis—For Western blot analysis, cells were col-lected, lysed in the SDS sample buffer without bromphenol blue and2-mercaptoethanol, and sonicated for 4 s. The resulting lysates werecleared by centrifugation at 15,000 rpm for 10 min. After protein con-centration was determined by BCA kit (Pierce), 5% 2-mercaptoethanol(final concentration) was added. Aliquots of 20 �g of cell lysate were

FIG. 1. Characterization of binding region of Trip15/CSN2 to Nif3l1. A, yeast two-hybrid analysis of the Trip15/CSN2 domain required forbinding to Nif3l1. Various fragments of Trip15/CSN2 cDNA were ligated in frame with the GAL4DBD in pAS2–1 and used as baits. Nif3l1 cDNAcorresponding to amino acid residues 243–376 was ligated with the GAL4 activation domain in pACT-2. These Nif3l1 and Trip15/CSN2 expressionconstructs were simultaneously introduced into yeast strain Y153 cells. The cells were streaked on both selection (Leu�, Trp�, His�, and3-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 Tascouet al. (25). We found a putative Leu Zip (open box) sequence, which is commonly observed in a protein-protein interaction site (52). Underlineindicates the region of Nif3l1 used as a prey. C, schematic view of protein binding regions of Nif3l1 and Trip15/CSN2. The regions of Trip15/CSN2required for interaction with DAX-1, TR� (14), and COP9 signalosome (21–24) have been previously reported.

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heated at 95 °C for 5 min and subjected to 12% SDS-PAGE. The pro-teins were transferred to a Clear Blot Membrane (Atto). The membranewere blocked in TBST containing 1% nonfat dry milk for 1 h at roomtemperature and incubated overnight at 4 °C with anti-�-tubulin typeIII antibody (1:1000 dilution, Sigma) or anti-GFAP antibody (1:400dilution, Sigma). After washing three times with TBST, the membranewas incubated with anti-mouse IgG conjugated with horseradish per-oxidase (1:5000 dilution, Sigma). Signals were visualized with the ECLsystem (Amersham Biosciences) according to the protocol from themanufacturer. Densitometric analysis was performed using Image 1.62ppc program as described above.

Luciferase Reporter Assay—A luciferase reporter construct was gen-erated by the insertion of the thymidine kinase (TK) basal promoterderived from pMC1neoPolyA (Stratagene) into XhoI-BamHI site ofpTRE2-Luc (Clontech) containing luciferase gene. In addition, blunt-ended fragment containing five copies of GAL4 binding sequence wasinserted into blunt-ended XhoI sites of pTRE2-Luc. The plasmidwas designated as pGAL4-TK-Luc. The GAL4DBD expression vectorwas constructed by the insertion of the GAL4DBD sequence derivedfrom pAS2–1 into blunt-ended EcoRI sites of pcDNA3. The plasmid wasdesignated as pcDNA3GAL4DBD. The GAL4DBD-fused Nif3l1 andTrip15/CSN2 expression vectors were constructed by the insertions ofentire coding region of Nif3l1 and Trip15/CSN2 in frame into thedownstream of GAL4DBD-coding sequence in pcDNA3GAL4DBD anddesignated as pGAL4DBD-Nif3l1 and pGAL4DBD-Trip15/CSN2, re-spectively. Trip15/CSN2 expression vector was constructed by insertionof the BamHI-EcoRI fragment containing entire ORF of pGEM7zf(�)-Trip15/CSN2 into the BamHI-EcoRI sites of pcDNA3 and designated aspcDNA3-Trip15/CSN2 (11). For each transfection, 2 � 105 P19 cells/35-mm diameter dish was transfected with 1 �g of pGAL4-TK-Luc, 0.5�g of �-gal expression vector pcDNA3.1/Myc-His/lacZ (Invitrogen), 0.5�g of GAL4DBD-fused Nif3l1 or Trip15/CSN2 expression vector orempty expression vector (pcDNA3) which indicated, 0.5 �g of Trip15/CSN2 expression vector was also cotransfected. At 48 h after transfec-tion, cells were lysed with the lysis buffer of luciferase assay kit (Pro-mega), and luciferase activities were determined according to theinstructions 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 con-structed by the insertions of entire cording region of Nif3l1 and Trip15/CSN2 into the downstream of EGFP-coding sequence in frame inpEGFP-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 combi-nations using DOTAP. pEF/Myc/ER/GFP (Invitrogen) having a target-ing signal of endoplasmic reticulum (ER) was also transfected as amarker of ER. After 36 h, the cells were fixed with 4% paraformalde-hyde and washed with PBS(�). To see the cellular localization of Nif3l1,P19 cells (1 � 106 cells/10-cm dish) were transfected with pEGFP-Nif3l1expression vector using LipofectAMINE PLUSTM (Invitrogen). After12 h, the cells were treated with 5 � 10�7 M RA and cultivated forvarious times. The cells were then fixed with 4% paraformaldehyde andsubsequently washed with PBS(�). The nuclei were stained with 1�g/ml Hoechst 33258. The cells were observed under a fluorescentmicroscope (Axioplan2, Carl Zeiss, Oberkochen, Germany).

RESULTS

Identification of Trip15/CSN2-binding Proteins—The N-ter-minal region of Trip15/CSN2 has been shown to be sufficientfor its effective function and for association with its bindingpartners including TR and DAX-1 via its N-terminal region(12–15). To analyze the role of Trip15/CSN2 in the process ofneuronal differentiation, cDNA clones encoding novel Trip15/CSN2-binding proteins were searched for by using a yeasttwo-hybrid screening system. A yeast strain Y153 (His�, Trp�,Leu�) was cotransfected with the bait vector, which expressesa fusion protein composed of GAL4DBD and the N-terminalregion (amino acids 1–275) of rat Trip15/CSN2, and a RA-treated P19 cell cDNA library, which direct the synthesis offusion proteins composed of cDNA-encoded proteins and theGAL4 transcriptional activation domain. Y153 strain containsthe His3 and LacZ genes linked to the GAL4 promoter. By the

transfection of 3 � 106 Y153 cells, 52 His� colonies were de-veloped and 17 of 52 colonies were �-gal-positive. Sequenceanalysis of these clones showed that 5 of 17 clones encode theC-terminal region (amino acids 243–376) of mouse Nif3l1(Ngg1-interacting factor 3 like-1), which possesses a high ho-mology to yeast Ngg1-interacting factor 3 homolog (25). Basedon this finding, we focused on the functional analysis of Nif3l1in neural differentiation. Full-length Nif3l1 cDNA was ampli-fied by RT-PCR using total RNA from mouse P19 cells andNif3l1-specific primers (25).

Interaction of Nif3l1 with Trip15/CSN2 in Vitro and inVivo—We found that Nif3l1 possesses two putative leucinezipper (Leu Zip) motifs (210–224 and 264–278) mediating aprotein-protein interaction (Fig. 1B) (52). To determine thebinding region of Trip15/CSN2 to the C-terminal region (243–376) of Nif3l1, various regions of Trip15/CSN2 were insertedinto pAS2–1 and obtained the bait constructs that expressfusion 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), areshown in Fig. 1A. These bait constructs were transfected intoyeast strain Y153 cells, which allow selection for two differentmarkers: His and �-gal. Cotransformation with the vector forGAL4 activation domain-fused C-terminal region of Nif3l1

FIG. 2. Interaction between Nif3l1 and Trip15/CSN2 in vivoand in vitro. A, binding of Trip15/CSN2 with Nif3l1 analyzed by GSTpull-down assay. Bacterially expressed GST-Trip15/CSN2 or GST werepreloaded to glutathione-Sepharose beads. The beads were incubatedwith [35S]methionine-labeled Nif3l1 protein. The retained proteinswere analyzed by 12% SDS-PAGE. B, interaction between Trip15/CSN2and Nif3l1 in COS-7 cells. COS-7 cells were transfected with pFlag-CMV2-Nif3l1, pcDNA3-HA-Trip15/CSN2, or both, and the cell lysatesprepared after 48 h were subjected to immunoprecipitation with amonoclonal anti-Flag antibody. The immunoprecipitates were analyzedby Western blot with anti-HA antibody (upper panel). Expression ofFlag-Nif3l1 and HA-Trip15 was confirmed in the cells transfected withthe corresponding vector alone by Western blot (middle and lowerpanels, 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, immuno-precipitated with anti-Flag antibody, and subjected to Western blotwith anti-Trip15/CSN2 antibody (upper panel; Ref. 11) and anti-Flagantibody (lower panel). The cell lysate from parental P19 cells treatedwith RA for 12 h was also analyzed to confirm the induction level ofTrip15/CSN2.

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(243–376) showed that the N2 region selectively bound to theC-terminal region of Nif3l1 (Fig. 1A). The full-length Trip15/CSN2 bound to Nif3l1 weakly. The result suggests that the N2region of Trip15/CSN2 containing a putative Leu Zip, nuclearlocalization signal (NLS), and corepressor motif ((I/L)XX(I/V)I;Ref. 53), which is required for binding to a nuclear hormonereceptor DAX-1 (14), is necessary and sufficient for interactionwith the C-terminal region of Nif3l1 containing a putative LeuZip (Fig. 1C). It seems likely that these putative motifs partic-ipate in the interaction between Trip15/CSN2 and Nif3l1 andthe weak interaction of full-length Trip15/CSN2 with Nif3l1 isrelated to the transcriptional corepressor activity of Trip15/CSN2 (12–14).

To further confirm the interaction between the full-lengthNif3l1 and Trip15/CSN2 in vitro, GST pull-down assay wasperformed. GST-fused Trip15/CSN2 protein was bound to glu-tathione-Sepharose beads, and then 35S-labeled Nif3l1 synthe-sized by a in vitro translation was added to the beads. Afterwashing with the binding buffer, 35S-labeled Nif3l1 retained inthe beads was eluted with the SDS sample buffer and analyzedby 12% SDS-PAGE. As shown in Fig. 2A, the 42-kDa Nif3l1protein specifically bound to GST-Trip15/CSN2 fusion protein.

To demonstrate that the interaction between these two pro-teins also occurs in vivo, COS7 cells were transfected withexpression 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 celllysates were prepared and subjected to Western blot analysis.

The transient expression detected two proteins of 43 and 53kDa that correspond to Flag-tagged Nif3l1 and HA-taggedTrip15/CSN2, respectively, judging from the estimated molec-ular mass of these constructs (Fig. 2B, middle and lowerpanels, respectively). Immunoprecipitation of the extracts pre-pared 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 cellstransfected with either one of expression vectors. No effect onthe expression level of either Trip15/CSN2 or Nif3l1 was ob-served in the cotransfected cells as determined by Western blotanalysis. To examine further the interaction between Nif3l1and endogenous Trip15/CSN2, P19 cells were transfected withpFlag-CMV2-Nif3l1 vector and then treated with RA to inducethe Trip15/CSN2 gene expression. After 12 h when the maxi-mal induction of Trip15/CSN2 protein was observed (11), thecell lysate was prepared, immunoprecipitated with anti-Flagantibody and subjected to Western blot with polyclonal anti-Trip15/CSN2 antibody (11). As shown in Fig. 2C, endogenousTrip15/CSN2 was coimmunoprecipitated with Nif3l1. Theseresults indicate that the interaction between Nif3l1 andTrip15/CSN2 occurs in vivo.

Expression Pattern of the Nif3l1 Gene during Neural Differ-entiation—To examine the expression level of Nif3l1 mRNAduring the neural differentiation of P19 cells, total RNAs wereextracted from the aggregated P19 cells treated with 5 � 10�7

M RA for various times and analyzed by Northern blotting withthe 0.6-kb EcoRI-DraI fragment of pGEM-5zf(�)-Nif3l1 as aprobe. As shown in Fig. 3A, the Nif3l1 gene was mainly ex-pressed as 1.85-kb mRNA and the levels of two additionaltranscripts of 2.4 and 3.4 kb were very low. The expressionpattern of the Nif3l1 gene in P19 cells resembles those of thespermatogonia-derived GC-1 spg cells and the mouse terato-carcinoma F9 cells (25). Three species of mRNAs might begenerated by alternative splicing, because the Nif3l1 gene hasbeen shown to be a single-copy gene (25). During neural differ-entiation of RA-treated P19 cells, the level of Nif3l1 gene ex-

FIG. 3. Expression levels of Nif3l1 mRNA in RA-treated P19cells and in various mouse tissues. A, expression patterns of Nif3l1and Trip15/CSN2 mRNAs during RA-primed P19 cell neural differen-tiation. Total RNAs were extracted from the P19 cells treated with 5 �10�7 M RA for various times and analyzed by Northern blot using a0.6-kb EcoRI-DraI fragment of mouse Nif3l1 cDNA (upper panel) and a2.1-kb EcoRI fragment of rat Trip15/CSN2 cDNA (middle panel) asprobes. Expression levels of Nif3l1 mRNA in various adult mousetissues (B), embryonic mouse brain (C), and postnatal mouse brain (D)were similarly analyzed. Pictures of ethidium bromide-stained 28 Sribosomal RNA are included for comparison of total amount of RNAemployed.

FIG. 4. Induction of sense and antisense Nif3l1 RNAs by Tetremoval. Expression levels of Nif3l1 mRNA in R13 cells in the pres-ence 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 endogenousNif3l1 mRNA expression in R13NifA cells after removal of Tet. Expres-sion levels of endogenous Nif3l1 mRNA were analyzed by RT-PCR. Theprimers 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 CTCCAG TAC CTG CTC CGA G-3�. Expression levels of PO mRNA werealso analyzed by RT-PCR as an internal control using the followingprimers. 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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pression was not changed. On the other hand, the expression ofTrip15/CSN2 mRNA was induced shortly after the addition ofRA reaching a maximal level at 3 h after the treatment. Thelevel then decreased and became barely detectable after 12 h.However, Trip15/CSN2 seemed to be accumulated during thisperiod, and its ability to translocate Nif3l1 into the nuclei wasaugmented along with the progression of neural differentiationas shown below (see Fig. 10B). The result suggested that Nif3l1interacts with Trip15/CSN2 for only the limited period duringthe early stage of neuronal differentiation. To analyze thetissue-specific expression of the Nif3l1 gene, total RNAs wereextracted from various adult mouse tissues and analyzed byNorthern blot. The Nif3l1 gene was expressed as 1.85-, 2.4-,and 3.4-kb mRNAs in all the tissues so far examined, but theexpression levels vary among these tissues (Fig. 3B). Higherlevels of expression were observed in cerebellum, heart, andkidney and low levels of expression in spleen and muscle. Thelevels of expression in cerebrum, lung, and liver were interme-diate. In cerebellum and kidney, the levels of 2.4-kb mRNAwere higher than those of other tissues. We also analyzed theNif3l1 gene expression during the development of mouse brain.A high level of Nif3l1 expression as 2.4- and 1.85-kb mRNAswas already detectable in the brain at E10.5 and continueduntil 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 did2.4- 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 changesof expression among these splicing variants. The reason is notclear at present. These results support the idea that the Nif3l1gene plays an important role in neural differentiation anddevelopment, and in maintenance of neural function.

Implication of Nif3l1 in Neural Differentiation—To demon-strate the implication of Nif3l1 in neural differentiation, weestablished P19 derivative cell lines in which the exogenousexpressions of sense and antisense mouse Nif3l1 RNAs couldbe initiated by the withdrawal of Tet. Briefly, pTRE2-senseNif3l1 and pTRE2-antisense Nif3l1 vectors were transfectedinto the pTet-Off vector-introduced P19 (termed as R13) cellsas described under “Experimental Procedures.” The resultingstable transformants introduced with sense and antisenseNif3l1 vectors were designated as R13NifS and R13NifA, re-spectively. To examine whether the expression of exogenoussense and antisense Nif3l1 RNA could be induced by the re-moval of Tet, we analyzed the expression levels of Nif3l1mRNA in R13, R13NifS, and R13NifA cells in the presence andabsence of Tet (Fig. 4). The endogenous expression level ofNif3l1 mRNA in R13 cells was not changed in the Tet-freemedium (Fig. 4A). In R13NifS cells, the expression of Nif3l11.85-kb mRNA analyzed by Northern blot was substantiallyinduced by the withdrawal of Tet and after 12 h, the levelincreased to 6.5-fold higher than that expressed in the presenceof Tet (0 h) (Fig. 4B). In contrast, the expression level ofendogenous 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

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 �m. B,quantification of effects of sense and antisense Nif3l1 RNAs on differentiation to �-tubulin III-positive neurons. �-Tubulin III-positive neuronswere counted at least 5 fields/slide under a microscope (original magnification, �100) and estimated the Tet(�)/Tet(�) ratios. Each value is theaverage � S.E. of triplicate chamber slides. *, p � 0.001; **, p � 0.05 compared with the control R13 cells. C, effects of Tet-controlled expressionof 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) cellswere 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 7days after replating. Scale bar presents 500 �m. D, quantification of effects of sense and antisense Nif3l1 RNAs on differentiation to GFAP-positiveglial 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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level of PO mRNA examined as an internal control in R13NifAcells was not changed by the removal of Tet, showing thatantisense Nif3l1 RNA specifically interacted with endogenousNif3l1 mRNA. These results indicate that the exogenous ex-pression of the sense and antisense Nif3l1 RNAs could really becontrolled by Tet.

Using these stable transformants, we analyzed the effect ofexogenous expression of Nif3l1 on the neural differentiation ofP19 cells immunocytochemically (Fig. 5). R13, R13NifS, andR13NifA cells could not differentiate into neurons after thesimple aggregation culture without the addition of RA, irre-spective of the absence of Tet (Fig. 5A (a, d, and g)). On theother hand, in the presence of RA all transformants weredifferentiated into �-tubulin III-positive neurons even if Tetwas added (Fig. 5A (b, e, and h)). Nonetheless, by the removalof Tet, the neuronal differentiation of R13NifS cells was signif-icantly stimulated (Fig. 5, A (f) and B). In contrast, the neuro-nal differentiation of R13NifA cells was slightly decreased (Fig.5, A (i) and B). In R13 cells, the effect of Tet removal onRA-primed neuronal differentiation was not observed (Fig. 5, A(c) and B), showing that Tet itself did not affect the RA-primedneuronal differentiation.

The effect of Nif3l1 on the GFAP-positive glial cell differen-tiation was also examined. Although differentiation of alltransformants to GFAP-positive glial cells was not observedwithout the addition of RA, as was the case in neural differen-tiation (data not shown), differentiation of R13NifS cells to glialcells induced by RA was stimulated by the removal of Tet (Fig.5C, compare m with l). In contrast, the glial differentiation ofR13NifA cells induced by RA was significantly suppressed afterTet removal (Fig. 5C, compare o with n), whereas in R13 cells,the effects of Tet removal was not evident (Fig. 5C (k)). Theseresults 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 ofNif3l1 on the expression of neuron and glial cell markers byWestern blotting with anti-�-tubulin III and anti-GFAP anti-bodies. In aggregated R13NifS cells, the expression levels of�-tubulin III and GFAP were not induced by the withdrawal ofTet in the absence of RA (data not shown). In the presenceof RA, the additive effect of the exogenous expression of Nif3l1was observed in aggregated R13NifS cells on the expression of�-tubulin III (Fig. 6, A and C) and GFAP (Fig. 6, B and D). Onthe other hand, the expression of �-tubulin III and GFAP inR13NifA cells was conspicuously reduced by the expression ofantisense Nif3l1 RNA in the presence of RA. These resultswere consistent with the data obtained from immunocytochem-ical analysis as shown in Fig. 5. Taken together, it seems likelythat the Nif3l1 gene plays a crucial role in the RA-primedneural differentiation signal pathway.

Implication of Nif3l1 Gene in Early Commitment of P19 Cellsto Neural Lineage—The enforced expression of sense Nif3l1RNA in R13NifS cells by the removal of Tet enhanced differ-entiation into �-tubulin III-positive neurons in the presence of

FIG. 6. Effects of enforced expression of sense and antisenseNif3l1 RNAs on expression of neuron and glial cell marker pro-teins. R13, R13NifS, and R13NifA cells were treated with RA for 4 daysin the presence or absence of Tet. Expression levels of �-tubulin III (A)and GFAP (B) were analyzed by Western blot with corresponding an-tibodies at 3 and 7 days after plating, respectively. C, the graph showsthe Tet(�)/Tet(�) ratio of �-tubulin III expression levels. Values areshown as the mean � S.E. of four experiments. *, p � 0.004; **, p �0.002 compared with the control R13 cells. D, the graph shows theTet(�)/Tet(�) ratio of GFAP expression levels. Values are shown as themean � S.E. of four experiments. *, p � 0.001; **, p � 0.006 comparedwith the control R13 cells.

FIG. 7. Effect of Tet-controlled expression periods of senseNif3l1 RNA on neuronal differentiation. R13NifS cells weretreated 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 RAand 2 �g/ml Tet up to 4 days. Immunocytochemical analysis was per-formed using antibody against �-tubulin III at 3 days after replating.Scale bar presents 500 �m. E, effect of enforced expression periods ofsense Nif3l1 RNA on the fraction of R13NifS cells differentiated to�-tubulin III-positive neurons. The differentiated neurons were countedat least 5 fields/slide. Values are the average � S.E. of three independ-ent chamber slides. *, p � 0.05 compared with the Tet(�) control. F,effect of enforced expression periods of sense Nif3l1 RNA on �-tubulinIII expression. Cell lysates were prepared at 3 days after replating andanalyzed by Western blot. G, the result obtained in F was quantitativelyshown as relative expression levels. The �-tubulin III level expressed attime 0 was taken as 1. Values are the average � S.E. of three experi-ments. *, p � 0.001 compared with the Tet(�) control. H, rapid induc-tion of sense Nif3l1 RNA in R13NifS cells by Tet removal. R13NifS cellswere cultivated in the absence of Tet for various times, and expressionlevels of Nif3l1 RNA were analyzed by Northern blot.

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RA (Fig. 5A). To investigate which periods of sense Nif3l1 RNAexpression are effective for RA-primed neuronal differentia-tion, we removed Tet for various time periods and the extentsof cell differentiation and the levels of �-tubulin III were ana-lyzed by immunocytochemistry and Western blot, respectively.We confirmed that sense Nif3l1 RNA expression was inducedjust after 30 min of the Tet removal (Fig. 7H). Immunocyto-chemical analysis revealed that neuronal differentiation ofR13NifS cells was significantly stimulated by Tet removal, onlyfor 3 h (Fig. 7, B and E). Longer removal of 12 h stimulated thedifferentiation further, but only to a small extent, and theextent was even slightly decreased by removal for 24 h (Fig. 7,C, D, and E). Western blot analysis also showed that theexpression of �-tubulin III was induced to near maximal levelduring 3 h of Tet removal (Fig. 7, F and G). These resultsindicate that Nif3l1 acts on the very early stage of RA-primedneuronal differentiation.

Effect of Nif3l1 on Oct-3/4 and Neurogenic Gene Expres-sions—Transcription factors regulate expression of specificgenes to control cellular phenotype. Oct-3/4 is a transcriptionfactor and acts to maintain the undifferentiated state of P19cells as well as embryonic stem cells. Its mRNA expression isdramatically diminished within 24 h of the RA treatment (3).Thus, the down-regulation of Oct-3/4 is required for the onset ofneural differentiation. In addition, Nif3l1 and Trip15/CSN2 areknown as transcriptional regulator and transcriptional core-pressor, respectively (12–14, 25, 26). On the basis of these facts,whether Nif3l1 participates in neural differentiation throughthe down-regulation of Oct-3/4 gene expression was analyzed inaggregated R13NifS cells after the removal of Tet in the pres-ence and absence of RA by Northern blot (Fig. 8A). When thecells were treated with RA in the absence of Tet, the level ofOct-3/4 mRNA began to decrease after 36 h and lowered to analmost undetectable level after 60 h (Fig. 8A). The reduction ofOct-3/4 mRNA level after 60 h, however, was not observed inthe presence of Tet, suggesting that Nif3l1 is involved in the

repression of the Oct-3/4 gene and the effect became visibleafter 60 h. This residual effect of Nif3l1 was also seen in theabsence of RA, and the level was reduced to 75% of the originallevel at 96 h after removal of Tet (Fig. 8A).

We further analyzed the expression level of neurogenicMash-1 gene during neural differentiation, because it plays akey 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 washardly detected in R13NifS cells not treated with RA. By theaddition of RA, expression of Mash-1 mRNA became detectableafter 60 h when the down-regulation of Oct-3/4 mRNA wasenhanced. This induction of Mash-1 gene expression seemed tobe influenced by exogenously expressed Nif3l1, although theeffects was not so evident. Under these conditions, the level ofPO mRNA, analyzed as an internal control, was not signifi-cantly altered, although some bias was observed (Fig. 8C).

Nif3l1 Harbors an Autonomous Silencing Function and Co-operates with Trip15/CSN2—Trip15/CSN2 interacts with asubset of nuclear hormone receptors such as DAX-1, ecdysonereceptor, COUP-TF1, Ftz-F1, and TRs, but not with retinoicacid receptors and retinoid X receptors (12–14). Trip15/CSN2interacts with TR� and harbors an autonomous silencing func-tion. This interaction is inhibited by increasing amounts ofthyroid hormone (13). Thus, Trip15/CSN2 represents a mem-ber of novel class of corepressors specific for a selected memberof the nuclear hormone receptor family (13, 14). In addition,Nif3l1 interacts with transcriptional activator/repressor pro-tein ADA3/NGG1 (26, 29, 30). Therefore, if Nif3l1 is involved ina transcriptional silencing, we expected that Nif3l1 shouldharbor an autonomous silencing function and cooperate withTrip15/CSN2 in a transcriptional silencing.

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) orpGAL4DBD-Nif3l1 (middle panel) and stained with anti-GAL4 anti-body after 48 h (Santa Cruz Biotechnology, Santa Cruz, CA). Nucleiwere stained with hematoxylin (lower panel). Scale bar presents 20 �m.B, both luciferase reporter plasmid pGAL4-TK-Luc (1 �g) and �-galexpression plasmid pcDNA3.1/Myc-His/lacZ (0.5 �g) were transfectedinto P19 cells together with 0.5 �g of pGAL4DBD, pGAL4DBD-Nif3l1,or pGAL4DBD-Trip15/CSN2 in combination with 0.5 �g ofpcDNA3-Trip15/CSN2 or empty pcDNA3. At 48 h after transfection, thecells were lysed and assayed for luciferase activity. Luciferase activitieswere normalized using �-gal activity as an internal control, and theactivity expressed by cotransfection with pGAL4DBD as a control wastaken as 1. Each value is shown as average � S.E. of triplicate culturedishes. *, p � 0.002; **, p � 0.001; ***, p � 0.001 compared with thepGAL4DBD control.

FIG. 8. Effect of enforced expression of Nif3l1 mRNA on thelevels of Oct-3/4 and Mash-1 mRNAs. R13NifS cells were treated oruntreated 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 analyzedby Northern blot. PO mRNA expression (C) was analyzed simulta-neously as an internal control.

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To localize Nif3l1 and Trip15/CSN2 effectively in nuclei,expression vectors containing the full-length Nif3l1 andTrip15/CSN2 cDNAs fused to the GAL4DBD coding sequencedownstream of the CMV promoter were constructed, becauseGAL4DBD possesses PKTKRSP sequence as a NLS.GAL4DBD itself and GAL4DBD-fused Nif3l1 transiently ex-pressed in P19 cells were actually localized in nuclei (Fig. 9A).The effect of Nif3l1 and Trip15/CSN2 on the suppression of TKpromoter activity in P19 cells was studied using these vectorsand a luciferase reporter construct, pGAL4-TK-Luc which con-tains GAL4 binding sequence upstream of the TK basal pro-moter. P19 cells were transfected with pGAL4-TK-Luc togetherwith expression vectors of pGAL4DBD, pGAL4DBD-Nif3l1,pGAL4DBD-Trip15/CSN2, pcDNA3-Trip15/CSN2, or a combi-nation of these expression vectors. As shown in Fig. 9B, Nif3l1significantly suppressed the TK promoter activity to one eighthof the activity expressed by cotransfection with pGAL4DBD,which expresses GAL4DBD alone. Trip15/CSN2 also sup-pressed the TK promoter activity but to a much lesser extent.When Nif3l1 was coexpressed with Trip15/CSN2 by cotransfec-tion with pcDNA3-Trip15/CSN2, additive effect was observedon the suppression of TK promoter activity. These results sup-port the idea that a transcriptional silencer Nif3l1 cooperateswith Trip15/CSN2 for the suppression of the TK promoteractivity and perhaps implicates in the down-regulation of Oct-3/4 gene.

Cellular Localization of Nif3l1—To investigate the subcellu-lar localization of Nif3l1, we constructed a EGFP-fused Nif3l1expression vector by the insertion of full-length Nif3l1 cDNAinto pEGFP-C1 downstream of EGFP coding sequence in

frame. The EGFP-fused Nif3l1 expression vector was intro-duced into COS-7 cells, and subcellular distribution of thefused protein was observed after 48 h. As shown in Fig. 10A (a),EGFP-Nif3l1 fusion protein was localized predominantly in thecytoplasm, although fluorescence was homogenously detectedboth in the nuclei and cytoplasm when only EGFP protein wasexpressed (data not shown). The localization of EGFP-Nif3l1was different from that of ER retention signal-tagged GFP,which was concentrated around the nuclei (Fig. 10A (e)). On theother hand, EGFP-fused Trip15/CSN2 was localized in thenuclei predominantly (Fig. 10A (c)) and showed an image sim-ilar to the nuclei that are stained with Hoechst 33258, whichintercalate to DNA (Fig. 10A (c and f)). For the expression oftranscriptional silencing activity, however, the translocation ofNif3l1 from the cytoplasm into the nuclei is absolutely re-quired. As described above, Nif3l1 bound to Trip15/CSN2 invivo and in vitro, and cooperated with Trip15/CSN2 for thesuppression of the TK promoter activity, suggesting thatTrip15/CSN2 participates in the translocation of Nif3l1 fromthe cytoplasm to the nuclei. To confirm this assumption, theeffect of Trip15/CSN2 on the intracellular localization of Nif3l1was examined by cotransfection of COS-7 cells with pEGFP-Nif3l1 and pcDNA3-Trip15/CSN2 and the cells exhibiting dis-tinct fluorescence in the nuclei or cytoplasm were counted. Thefraction of EGFP-fused Nif3l1 localized predominantly in thenuclei was increased depending on the increase in the amountof 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 theabsence of Trip15/CSN2 (Fig. 10A (g)). In contrast, the inter-

FIG. 10. Nuclear localization of Nif3l1 controlled by Trip15/CSN2. A, implication of Trip15/CSN2 in translocation of Nif3l1. COS-7 cellswas 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 withpEGFP-Trip15/CSN2 alone (c) or together with pcDNA3-Nif3l1 in the ratio of 1:9 (d). Transfection with pEF/Myc/ER/GFP (Invitrogen) wasperformed as a marker of ER (e). Nuclei were stained with Hoechst 33258 (f). At 48 h after transfection, the cells were observed under a fluorescentoptics (Axioplan2; Carl Zeiss). Scale bar presents 40 �m. g, quantitative analysis of the effect of Trip15/CSN2 on intracellular localization ofEGFP-fused Nif3l1. The cells exhibiting distinct fluorescence in the cytoplasm and nuclei as shown in a and b, respectively, were counted. Eachvalue is the average � S.E. of triplicate culture dishes. *, p � 0.004 compared with the cells transfected with pEGFP-Nif3l1 alone. h, quantitativeanalysis of the effect of coexpression of Nif3l1 on intracellular localization of EGFP-fused Trip15/CSN2. Each value is the average � S.E. oftriplicate culture dishes. B, translocation of Nif3l1 during neural differentiation. The pEGFP-Nif3l1-transfected P19 cells were cultivated in theabsence 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 Hoechst33258 (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 �m. g, time-dependent translocation of EGFP-fused Nif3l1 during RA-primed P19 cell neural differentiation. Values arepresented as the average � S.E. of four independent dishes. *, p � 0.03 compared with of RA(�) control.

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cellular localization of EGFP-fused Trip15/CSN2 was not af-fected by the coexpression of Nif3l1 (Fig. 10A (d and h)).

Because the Trip15/CSN2 gene was highly expressed in anearly stage of neural differentiation of RA-treated P19 cells(Fig. 3A), we assumed that the translocation of Ni3fl1 from thecytoplasm into the nuclei might be enhanced at this early stageof neuronal differentiation. To confirm this assumption, P19cells were transfected with pEGFP-Nif31l and cultivated withand without RA for various times and the intracellular local-ization of EGFP-fused Nif3l1 was examined. The localization inthe nuclei was confirmed by staining the cells with Hoechst33258. Although in the absence of RA, EGFP-fused Nif3l1 wasmainly detected in the cytoplasm (Fig. 10B (a–c)), the fractionof cells exhibiting EGFP-fused Nif3l1 predominantly in thenuclei was augmented by the treatment with RA in a time-de-pendent manner and, after 24 h, increased to �3.5-fold of thatobserved in the absence of RA (Fig. 10B (d–g)). The merge oftwo images obtained by the EGFP fluorescence and by stainingwith Hoechst 33258 showed that Nif3l1 localizes as nuclearpatches (Fig. 10B (f)).

DISCUSSION

The data presented here provide the first insight into themolecular mechanism underlying the involvement of Nif3l1in neural differentiation through the association withTrip15/CSN2.

Using a yeast two-hybrid method, we found that the N-terminal region (amino acids 1–275) of Trip15/CSN2 bound tothe C-terminal region (amino acids 243–376) of Nif3l1 (Fig. 1).The interaction was confirmed by a pull-down assay and anepitope-tagged coimmunoprecipitation (Fig. 2, A and B). Inaddition, the interaction between Nif3l1 and endogenousTrip15/CSN2 in RA-primed P19 cells was also confirmed (Fig.2C). Trip15/CSN2 possesses a putative NLS and Leu Zip se-quence and is functioning in the nucleus (54), whereas itsbinding partner Nif3l1 does not contain a NLS (25). One expla-nation could be that Nif3l1 binds to Trip15/CSN2 in the cyto-plasm and subsequently enters the nucleus via cotransport asreported for interleukin-5 and its receptor subunit (55). In fact,this assumption was demonstrated based on the finding thatthe translocation of Nif3l1 from the cytoplasm to the nucleuswas dependent on the expression level of Trip15/CSN2 protein,whereas the nuclear localization of Trip15/CSN2 was not af-fected by the coexpression of Nif3l1 protein (Fig. 10A). Further-more, the nuclear localization of Nif3l1 was essentially re-quired for its functional expression, because Nif3l1 acted as atranscriptional silencer and synergized with Trip15/CSN2 forrepression of TK the promoter activity of a reporter construct,pGAL4-TK-Luc (Fig. 9).

Yeast Nif3 was originally identified as a NGG1-interactingprotein by a yeast two-hybrid screening (26). NGG1 was iso-lated based on its requirement for the inhibition of transcrip-tional activation of the genes involved in the utilization ofgalactose by GAL4 protein when yeast was grown in glucosemedium (27). ADA3/NGG1 was also isolated based on its abilityto suppress mutations and the toxic effects of overexpression ofthe viral activator protein, VP16 in yeast (28), suggesting thatADA3/NGG1 is involved in transcriptional activation and re-pression by Nif3l1 (26, 29, 30). Genetic studies on the transcrip-tional activation in yeast have identified ADA3/NGG1 as acritical component of coactivator complexes that link to tran-scriptional activators (56). ADA3/NGG1 and its associatedadapter ADA2 form a complex that recruit a histone acetyl-transferase GCN5 to promoters (35, 37, 57, 58). Through asimilar mechanism, coactivator complexes could also implicatein 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 luciferasereporter assay, we revealed that a Trip15/CSN2 binding part-ner, Nif3l1, possesses a transcriptional silencing function (Fig.9). Therefore, we propose that Nif3l1 and Trip15/CSN2 form anovel corepressor complex that might recruits an inhibitor ofhistone acetyltransferase or a histone deacetylase to targetpromoter(s) during neural differentiation, although direct evi-dence is needed.

Enforced expression of sense Nif3l1 RNA by the Tet expres-sion system caused a small but significant enhancement ofRA-primed neural differentiation of P19 cells into �-tubulinIII-positive neurons and GFAP-positive glial cells (Figs. 5 and6). Interestingly, the enforced expression for only up to 3 h wasnearly sufficient for the enhancement (Fig. 7). This time corre-sponds to that when the level of Trip15/CSN2 induced by thetreatment with RA becomes nearly maximal in accordance withthe cooperative action of Nif3l1 and Trip15/CSN2 for inductionof neural differentiation. Moreover, in relation to the intensedown-regulation of Oct-3/4, which maintains an undifferenti-ated state of P19 cells (3), the expression of neurogenic Mash-1gene was rapidly induced by the enforced expression of Nif3l1mRNA (Fig. 8). It appeared that Nif3l1-Trip15/CSN2 complexcould be implicated in the commitment of multipotent P19 cellsto neural lineage via the Oct-3/4 down-regulation. In the de-veloping brain, a common cortical progenitor cell gives rise firstto a variety of layer-specific neurons and then switches toproducing astrocytes and ultimately oligodendrocytes (59, 60).As in the case of neurogenesis in vivo, neurons appear earlierthan glial cells during RA-primed P19 cell neural differentia-tion (61). Therefore, the enhancement of both neurogenesis andgliogenesis by Nif3l1 might be caused by the increase in thefraction of cells committed to differentiate to neural lineage,although the precise estimation of the fraction of neural lineagewas difficult because of the occurrence of the cells that under-went proliferation and apoptosis during P19 cell neural differ-entiation (62).

The mouse Nif3l1 gene has been isolated by a suppressionsubtractive hybridization between the spermatogonia-derivedcell line GC-1 spg and spermatocyte-derived cell line GC-4 spc(25). Yeast Nif3 and NGG1 form a complex for effective inhibi-tion of the transcriptional activation by GAL4 (27). The Nif3l1gene was expressed throughout embryonic development and inthe various adult mouse tissues ubiquitously accompanying theputative alternative splicing (Fig. 3). The widespread expres-sion of Nif3l1 gene suggests that the gene acts as a commonrepressor and is unlikely to be restricted to specify neuralidentities by itself. The expression of Nif3l1 gene was alsoobserved in the undifferentiated P19 cells, and the expressionlevel was not changed significantly during RA-primed neuraldifferentiation. On the other hand, a binding partner Trip15/CSN2 gene expression was markedly induced by the treatmentof RA (Fig. 3A; Ref. 11). These observations suggest that Nif3l1-Trip15/CSN2 complex is specifically required for the earlystage of neural differentiation and various regulatory factorsdistinct from Trip15/CSN2 cooperate with Nif3l1 in the processof the important cellular events such as spermatogenesis andmaintenance of cell type-specific functions.

P19 cells possess many properties similar to embryonic stemcells isolated from mice and humans (63). Therefore, it may bepossible that the expression system of Nif3l1-Trip15/CSN2complex can be utilized for the production of large amount ofneurons from human embryonic stem cells in combination withthe expression of key neurogenic genes.

Acknowledgment—We thank Prof. K. Oda for encouragementthroughout the experiment and critical reading of the manuscript.

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Sugiyama and Fumio TashiroHirotada Akiyama, Naoko Fujisawa, Yousuke Tashiro, Natsuko Takanabe, Akinori

Differentiation via Cooperation with Trip15/CSN2The Role of Transcriptional Corepressor Nif3l1 in Early Stage of Neural

doi: 10.1074/jbc.M209856200 originally published online January 8, 20032003, 278:10752-10762.J. Biol. Chem. 

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