Biochemical and Biophysical Research Communications 291, 775–779 (2002)

doi:10.1006/bbrc.2002.6528, available online at http://www.idealibrary.com on

Functional Diversity among Notch1, Notch2,and Notch3 Receptors

Kiyoshi Shimizu,*,† Shigeru Chiba,*,† Toshiki Saito,*,† Keiki Kumano,*,†Yoshio Hamada,‡ and Hisamaru Hirai*,†,1

*Department of Hematology and Oncology and †Department of Cell Therapy and Transplantation Medicine, GraduateSchool of Medicine, University of Tokyo, Tokyo, Japan; and ‡National Institute of Basic Biology, Okazaki, Japan

Received January 24, 2002

To clarify functional diversities among the Notchreceptors, we generated truncated forms of Notch1,Notch2, and Notch3 comprising the intracellular do-main (aN1, aN2, and aN3) and investigated their tran-scriptional activities for HES1 and HES5 promotersdriving the luciferase reporter gene (HES1-Luc andHES5-Luc). The reporter assays demonstrated that thetranscriptional activities of aNs were markedly differ-ent from each other and dependent on the promotersexamined. Furthermore, relative activities betweensome aN and another for each promoter were alteredby the expression level of RBP-J�. We also found thatthe activities of aN1 and aN3 were reduced by coex-pression of aN2. These observations suggest that eachNotch receptor has a diverse role in the downstreamgene expression and that the levels of HES1 and HES5gene expression are complexly determined by variousfactors, such as the type and combination of the Notchreceptors which confer the downstream signals andthe expression level of RBP-J�. © 2002 Elsevier Science (USA)

Key Words: Notch; activated Notch; HES1; HES5;RBP-J�; reporter assay.

The Notch signaling pathway is evolutionarily con-served and proves to regulate cell fate decision in var-ious cell types during and after development (1–3).Notch is initially synthesized as about a 300-kDa pro-tein which is then processed by a furin protease in theGolgi-network, generating an extracellular fragment(NEC) and a transmembrane fragment (NTM) (4–5).These fragments are reassembled in the trans-Golginetwork and are presented as a heterodimeric, maturereceptor at the cell surface (4–5). Binding of the Notchligand (NL) to NEC induces cleavage of the Notch re-

1 To whom correspondence should be addressed at Department ofCell Therapy and Transplantation Medicine, University of TokyoHospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Fax:�81-3-5689-7286. E-mail: hhirai-tky@umin.ac.jp.

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of Notch (NICD) from NTM (6–8). NICD is subsequentlytranslocated into the nucleus, where functioning as atranscriptional regulator in concert with a transcrip-tional factor, RBP-J� (also called CBF1) (6–9). It isproposed that NICD-unbound RBP-J� mediates gene re-pression in conjunction with the histone-deacetylasecomplex SMRT/sin3/HDAC and that the binding ofNICD to RBP-J� displaces the corepressor complexesfrom RBP-J� and turn RBP-J� into a transcriptionalactivator by conveying histone acetyl transferases (10–11). The activated RBP-J� protein stimulates expres-sion of HES genes that belongs to a basic helix-loop-helix protein family, which serves as transcriptionalrepressors for ligand-restricted key genes involved incell fate decisions (10–12).

In higher vertebrates, multiple Notch homologs havebeen to date identified, including Notch1 throughNotch4 in rodents and human (13–18). Notch1 andNotch2 have the highest homology with each other,while Notch3 and Notch4 are structurally slightly di-verged from Notch1 and Notch2, both in the extracel-lular and intracellular domains. Truncated Notch pro-teins consisting only of its intracellular domain areknown to act as a constitutive transcriptional activa-tor, and are often called as activated Notch (aN) andwidely used to investigate the function of Notch recep-tors and the mechanism of the Notch-mediated signal-ing (19–23).

Multiple ligands for the Notch receptors are alsoidentified in mammals (Jagged1/Serrate1 Jagged2/Serrate2, Delta1, Delta3, and Delta4). At least, Delta1,Jagged1, and Jagged2 have been characterized as li-gands for Notch1, Notch2, and Notch3 receptors (8,24–28). The presence of multiple Notch ligands (NLs)and Notch receptors raises a question whether theyplay some distinct roles. Recently, it was reported thatDelta1 but not Jagged1 blocks early B-cell develop-ment from CD34�CD38� cord blood cells (29). An-other important view is that signal transducing activ-

ceptor to cause the release of the intracellular domain

0006-291X/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

ities of NLs are differentially modulated by the fringeprotein-induced modification of the Notch extracellulardomain (30, 31). Several proposals have also beenraised with regard to the functional diversity amongthe Notch receptors, such that transcription from theHES1 promoter is not activated by aN3, but by aN1(32, 33), and that myeloid differentiation of 32D cells inresponse to granulocyte colony-stimulating factor isblocked only by aN1 but not by aN2 (34). RegardingaN3, however, controversial data have been reportedthat it strongly activated HES1 in aN3-transgenicmice (35). Therefore, further investigation has beenawaited to address how individual Notch receptors ex-ecute different functions.

In this study, we describe that functions of aN1, aN2,and aN3 are clearly separated and that they depend onthe context such as the promoters targeted and theexpression level of RBP-J�.

MATERIALS AND METHODS

Plasmid construction. Mouse Notch1 (mNotch1) cDNA was akind gift from J. S. Nye. mNotch2 cDNA was isolated as described(8). To generate activated Notch1 (aN1) and aN2, cDNAs correspond-ing to their intracellular domain were amplified by PCR (Notch1[from serine (1,747th amino acid (a.a.)) to lysine (2,531th a.a.)],Notch2 [from alanine (1,700th a.a.) to alanine (2,470th a.a.)]. AcDNA for aN3 comprising the region from alanine (1,666th a.a.) toalanine (2,318th a.a.) was originally isolated using PCR from amouse embryo library (Clontech). The resulting aN cDNAs wereconstructed in an expression vector pTraserCMV (Clontech). Theplasmid for RBP-J� expression was a kind gift from T. Honjo.

Transient reporter assay. CHO (r) cells were inoculated at 4 � 104

in a 24-well plate and were transfected with activated Notch cDNAand either of the three kinds of reporter plasmids (0.2 �g), TP1-luc(pGa986-1, a gift from T. Honjo), HES1-luc and HES5-luc (gifts fromR. Kageyama), by a liposome-based method (SuperFect, Qiagen).Following transfection, the cells were cultured for 30–40 h. Lucif-

erase activity in the mixture of CHO (r) cells was then measuredusing a luminometer.

RESULTS

A Functional Diversity among aN1, aN2, and aN3

To investigate functional diversities among themammalian Notch receptors, we generated activatedforms of the three Notch receptors, Notch1, Notch2,and Notch3, comprising the entire intracellular do-main, which are capable of transducing downstreamsignals autonomously (19–23). We assessed their tran-scriptional activities for three kinds of promoters driv-ing a luciferase gene (TP1-luc, HES1-luc, and HES5-luc). TP1-luc is a reporter plasmid containing multiplerepeats of an RBP-J�-binding sequence from the TP1promoter of Epstein–Barr virus (36). HES1-luc andHES5-luc are reporter plasmids containing a promoterof the HES1 and HES5 genes, respectively (37, 38).When TP1-luc was used as a reporter, we found that allaNs strongly stimulated transcription of the reportergene, with aN1 showing the highest activity (Fig. 1). Incontrast, when HES1-luc and HES5-luc were used, theability of individual aN to transactivate each reporterwas significantly different. For HES1-luc, aN3 showedthe strongest transactivation, followed by aN1 and aN2in this order, while HES5-luc gene was most stronglytransactivated by aN1, followed by aN3 and aN2 (Fig.1). These results indicate that each Notch protein hasa specificity for transcription of the target genes.

We then investigated the effect of RBP-J� on eachaN protein-mediated transcription of the reportergenes. RBP-J� is a Notch signaling mediator, whichbinds to aN protein and is involved in expression of thedownstream genes, HES1 and HES5 (12). We found

FIG. 1. Comparison of the transcriptional activating activity among three kinds of activated Notch proteins, aN1, aN2, and aN3. EachcDNA (0.1 �g) was transiently co-transfected into CHO (r) cells with one of the three reporter genes, TP-1-luc, HES1-luc, and HES5-luc. After1.5 days, the luciferase activity in these cells was measured. Fold induction of the luciferase activity for each sample (mean of tripletmeasurements with standard deviation) was calculated against the control.

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that aN1- and aN2-induced transactivation was inhib-ited for all the three reporters when RBP-J� was over-expressed, while only aN3-mediated HES5-luc, but notTP1-luc or HES1-luc, was enhanced by RBP-J� over-expression (Fig. 2). In contrast, in the absence of aN, itwas shown that overexpression of RBP-J� resulted in alow level of transactivation from all the three reporters(Fig. 2). These data demonstrated that HES1 andHES5 gene expression was complexly regulated by thetype and the combination of aN receptors that conferthe downstream signals, and the expression level ofRBP-J�.

N2 Signal Inhibits aN1- or aN3-MediatedTranscriptional Activation of HES1and HES5 Reporter Genes

Next, we investigated the influence of the coexpres-sion of aN2, which showed the lowest activity amongthe three aNs for both HES1 and HES5 promoters, onthe aN1 and aN3 signals. Cotransfection of aN2 cDNAreduced the aN1- or aN3-mediated transcription ofHES1 and HES5 reporter genes (Fig. 3). The reductionwas dependent on the dose of aN2 (data not shown).These observations suggest that overexpression of N2negatively affects against N1 and N3 signals and thatN2 signal can be a negative regulator for HES1 andHES5 gene expression.

DISCUSSION

In this study, we identified a functional diversityamong the three Notch receptors. We demonstratedthat their relative signal-transducing activities for thetargeted promoters, i.e., HES1 and HES5, are variableand that the intensity of signaling from each Notchreceptor was affected by the expression level of RBP-

J�. Furthermore, we also showed that aN2 inhibitedaN1- and aN3-mediated gene expression.

Experiments with retrovirally expressed aN1 dem-onstrated that HES1 and HES5 are the target genesand essential effectors of Notch1 in regulation of mam-malian neuronal differentiation (39). Yet, it was notfully elucidated whether Notch1 has a preference forindividual HES gene. In addition, it was not fully un-derstood whether there is a difference in the transcrip-tional activity among the Notch receptors againstHES1 or HES5 gene. In addition, conclusions reportedabout the Notch3 were controversial (32, 35). To clarifythese issues, we used constitutively active forms of thethree Notch proteins, Notch1, Notch2, and Notch3, andinvestigated their transcriptional activity for HES1-luc

FIG. 2. An effect of exogenous RBP-J� protein on HES1-luc or HES5-luc gene activation mediated by individual activated Notch (aN).Transcriptional activating activity of each aN protein against TP-1-luc, HES1-luc, and HES5-luc reporter genes was examined with orwithout the transfection of 0.2 �g of RBP-J� cDNA.

FIG. 3. Activated N2 (aN2) inhibits aN1- or aN3-mediated tran-scriptional activation of HES1 and HES5 reporter genes. aN1 or aN3cDNA (0.1 �g) in the presence or the absence of 0.1 �g of aN2 cDNAwas transfected into the CHO (r) cells and the effect of aN2 on aN1or aN3-mediated transcription of HES1-luc and HES5-luc was inves-tigated.

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and HES5-luc reporter genes. The results suggestedthe existence of a functional diversity among the threeNotch receptors (Fig. 1). Our observation that aN3strongly activated transcription of the HES1-luc gene(Fig. 1) coordinated with the previous result obtainedfrom the analysis of aN3 transgenic mice (35), while itwas contradictory to the report that aN3 hardly acti-vated HES1 reporter transcript (32). Regarding thediscrepancy, we suppose that it may be due to a differ-ence in the RBP-J� expression level in the cells used inthe experiments, given the fact that the expressionlevel of the RBP-J� gene modulated the activity of aN3for the HES1-luc gene expression (Fig. 2). Alterna-tively, it may be explained by a difference in the ex-pression levels of other genes.

We also demonstrated that the transcriptional activ-ity of each aN for the individual HES gene is differen-tially modulated by the expression level of RBP-J�.Except for the aN3-induced HES5-luc activation, tran-scription of both HES1-luc and HES5-luc genes trig-gered by all the active Notch was inhibited by theintroduction of exogenous RBP-J� (Fig. 2), which isconsistent with the previous reports (40, 41). Activa-tion of the HES5-luc gene by aN3 was exceptionallyenhanced by the presence of exogenous RBP-J� (Fig.2). Moreover, it was also shown that expression ofexogenous RBP-J� in the absence of the aN proteinsdirectly stimulated HES1-luc and HES5-luc transcrip-tion (Fig. 2), being compatible with recent reports (42,43). These data indicate that the transcriptional levelsof HES1 and HES5 gene were complexly controlled bythe individual Notch receptor and the expression levelof RBP-J�. We speculate that the exceptional activityshown in aN3 can be related to the fact that Notch3 hasunique structural characteristics among Notch1,Notch2, and Notch3 proteins, which in turn may resultin conveying unique binding partners to RBP-J�.

We also showed that cotransfection of aN2 cDNA pre-vented aN1- and aN3-mediated transcription of the usedreporter genes (Fig. 3), suggesting that N2 functions as anegative regulator against Notch1 and Notch3 signals.Previously, an inhibitory action of aN3 was also reportedby another group (32, 33). However, we note that, whenassessing it in our systems, the inhibitory activity of aN3against other aNs was much lower than that of aN2 (datanot shown). It was recently reported that the EP domain,a three amino acid stretch present in the immediatelyC-terminal region of the ankyrin repeats, is a binding sitefor the transcriptional coactivator, p300 and is importantfor Notch1 signaling (44). Another group revealed thatthe region surrounding this three amino acid stretchfunctions as a transcriptional activating domain in aN1,while it does as an inhibitory domain in aN3 (33), despitethe fact that EP domain is conserved in Notch1, Notch2,and Notch3. In the case of aN2, it is likely that functionsof its EP domain and the flanking region varies on de-pendence of the promoter sequence targeted by aN2

(Figs. 1 and 3). The inhibition by aN2 may be explainedby competition for p300, which would be present in alimiting amount, and failure of an ability of p300-boundaN2 to activate HES1 and HES5 transcript, as discussedin the case of aN3 (33). As another possibility for themechanism of such an inhibitory action of aN2, we couldpropose that aN2 forms heterodimer complexes withother aN proteins, and as a consequence, the activity ofthe other aN protein is inhibited.

In summary, we demonstrate a functional diversityamong the Notch receptors. Furthermore, our data in-dicate that activation of HES1 and HES5 promoters iscomplexly controlled by the individual Notch receptorexpressed on the cells, a combination of the expressedNotch receptors, and the expression level of RBP-J�.

ACKNOWLEDGMENTS

We thank J. S. Nye for mNotch1 cDNA, T. Honjo for pGa981-6 andRBP-J� plasmids, R. Kageyama for HES1-luc and HES5-Luc plas-mids, and S. Shirahata for CHO ras clone-I cells. This work wassupported by grants-in-aid from the Ministry of Education, Science,Sport, Culture, and Technology of Japan and the Ministry of Health,Welfare, and Labor of Japan.

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