Cooperative assembly of higher-order Notchcomplexes functions as a switch toinduce transcriptionYunsun Nam*, Piotr Sliz†, Warren S. Pear‡, Jon C. Aster*, and Stephen C. Blacklow*§

*Department of Pathology, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA 02115; †Department of Biological Chemistryand Molecular Pharmacology, Harvard Medical School, and Howard Hughes Medical Institute, Boston, MA 02115; and ‡Department of Pathologyand Laboratory Medicine, Abramson Family Cancer Research Institute, Institute for Medicine and Engineering, University of Pennsylvania,Philadelphia, PA 19104

Communicated by Elliott D. Kieff, Harvard University, Boston, MA, December 13, 2006 (received for review November 1, 2006)

Notch receptors control differentiation and contribute to patho-logic states such as cancer by interacting directly with a transcrip-tion factor called CSL (for CBF-1/Suppressor of Hairless/Lag-1) toinduce expression of target genes. A number of Notch-regulatedtargets, including genes of the hairy/enhancer-of-split family inorganisms ranging from Drosophila to humans, are characterizedby paired CSL-binding sites in a characteristic head-to-head ar-rangement. Using a combination of structural and molecular ap-proaches, we establish here that cooperative formation of dimericNotch transcription complexes on promoters with paired sites isrequired to activate transcription. Our findings identify a mecha-nistic step that can account for the exquisite sensitivity of Notchtarget genes to variation in signal strength and developmentalcontext, enable new strategies for sensitive and reliable identifi-cation of Notch target genes, and lay the groundwork for thedevelopment of Notch pathway inhibitors that are active on targetgenes containing paired sites.

Mastermind � Notch signaling � signal transduction � dimerization �gene expression

Notch proteins constitute the receptors of essential signalingpathways that influence a broad range of cell fate decisions

in metazoan organisms (1, 2). Notch signals are used iterativelyat different decision points and have functional outcomes thatdepend heavily on gene dose and context. Both deficiencies andabnormal increases of Notch signaling are associated with hu-man developmental abnormalities and cancer, emphasizing theimportance of precisely regulating Notch signal strength (3–9).

The intracellular portion of the Notch receptor (ICN) is theprimary effector of canonical Notch signaling. ICN is proteo-lytically released from the membrane upon ligand stimulation,translocates to the nucleus, and induces transcription of targetgenes by driving the assembly a Notch transcriptional activationcomplex (NTC) that includes a DNA-bound transcription factorCBF-1/Suppressor of Hairless/Lag-1 (CSL) (10) and a coactiva-tor protein of the Mastermind-like (MAML) family (11, 12).Structures of NTC cores, now solved for both human Notch1 andCaenorhabditis elegans LIN12, reveal that the Notch ankyrin-repeat (ANK) domain and a Rel-homology region (RHR) withinCSL combine to create a groove that binds the N-terminal partof MAML-1 as a kinked, 70-Å helix (13, 14). The CSL/ICN/MAML ternary complex then recruits additional factors throughthe C-terminal portion of MAML, such as p300/CBP, PCAF/GCN5, and the CDK8/mediator complex (15–18), that drive thetranscription of target genes.

A key unresolved question in Notch signaling is to determinethe factors that govern target gene activation in different devel-opmental, physiologic, and pathophysiologic contexts. One clueis found in the promoter regions of a number of well character-ized Notch-responsive genes (19), such as the hairy/enhancer-of-split genes in Drosophila and a number of their mammalian

homologues, which contain dual ‘‘sequence-paired’’ binding sites(SPSs) (20, 21). SPSs consist of two CSL-binding sites orientedhead-to-head that typically are separated by 16 or 17 nt, de-pending on the species examined [supporting information (SI)Fig. 6]. Both the directionality and the spacing between thepaired sites influence Notch-dependent transcription in cell-based assays and transgenic flies, suggesting that the SPS is aspecialized response element that modulates or tunes the re-sponse of target genes to varying doses of activated Notch (22,23). Accordingly, the promoter of the HES-1 gene, whichcontains an SPS, responds to lower doses of ICN than does theHES-5 promoter, which lacks an SPS (23).

The evolutionary conservation of paired CSL recognition sitessuggests that cooperative assembly of higher-order Notch com-plexes might have a role in regulating transcription of certain keyNotch target genes. Yet, a molecular model for assembly ofhigher-order complexes has remained elusive. CSL does notdimerize on cognate DNA sites (22, 24, 25), and previous studieshave failed to detect any evidence of cooperativity in the loadingof intracellular Notch-derived polypeptides onto CSL/DNAcomplexes at SPSs (23).

We now find that assembled NTCs cooperatively dimerize onSPSs. Dimerization requires both CSL and MAML, depends onconserved residues from the convex face of the Notch ANKdomain and is a general feature of the four mammalian Notchreceptors. Dimerization-defective forms of ICN1 have a dimin-ished capacity to activate promoter elements containing SPSsites and interfere with transcriptional induction by normalICN1. These findings suggest that site cooperativity serves as aregulatory switch that controls the transcription of certain Notchtarget genes containing SPS elements and provide a model forcontext- and dose-dependent variation in the sensitivity ofdifferent Notch-responsive genes to Notch signals.

ResultsCrystal Contacts Suggest a Mode of Dimerization at SPS Elements.Cocrystals of a human NTC core (13), which consists of anN-terminal MAML-1 peptide, the ANK domain of humanNotch1, and CSL on a DNA duplex derived from the HES-1

Author contributions: Y.N., P.S., J.C.A., and S.C.B. designed research; Y.N., P.S., and J.C.A.performed research; Y.N., P.S., W.S.P., J.C.A., and S.C.B. analyzed data; and Y.N. and S.C.B.wrote the paper.

The authors declare no conflict of interest.

Abbreviations: ICN, intracellular portion of the Notch receptor; NTC, Notch transcriptionalactivation complex; CSL, CBF-1/Suppressor of Hairless/Lag-1; MAML, Mastermind-like; ANKdomain, ankyrin-repeat domain; RHR, Rel-homology region; SPS, ‘‘sequence-paired’’ bind-ing site.

§To whom correspondence should be addressed. E-mail: sblacklow@rics.bwh.harvard.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0611092104/DC1.

© 2007 by The National Academy of Sciences of the USA

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promoter, contain contacts between the convex surfaces of ANKdomains from adjacent unit cells that also are seen in crystals ofthe ANK domain solved in isolation in several different crystal-lization conditions (13, 26). These contacts lie near a twofoldsymmetry axis in the crystals, such that the interacting complexesare positioned head-to-head at a distance roughly equal to thatneeded to occupy both recognition elements of an SPS (Fig. 1A).Primary sequence alignment of Notch ANK domains fromdifferent homologs shows that the key contacts are evolutionarilyconserved (Fig. 1B). These conserved residues are not engagedin contacts within an individual MAML1/ANK/CSL/DNA com-plex, suggesting that the observed conservation reflects func-tional importance in mediating dimerization at SPS sites. Theconservation among the four mammalian Notch receptors also

predicts that each receptor should be capable of making inter-actions like those between the adjacent Notch1 complexesillustrated here (Fig. 1).

The ANK–ANK contacts primarily are electrostatic and lie in thesecond and third ankyrin repeats. Key interactions consist ofcontacts between the guanidino group of Arg-1985 and at leastthree backbone carbonyl oxygen atoms, as well as interactionsbetween Glu-1950 and Lys-1946 (Fig. 1C). Arg-1983 also formshydrogen bonds with Ser-1952 and a backbone carbonyl. In additionto homotypic interactions between the ANK domains, unmodeledelectron density in the MAML-1/ANK/CSL/DNA complex (13)also suggests the existence of interactions between the ANKdomain of one complex and the N-terminal end of MAML-1 in thesecond complex (SI Fig. 7). Based on the architecture of thecomplex, and the evolutionary conservation of SPSs and the crystalcontact residues, we postulated that the ANK domains of Notchreceptors mediate dimerization of ternary complexes on SPSsfound in Notch target gene promoters.

ANK–ANK Interactions Drive Dimerization of NTCs. To test whetherresidues engaged in ANK–ANK contacts in the crystal contrib-ute to transcriptional activation of SPS-bearing promoters, wecompared the ability of different forms of ICN to inducetranscription of a luciferase gene under control of the HES-1promoter, which has a functionally important SPS element (22,23). In contrast to normal ICN1, mutations that disrupt thepredicted dimerization interface either abrogate (R1985A) ordiminish (K1946E and E1950K) the ability of ICN1 to induceexpression of the HES-1 reporter gene (Fig. 2A). Combining theK1946E and E1950K mutations in cis, however, rescues thedefect in transcriptional activation, indicating that the putativedimerization interface is functionally important in regulatingtranscriptional activity at a promoter that contains an SPS. Inaddition, when coexpressed with ICN1, the R1985A mutationdominantly interferes with activation of the HES-1 promoterelement by normal ICN1 (Fig. 2B). Importantly, when thesemutants are scored on an artificial reporter that contains fourCSL-binding sites oriented in the same direction and in tandem,there is no change in the ability of the mutants to activatetranscription (SI Fig. 8). Moreover, in cotransfected cells, allICN1 polypeptides with mutations that disrupt the predicteddimerization interface are expressed at similar levels to normalICN1, and they coimmunoprecipitate in similar amounts withCSL and MAML-1 (Fig. 2C). Together, these findings indicatethat the ability to form monomeric ternary complexes withMAML-1 and CSL is not affected by these mutations.

To establish directly whether NTCs (consisting of one mole-cule each of MAML-1, ICN, and CSL) can cooperativelydimerize on DNA, we carried out electrophoretic mobility shiftassays (EMSAs) on an oligonucleotide probe containing theHES-1 promoter SPS (Fig. 3A). Without Notch or MAML-1,CSL binds to each of the two sites independently. When presentin excess, most probes bind a single CSL molecule (Fig. 3A), afinding consistent with previous studies showing that CSL bindsits recognition element as a monomer without detectable coop-erativity at paired sites (23–25). Adding RAMANK from Notch1does not change the stoichiometric distribution of complexesbound per probe molecule. However, when MAML-1 is added,the stoichiometric distribution of the complexes changes dra-matically: all of the probe is either free or bound by NTC dimers,indicating that NTC loading at one site leads to cooperativeloading of the second site. As predicted, cooperative loading isabrogated by the R1985A mutation, which instead produces asmear corresponding to an ensemble of species that likely resultsfrom a weak residual tendency to self-associate. In contrast, theR1985A mutation does not detectably affect ternary complexformation on a probe containing only a single CSL-binding site(Fig. 3A Right), indicating that the R1985A mutation is a

Fig. 1. ANK–ANK interactions between conserved residues in the crystallattice. (A) Structure of two copies of the MAML-1/ANK/CSL/DNA complexrelated by crystallographic symmetry. The protein subunits are rendered asribbons (ANK, purple; CSL, gold; and MAML-1, green), and the DNA is ren-dered as blue sticks. Residues of ANK engaged in lattice contacts are shown ascyan sticks. (B) Sequence alignment of ANK in the region of the dimerinterface. Key residues that participate in Notch1 lattice contacts are high-lighted in yellow. (C) Specific contacts observed between two symmetry-related ANK molecules (one in gray and the other in cyan) in the crystalstructure. (Left) Salt bridges in ankyrin repeat two between K1946 of one ANKsubunit and E1950 of the other. (Right) Interactions in ankyrin repeat threebetween R1985 of one ANK subunit and three backbone carbonyl groups ofthe other.

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cooperativity mutant that specifically interferes with dimeriza-tion. The partial loss of activity of the K1946E and E1950Kmutants in the HES-1 reporter assays is echoed in EMSA

titrations, where the proteins undergo a cooperative transition toform dimers at a concentration �4-fold greater than normalICN1 or the K1946E/E1950K double mutant (SI Fig. 9).

Proper Spacing and Orientation of CSL-Binding Sites Are Necessary forCooperative Dimerization. To test whether higher-order complexesexhibit specificity for the SPS architecture, we carried outadditional EMSA assays on variant DNA sequences that elim-inate the integrity of one of the SPS sites, f lip the site orientation,

Fig. 2. HES-1 reporter assays depend on an intact dimerization interface. (A)Effect of dimerization mutations on the ability of ICN1 to stimulate a HES-1reporter gene. (B) Dominant negative activity of R1985A. In A, U2OS cells werecotransfected with the indicated pcDNA3 plasmids (1 ng per well), whereas inB, the cells were transfected with 10 ng of normal ICN1 plasmid and/or 100 ngof ICN1 R1985A plasmid. In both A and B, fold stimulation is expressed relativeto the activity of the empty vector control, which is arbitrarily given a value of1. All data points were obtained in triplicate; error bars correspond to stan-dard deviations. (C) ICN1 polypeptides bearing dimerization mutations formstable complexes with CSL and MAML-1. 293 cells were cotransfected withplasmids encoding the indicated forms of ICN1, MAML-1 fused to GFP, andMyc-tagged CSL. Complexes were recovered from whole-cell lysates with ananti-Myc antibody and blotted for the presence of GFP-MAML-1, ICN1, andCSL-Myc as indicated.

Fig. 3. Cooperative dimerization of NTCs on HES-1 promoter DNA. (A)EMSAs performed with either normal or R1985A forms of human RAMANK1.(Left) Probe containing the SPS element of the human HES-1 promoter. (Right)Single-site probe. (B) Sequences used in EMSAs to determine the importanceof DNA site integrity, spacing, and orientation in promoting cooperativedimerization of ternary complexes. The S and SPS probes from the humanHES-1 promoter sequence are identical to those used in A. The consensus siteis underlined, and alterations are noted with lowercase in red. (C) EMSAsperformed by using RAMMANK-1, CSL, and MAML-1 on the DNA probes listedin B. Mutation of one site (mutA or mutB), inversion of the second site (invB),or an insertion of 5 bp between the two sites (ins) prevents cooperativedimerization of ternary complexes. The band that contains the dimer ofternary complexes on DNA is indicated to the left of the gel. (D) Ability of NTCsto undergo cooperative dimerization as a function of intersite spacer length.Oligonucleotide probes used to scan spacer length are listed in SI Table 1.

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or alter the spacing between the sites by a half-turn of helix (Fig.3B). When either site A or site B is mutated so that it no longercorresponds to a CSL consensus site (YGTGDGAA) (27),cooperative assembly of the dimer is no longer observed (Fig.3C, mutA and mutB). Moreover, cooperative dimerization is nolonger detected when the second site is inverted (Fig. 3C, invB),and it is dramatically diminished when the second site is movedby a 5-base insertion (ins). Because the intrinsic affinity of asingle ternary complex for DNA is not altered under theconditions of inversion or insertion, these studies show that theproper spatial arrangement of the two individual binding sites isneeded for cooperative dimerization to occur.

We next asked what range of spacer lengths between sites iscompatible with cooperative loading of dimeric complexes (Fig.3D). The optimal spacing between consensus sites for cooper-ative dimerization is 16 bp, but cooperative dimerization still canoccur on templates with spacers varying from 15 to 19 bp,implying that two NTCs can adjust their positions relative to eachother (see below) to accommodate a modest range of spacerlengths between sites. This inferred flexibility is consistent withthe different conformations of CSL seen in the crystal structuresof the Notch ternary complexes formed with the human andworm proteins (13, 14) and with the enrichment of adenosineand thymidine in the spacer between the paired sites.

To determine whether the assembly of NTCs and theircooperative dimers is general among the human Notch homo-logues, we tested the ability of the RAMANK domains ofNotch1–4 to form complexes on single and sequence-paired sites(Fig. 4). Despite qualitative differences in mobility on theEMSA, all four purified RAMANK polypeptides bind to CSLindependent of MAML-1 and then recruit MAML-1 to ternarycomplexes on a single site probe. When the longer, paired siteprobe is provided, all RAMANK polypeptides mediate coop-erative dimerization, as predicted from the conservation inprimary sequence at the dimerization interface (Fig. 1B). Thus,a similar series of events takes place to assemble single anddimeric NTCs in all four mammalian Notch homologues.

DiscussionGene transcription in eukaryotes relies on the combinatorialintegration of inputs from different biological signals. Cooper-ative interactions among transcription factors and their associ-ated proteins on promoter DNA constitutes one mechanism bywhich signal integration is achieved (28). The studies reportedhere elucidate a new mechanistic step in the regulation andassembly of Notch transcription complexes: the cooperativeformation of NTC dimers when two CSL-binding sites arearranged in an SPS architecture. Protein–protein interactionsthat stabilize the dimer include a conserved region on the convexsurface of the ANK domain, which is on the opposite face from

the surface engaged in interactions with CSL and MAML-1.Additional interactions among ICN, CSL, and MAML-1 mayexist in the context of the full-length proteins. Cooperativeformation of NTC dimers is seen with all four human Notchproteins, and the evolutionary conservation of both the dimer-ization interface and the SPS elements suggests that cooperat-ivity between two NTCs on DNA represents a mechanism formodulation of NTC activity that is conserved among a numberof metazoan organisms (Fig. 1B).

Previous studies examining whether isolated ANK domainsfrom various Notch receptors form dimers yielded conflictingresults, which now are reconciled by the findings reported here.Two-hybrid studies suggested that the ANK domains of Dro-sophila Notch and C. elegans GLP-1 are capable of self-association (29, 30), yet when isolated and purified, DrosophilaANK and the ANK domain from human Notch1 can remainmonomeric at concentrations as high as 200 �M (31) and 6 �M(25) respectively, indicating that any weak intrinsic tendency ofthe ANK domain to self-associate in isolation readily is sup-pressed by commonly used solution conditions (probably be-cause the contacts are predominantly electrostatic and thesurface area buried in the ANK–ANK interface is only �500 Å2).When CSL, MAML-1, and ICN all are present in a singlecomplex, contacts with MAML and CSL restrict the conforma-tional space accessible to ANK, positioning it to participate inhomotypic interactions. Thus, when two NTCs are orientedproperly and adjacent to each other on DNA, the effectiveconcentration of one ANK domain for the second increasesdramatically, revealing a capacity for self-association that is notapparent from bulk solution experiments. Indeed, the ANKresidues implicated in cooperative dimerization of humanNotch1 NTCs form crystal contacts both in the NTCs themselvesand in several structures of isolated Notch ANK domains solvedindependently, consistent with the idea that the context of thecrystal lattice may more effectively mimic the constrained en-vironment on DNA than bulk solution. Unmodeled electrondensity in the crystallized NTC core complex (SI Fig. 7) and theproximity of modeled atoms suggest that additional interactionsbetween MAML-1 of one NTC and ANK from the other NTCalso contribute to stabilization of dimers and are consistent withthe biochemical observation that cooperative dimerization is notseen until after addition of the MAML-1 polypeptide. Theconservation of the ANK–ANK interface among the four humanNotches also suggests that NTCs may form heterodimers thatdepend on the promoter context and the cellular concentrationsof the various Notch homologues.

Dependence of NTC Dimerization on CSL and DNA Flexibility. Al-though the optimum distance between CSL sites is 16 bp, allspacer lengths from 15 to 19 bp can support cooperativedimerization of NTCs (Fig. 3D). The existence of length varia-tion in the intersite distance between individual CSL-bindingsites in human and Drosophila SPS elements (SI Fig. 6), as wellas the range of DNA sequences that can accommodate NTCdimers (Fig. 3D), implies a requirement for intrinsic f lexibility inone or more of the components of the complex.

Some of the flexibility to accommodate different spacerlengths probably comes from CSL itself. CSL, which is relatedstructurally to the Rel-homology family of transcription factors,has three domains, and the linkers connecting the domains maybe the source of flexibility. In canonical Rel-family members likenuclear factor of activated T cells 1 (NFAT1), f lexibility in thelinkers connecting the RHR-N and RHR-C domains enablesadaptation to different environments to bind different DNAsequences and cooperate with other factors on DNA (32–34).The ability of the C. elegans homolog LAG-1 to undergo adomain shift upon complexation with ICN and MAML (14)substantiates the notion that the worm CSL protein also can

Fig. 4. Comparison of complexes formed by the RAMANK regions of humanNotches 1–4. Equal concentrations of RAMANK and CSL were mixed with orwithout the MAML-1 polypeptide, and EMSAs were performed on a shortprobe with a single CSL-binding site (Left and Center) or the SPS element fromthe HES-1 promoter (Right).

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adapt to different environments (for example, different spacerlengths between paired sites) by domain movement withoutlosing the ability to bind consensus site DNA, although thedivergence of the C. elegans notches (Fig. 1B) also raises thepossibility that they may not cooperatively dimerize like thehuman proteins. Further support for this proposal emergesdirectly from comparison of the human (13) and C. elegans (14)NTC structures: when the �-trefoil domains (BTDs) of the twostructures are superimposed, the rest of the protein domains(RHR-N, RHR-C, ANK, and MAML) rotate as a single rigidbody around a hinge region between the BTD and RHR-N (35).

Flexibility also is likely to derive from the DNA thatintervenes between the two sites of an SPS. If the DNA wererigid B-form DNA, a change in spacer length of 5 bp wouldresult in a rotation of �180° around the DNA helix, a changethat would be difficult to accommodate even with intrinsicprotein f lexibility. Thus, the length and the intrinsic f lexibilityof a particular spacer DNA, in addition to the individualaffinities of the two CSL-binding sites for CSL, also ought tocontribute to the likelihood that a given promoter DNAsequence will recruit an NTC dimer. Dimerization-inducedbending or distortion of DNA also could inf luence occupancyof neighboring transcription factor binding sites without re-quiring direct protein–protein interactions (36).

Implications for Notch-Dependent Gene Expression. Notch-regulated genes known to contain SPS elements in theirpromoters include basic helix–loop–helix genes from the en-hancer-of-split complex in Drosophila, as well as homologoushairy/enhancer-of-split genes in Xenopus, mouse, and humans(20–23, 37, 38). The cooperative homodimerization of NTCsat such paired sites provides a molecular mechanism toaccount for the varying sensitivity of different genes to Notchdose and context (Fig. 5). The sensitivity of a particularpromoter to a given dose of Notch signal may be inf luenced bythe affinity of individual binding sites for CSL, the length andintrinsic f lexibility of the spacer DNA at SPS elements, and theavailability of neighboring cis-regulatory elements that cancooperate with NTCs. NTC dimers can assemble on promoters

with SPS elements according to a cooperative dose–responsecurve that resembles the genetic switch exhibited by proteinslike lambda repressor (39). Cooperativity in the response toNotch at these promoters thus will ensure tight regulation oftranscription, with activated Notch rapidly f lipping the switchfrom off to on. On the other hand, promoters with only oneCSL-binding site or multiple sites without the proper SPSarchitecture may exhibit a less sensitive and more gradedresponse to the dose of active Notch and instead depend moreheavily on cooperativity with other transcription factors (23).Of note, studies investigating the functional implications ofdimerization in a well established mouse model for leukemo-genesis show that normal ICN1 induces T cell acute lympho-blastic leukemia (T-ALL) with full penetrance by 12 weeks,whereas ICN1 bearing the R1985A mutation fails to induceleukemia (W.S.P., unpublished observations). Whether dimersexert their activating effect on transcription by increasingpromoter occupancy, by generating novel recognition sites forother transcription factors, or by recruiting basal factors moreeffectively is still an open question, the answer to which mayvary depending on the identity of the promoter. The experi-mental criteria for formation of dimeric NTCs reported hereshould facilitate identification of the most sensitive directtargets of activated Notch in both normal development anddisease pathogenesis. Knowledge of the specific interactionsthat dictate the assembly and regulation of dimeric NTCs alsoshould enable the identification of small molecules that abro-gate Notch activation of target genes containing specific typesof promoter response elements.

Experimental ProceduresMolecular Graphics. Fig. 1 A and C were prepared with theprogram PyMOL (DeLano Scientific, Palo Alto, CA). Thesymmetry related copy of the MAML-1/ANK/CSL/DNA com-plex in Fig. 1 A was rendered by applying the symexp operationto the coordinates of the complex (PDB ID code 2F8X; ref. 13)and then choosing the copy that approaches to within 5 Å ofresidue 1985 of the ANK domain of the initial complex.

EMSAs. EMSAs were performed as described in ref. 25. Forexpression of RAMANK, residues 1761–2127 of human Notch1,1701–2103 of human Notch2, 1666–2038 of human Notch3, and1473–1828 of human Notch4 were built into a modified pGEX-4T1 vector described in ref. 25. The mutations in the ANKdomain of Notch1 were introduced by QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). CSL,RAMANK, ANK, MAML, and RAM polypeptides were puri-fied as described (13, 25). The oligonucleotides used as probesare shown in SI Table 1. The final protein concentration for eachlane (total volume 15 �l) was 140 nM for CSL, 170 nM fordifferent versions of RAMANK, 240 nM for ANK, 440 nM forMAML-1, and 450 nM for RAM.

Reporter Gene Assays. Empty pcDNA3 or pcDNA3 expressingICN1 with indicated mutations were transiently cotransfectedin triplicate into human U2OS cells with (i) firef ly luciferasereporter genes driven by either an element derived from theHES-1 promoter (40) or an artificial element containing fourtandem repeats of the CSL-binding site (41) and (ii) aninternal Renilla luciferase reporter gene driven by an elementderived from the human thymidine kinase promoter. Lucif-erase assays were performed and analyzed �48 h posttrans-fection by using the Promega (Madison, WI) Dual LuciferaseKit. Firef ly luciferase activities were normalized by usinginternal Renilla luciferase control values and expressed relativeto the empty vector control lysate, which was arbitrarily givena mean value of 1.

Fig. 5. Model for assembly of higher-order Notch transcription complexes.RAM delivers ANK to CSL, while also competing against or displacing core-pressors. ANK binds to CSL with weak affinity, but recruitment of MAML-1locks it in place to form a stable ternary complex (NTC). Interactions betweentwo properly oriented complexes then promote cooperative formation of NTCdimers. The formation of both homodimers and heterodimers of differentNotch receptors is postulated, as is the potential for recruitment of additionalDNA-binding factor(s) that can cooperate with NTCs to activate transcription.

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Immunoprecipitation Assays. 293 cells in six-well dishes werecotransfected with pcDNA3-ICN1 (0.1 �g), pEGFP-MAML1(0.5 �g), and pcDNA3-CSL-Myc (1 �g) plasmids. Two daysposttransfection, cell lysates and immunoprecipitates wereprepared with anti-Myc (clone 9E10) antibody as described(42). Western blots were stained with antibodies against the

Myc epitope, GFP (clone JL8; Clontech, Mountain View, CA),and the intracellular domain of Notch1 (42).

We thank Gavin Histen for technical assistance. This work is supportedby National Institutes of Health grants to S.C.B. (R01 CA92433), W.S.P.,and J.C.A.

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