Molecular Cell, Vol. 5, 811–820, May, 2000, Copyright 2000 by Cell Press

Oligomerization of RAR and AML1Transcription Factors as a Novel Mechanismof Oncogenic Activation

of chimeric genes (Look, 1997; Tenen et al., 1997). Ex-pression of the corresponding fusion proteins blocksterminal differentiation of isolated hemopoietic precur-sors and causes leukemias in animal models (Grignani etal., 1996; Brown et al., 1997; Grisolano et al., 1997; Ruthardt

Saverio Minucci,1,7,8 Marco Maccarana,1,8

Mario Cioce,1 Pasquale De Luca,1,2 Vania Gelmetti,1,3

Simona Segalla,5 Luciano Di Croce,1 Sabrina Giavara,1

Cristian Matteucci,1 Alberto Gobbi,1

Andrea Bianchini,4 Emanuela Colombo,1

Ilaria Schiavoni,1,5 Gianfranco Badaracco,5 et al., 1997; Gelmetti et al., 1998; Du et al., 1999).Xiao Hu,6 Mitchell A. Lazar,6 Nicoletta Landsberger,5 One of the genes involved in the translocations en-Clara Nervi,4 and Pier Giuseppe Pelicci1,7 codes invariably for a transcription factor, such as reti-1European Institute of Oncology noic acid receptor a—RARa—in acute promyelocyticDepartment of Experimental Oncology leukemia (APL) or AML1 in acute myelogenous leukemia20141 Milan (Shivdasani and Orkin, 1996; Look, 1997; Tenen et al.,2Department of Genetics 1997). The differentiation block is assumed to be theUniversity “Federico II” consequence of the altered properties of the chimeric80134 Naples transcription factors (Shivdasani and Orkin, 1996; Look,3Istituto di Medicina Interna e Scienze Oncologiche 1997; Tenen et al., 1997).Policlinico Monteluce Unliganded RARs (as heterodimers with RXR) repressPerugia University transcription by recruiting the nuclear corepressor06100 Perugia (N-CoR)/histone deacetylase (HDAC) complex to the re-4Dipartimento di Istologia ed Embriologia Medica sponse element of RA-target genes. RA triggers dissoci-University “La Sapienza”

ation of the N-CoR/HDAC complex, recruitment of coacti-00161 Rome

vators (PCAF, p300/CBP, SRC-1), and transcriptional5Department of Structural and Functional Biologyactivation (Chambon, 1996; Minucci and Pelicci, 1999;University of InsubriaXu et al., 1999). AML1 is associated with p300/CBP21100 Varesehistone acetylases, while ETO interacts with N-CoR andItalyrecruits HDACs (Minucci and Pelicci, 1999). PML-RAR6Division of Endocrinology, Diabetes, and Metabolismand AML1-ETO associate with N-CoR/HDAC, and muta-Department of Medicinetion of the N-CoR binding site(s) impairs their biologicalUniversity of Pennsylvania School of Medicineactivity, thus indicating that aberrant recruitment of his-Philadelphia, Pennsylvania 19104tone-modifying enzymes is essential for leukemogene-sis (Minucci and Pelicci, 1999).

Here, we investigate the mechanisms leading to ab-Summarynormal recruitment of the N-CoR/HDAC complex byPML-RAR and AML1-ETO. PML-RAR (unlike RAR) formsRAR and AML1 transcription factors are found in leu-oligomers in vivo, and the coiled-coil region of PML iskemias as fusion proteins with PML and ETO, respec-the structural determinant for oligomerization. A similartively. Association of PML-RAR and AML1-ETO withpotential to form oligomeric structures is found in otherthe nuclear corepressor (N-CoR)/histone deacetylaseAPL fusion proteins (PLZF-RAR and NPM-RAR). AML1-(HDAC) complex is required to block hematopoieticETO is also found in high molecular weight (HMW) com-differentiation. We show that PML-RAR and AML1-

ETO exist in vivo within high molecular weight (HMW) plexes, due to the ETO moiety of the fusion protein.nuclear complexes, reflecting their oligomeric state. Oligomerization is responsible, per se, for the increasedOligomerization requires PML or ETO coiled-coil re- recruitment of N-CoR, transcriptional repression, andgions and is responsible for abnormal recruitment of leukemogenic activation of the fusion proteins. TheseN-CoR, transcriptional repression, and impaired dif- results highlight oligomerization as a novel mechanismferentiation of primary hematopoietic precursors. Fu- for the oncogenic conversion of a transcription factorsion of RAR to a heterologous oligomerization domain in leukemias.recapitulated the properties of PML-RAR, indicating thatoligomerization per se is sufficient to achieve trans- Resultsforming potential. These results show that oligomeriza-tion of a transcription factor, imposing an altered interac- The Abnormal Recruitment of N-CoR by PML-RARtion with transcriptional coregulators, represents a novel Is Caused by the Coiled-Coil Region of PMLmechanism of oncogenic activation.

At low concentrations of RA (1–100 nM), the stability ofthe PML-RAR/N-CoR complex is higher than that of theIntroductionRAR/N-CoR complex (Grignani et al., 1998b; Lin et al.,1998). Pull-down assays were performed by incubationAcute myeloid leukemias (AMLs) are characterized byof in vitro translated, 35S-labeled PML-RAR or RAR withchromosomal translocations resulting in the generationGST-N-CoR coupled to agarose beads. PML-RARbound specifically even the lowest amounts of GST-N-7 To whom correspondence should be addressed (e-mail: pgpelicci@CoR, whereas 30-fold higher amounts of GST-N-CoRieo.it [P. G. P.], sminucci@ieo.it [S. M.]).

8 These authors contributed equally to this work. were required to obtain significant levels of RAR binding

Molecular Cell812

Figure 2. Role of the CC in Transcriptional Regulation by PML-RAR

(A) Transcriptional repression. HeLa cells were cotransfected withthe RARE-G5-TATA reporter in the absence (C) or presence of in-

Figure 1. Enhanced Recruitment of N-CoR by PML-RAR Is Due to creasing amounts (50, 100, 250, and 1000 ng) of the indicated ex-the CC Region of PML pression vectors (P-R, PML-RAR; DCC-PR: DCC-PML-RAR; CC-R:

CC-RAR; p53-R: p53-RAR). Transfection of the expression vectors(A-B) Decreasing amounts of GST-N-CoR (from 10 mg to 150 ng) oryielded comparable levels of protein expression (data not shown).GST as a control (10 mg; lane “-”) were incubated with the indicated(B) RA sensitivity. Cells were cotransfected with 500 ng of the indi-in vitro translated, 35S-labeled proteins. R, RING finger; and B, Bcated expression vectors. RA was added 24 hr after transfectionboxes. The input lanes (“I”) are loaded with the same (A) or 50% (B)(1, 10, 100 nM, 1, 10 mM).of the amount used in the assays.

The CC Region of PML Is Responsible for(Figure 1A). Deletion of the coiled-coil (CC) region of the Formation of PML-RAR OligomersPML (DCC-PML-RAR) dramatically decreased binding, The CC domain of PML is required for the biologicalwhile deleting other regions of PML (RING, B boxes) properties of PML-RAR (Grignani et al., 1996) and ishad no effect (Figure 1A; data not shown). Conversely, responsible for the appearance of PML-RAR within highfusion of the CC region of PML to RAR (CC-RAR) re- molecular weight complexes, as shown by size-exclu-sulted in enhanced binding to GST-N-CoR (Figure 1A). sion chromatography (SEC) analysis (Nervi et al., 1992;These results show that PML-RAR binds N-CoR with Grignani et al., 1999). Fusion of the CC to RAR mayhigher apparent affinity than RAR and that the determi- change the composition of RAR-associated factors,nant for this altered association is the CC region of PML. leading to oncogenic activity. To investigate this possi-

bility, we analyzed the molecular composition of thePML-RAR HMW complexes.The CC Region of PML Determines the Altered

Transcriptional Properties of PML-RAR SEC elution profile of unliganded PML-RAR from allcells examined (including fresh APL blasts and the pro-The enhanced binding of PML-RAR to N-CoR might

lead to enhanced transcriptional repression. To test this myelocytic NB4 cell line) revealed a peak with an appar-ent molecular weight of 700 kDa, identically to ligandedhypothesis, we designed the RARE-G5-TATA reporter,

which has five GAL4 response elements fused to a mini- PML-RAR (Figure 3A; data not shown; Nervi et al., 1992).In contrast, RAR eluted as a monomeric species (Figuremal promoter, and a RA responsive element (RARE) to

allow binding of RAR (or fusion proteins). GAL4-VP16 3A). Deletion of the CC region shifted the elution volumeto lower molecular weight (mono- or dimeric) species,strongly activated this reporter in HeLa cells (10- to 13-

fold). RAR overexpression decreased GAL4-VP16 acti- while CC-RAR was found in HMW complexes, confirm-ing that the CC domain of PML is necessary and suffi-vation (30%–40% repression, Figure 2A), and PML-RAR

behaved as a more potent transcriptional repressor cient for their formation (Figure 3A).In vitro translated or bacterially expressed PML-RAR(80%–90% repression, Figure 2A). DCC-PML-RAR (as

RAR) repressed GAL4-VP16 weakly, whereas CC-RAR were also found in HMW complexes (Figure 3A; datanot shown), suggesting that the nuclear complexes con-was comparable to PML-RAR (Figure 2A).

Higher concentrations of RA are required to dissociate sist of oligomeric PML-RAR. To test this hypothesis, wepurified PML-RAR from nuclear extracts. Our purifica-PML-RAR (as opposed to RAR) from N-CoR (Grignani

et al., 1998b). Near-physiological RA concentrations (up tion scheme included heparin-Sepharose, SEC, andDNA-affinity chromatography. A single, specific 120 kDato 100 nM) reverted RAR and DCC-PML-RAR repression,

whereas much higher concentrations (1–10 mM) were polypeptide (corresponding to PML-RAR by Westernblot) was observed after silver staining of the purifiedrequired to revert repression by PML-RAR and CC-RAR

(Figure 2B). material (Figure 3B). SEC analysis revealed that theeluted PML-RAR was still present in HMW complexesThese results show that PML-RAR is a stronger tran-

scriptional repressor than RAR and that its activity and (Figure 3B), indicating that it can be isolated from nu-clear extracts as oligomers.altered RA sensitivity require the CC region of PML.

Oligomerization of Transcription Factors in Leukemias813

Figure 3. PML-RAR Forms Oligomers In Vivo

(A) Role of the CC. Nuclear extracts from U937 clones expressing the indicated proteins, or recombinant PML-RAR from BL21 cells, werefractionated by SEC and analyzed by Western blotting using an anti-RAR antibody. Fraction number is indicated at the top of each lane.Elution fractions of molecular weight markers are indicated by arrows.(B) Biochemical purification of PML-RAR. Silver stain and Western blot analysis (upper panels) of highly purified PML-RAR after the final DNA-affinity chromatography: asterisks mark nonspecific bands. Western blot analysis of purified PML-RAR after SEC (lower panel).(C) In vivo cross-linking. Nuclear extracts from in vivo cross-linked, metabolically labeled U937-PML-RAR cells were analyzed by Westernblot after SDS PAGE in reducing (R) or nonreducing (NR) conditions: the arrows indicate the cross-linked species. Extracts were subjectedto SEC, and then HMW PML-RAR fractions were immunoprecipitated with anti-PML or control (C) antibodies and analyzed (before or after afurther SEC step) by SDS PAGE in reducing conditions, followed by autoradiography.(D) Characterization of the isolated CC domain of PML. (Upper panel) SEC of purified, bacterially expressed (BL21) CC. Fractions were analyzedby SDS-PAGE, followed by Western blotting using an anti-CC antibody. Silver stain (lower left panel) and Western blot analysis (lower rightpanel) of purified CC cross-linked in vitro with increasing concentrations of BS3. The positions of the mono-, di-, and trimeric CC are indicatedby arrowheads.

To investigate whether the PML-RAR oligomeric com- polypeptide (Figure 3C), recognized by anti-PML andanti-RAR antibodies, comigrating with PML-RAR inplexes exist in vivo, we performed in vivo cross-linking

experiments. Metabolically labeled cells were treated SDS-PAGE, and absent in control immunoprecipitates(data not shown). We conclude that the 35S-labeled poly-with the reversible cross-linking agent DTBP and ana-

lyzed by SDS-PAGE under nonreducing conditions (to peptide represents PML-RAR and that no other cellularproteins are stoichiometrically cross-linked under thesepreserve cross-linking) and Western blotting. In addition

to the 120 kDa PML-RAR polypeptide, anti-RAR anti- conditions. SEC analysis of the immunoprecipitateseluted by SDS 1% showed that PML-RAR was still pres-bodies recognized a more abundant polypeptide of ap-

proximately 350 kDa and less well-resolved polypep- ent in HMW complexes (Figure 3C). Together, these re-sults indicate that PML-RAR oligomers exist in vivo priortides of higher MW, which were absent in gels run in

reducing conditions (to revert cross-linking) or from non- to cell lysis and represent the natural form of PML-RARwithin the cell nucleus.cross-linked material (Figure 3C; data not shown). The

cross-linked material was still present in HMW com- Estimation of the molecular mass of the PML-RARoligomer by SEC has intrinsic limitations, since the CCplexes (data not shown). The anti-PML immunoprecipi-

tates from the fractions corresponding to HMW com- region of PML may influence the shape of PML-RAR. Bycentrifugation through a sucrose gradient, unligandedplexes contained exclusively one 120 kDa, 35S-labeled

Molecular Cell814

PML-RAR sedimented at approximately 700 kDa (datanot shown). Calculation of the molecular mass of theoligomeric complex based on the Stokes radius (fromgel filtration) and the sedimentation coefficient (fromsucrose gradients) is consistent with the formation of aPML-RAR hexamer. There are no known cases, how-ever, of CC domains forming hexamers (Lupas, 1996).Therefore, we analyzed the self-associating propertiesof the isolated CC domain of PML. By SEC analysis, the14 kDa CC eluted as a HMW peak ranging 60–150 kDa,confirming its capacity to oligomerize (Figure 3D, upperpanel). In vitro cross-linking with two different com-pounds (DTBP or BS3) resulted in the formation of higherMW bands, recognized by anti-CC antibodies and corre-sponding to di- and trimeric species of the CC (Figure3D, lower panels; data not shown) The CC domain can betherefore isolated from bacteria as a trimeric complex.Based on these findings, two possible hypotheses canbe formulated on the nature of the PML-RAR complex:(1) PML-RAR is a trimeric complex with different migra-tion properties with respect to the globular proteins usedas MW markers; (2) PML-RAR is a trimer–trimer complex,due to additional interactions mediated by other do-mains of PML or RAR. In support of this latter model,DCC-PML-RAR eluted as mono- and dimeric species(Figure 3A).

PML-RAR Oligomers Recruit N-CoRto RA-Responsive ElementsRecruitment of N-CoR and specific binding to DNA arecritical for the oncogenicity of PML-RAR (Minucci andPelicci, 1999).

Figure 4. PML-RAR Oligomers Associate with N-CoR and DNAN-CoR binds nuclear receptors through two interac-

(A) In vivo association of PML-RAR oligomers with N-CoR. Metaboli-tion domains (ID1 1 ID2, contained in the GST-N-CoR cally labeled U937 PML-RAR cells (I, input lane) were immunoprecip-described in Figure 1A; Hu and Lazar, 1999). In GST itated with anti-N-CoR (aN) or control (PI) antibodies (the PI lanepull-down assays, both RAR and PML-RAR showed a derives from approximately five times more material). The anti-N-

CoR immunoprecipitates were incubated in the presence of RA (10higher relative affinity for ID1 than ID2 (Figure 1B). PML-mM) for 2 hr at 48C (RA lane). Anti-PML immunoprecipitates (aP) areRAR was recruited more strongly than RAR to GST-ID1,shown in the last lane (upper panel). The RA-eluted material waswhile the associations of PML-RAR and RAR with GST-then analyzed by SEC, followed by SDS PAGE and autoradiography

ID2 were comparable (Figure 1B). Within the ID regions, (lower panel).short helical motifs directly associate with nuclear re- (B) Recruitment of multiple N-CoR molecules. Anti-N-CoR immuno-ceptors (CoRNR boxes 1 and 2; Hu and Lazar, 1999). precipitates from PML-RAR-expressing cells or control cells were

incubated with an in vitro translated, 35S-labeled N-CoR C-terminalBoth RAR and PML-RAR preferentially associated withfragment (VP16-N-CoR, aa 1944–2453: Hu and Lazar, 1999) and thenCoRNR-1, and PML-RAR showed the highest apparentanalyzed by SDS-PAGE followed by Western blot with anti-RARaffinity (data not shown). These results indicate that ID1antibodies (left panel) or autoradiography (right panel). I, input; C,

is sufficient for the increased recruitment of PML-RAR control cells; and PR, PML-RAR-expressing cells. Western blot anal-and suggest that ID2 is less accessible to the RAR bind- ysis with anti-N-CoR antibodies showed comparable amounts ofing site upon oligomerization. The increased stability of N-CoR immunoprecipitated from the two cell extracts (data not

shown). Note that the anti-N-CoR used in our assays is directedthe interaction of PML-RAR with N-CoR likely representsagainst the N terminus of N-CoR.the consequence of two intrinsically related mecha-(C) HMW PML-RAR complexes bind DNA. Mobility shift assays usingnisms, due to the oligomeric nature of PML-RAR: aviditythe RARE from the RARb2 promoter as a probe and extracts from

for ID1 and increase of the local concentration of N-CoR Xenopus oocytes programmed with mRNA for RAR, PML-RAR eitherbinding sites. singly or coinjected with mRNA for RXR. Lanes 3–4, 5–8, and 9–11

To investigate whether PML-RAR oligomers associate correspond to 0.03 (lane 5), 0.05 (lanes 6 and 9), 0.2 (lanes 3, 7, and10), and 0.5 (lanes 4, 8, and 11) oocyte equivalent extract amounts.with N-CoR in vivo, we analyzed anti-N-CoR immu-The empty arrow indicates the RXR-RAR-DNA complex.noprecipitates from metabolically labeled, PML-RAR-(D) HMW PML-RAR complexes recruit N-CoR on DNA. Mobility shiftexpressing cells. An approximately 120 kDa proteinassays of extracts from Xenopus oocytes coinjected with mRNA for

coprecipitated with N-CoR (Figure 4A), identifiable as PML-RAR and RXR. Extracts were incubated with the labeled RAREPML-RAR by (1) comigration with PML-RAR (in parallel in the presence of recombinant GST-N-CoR (aa 1782–2453) or GSTimmunoprecipitation with anti-PML or anti-RAR anti- as a control. Where indicated, RA (10 mM) was added during the

incubation. Empty arrow, PML-RAR/RXR/DNA complex; filled arrow,bodies), and absence in control immunoprecipitatesPML-RAR/RXR/N-CoR/DNA complex.(Figure 4A; data not shown) and (2) dissociation from

the anti-N-CoR immunoprecipitates by RA (Figure 4A).

Oligomerization of Transcription Factors in Leukemias815

PML-RAR was recovered in HMW complexes after SEC bind DNA (alone or in association with RXR) and thatanalysis of the RA-eluted fraction, demonstrating the they are able to recruit N-CoR to DNA. The associationexistence of an oligomeric PML-RAR/N-CoR complex of PML-RAR oligomers with N-CoR (and RXR) does notin vivo (Figure 4A). contradict our finding that the HMW complexes origi-

In the anti-N-CoR immunoprecipitates, the PML- nate from PML-RAR oligomerization, in the absence ofRAR/N-CoR ratio was .1 (Figure 4A), suggesting that other factors interacting stoichiometrically (Figure 3).more than one PML-RAR molecule is bound for each Neither N-CoR nor RXR cofractionated with PML-RARN-CoR molecule. Given the multimeric nature of PML- in SEC analyses, and the PML-RAR AHT mutant, unableRAR, two scenarios are possible: (1) recruitment of mul- to recruit the N-CoR/HDAC complex, has an elution pro-tiple N-CoR molecules in a .1:1 stoichiometric ratio; (2) file coinciding with PML-RAR (data not shown). It ap-recruitment of one N-CoR/HDAC complex per oligomer. pears, therefore, that PML-RAR oligomers represent theTo distinguish between these two possibilities, we “core” complex responsible for the interactions (at lowertested the capacity of immunoprecipitated N-CoR/PML- affinity and/or stoichiometry) with other cofactors.RAR complexes to mediate further recruitment of in vitrotranslated, 35S-labeled N-CoR(ID1 1 ID2) (Figure 4B). Fusion of RAR with a HeterologousN-CoR(ID1 1 ID2) did not bind to anti-N-CoR immuno- Oligomerization Domain Activatesprecipitates from control cells. In contrast, N-CoR/PML- Its Leukemogenetic PotentialRAR immunoprecipitates mediated further N-CoR(ID1 1 To determine whether oligomerization is per se the criti-ID2) recruitment (Figure 4B), suggesting a potential for cal function mediated by the CC domain of PML, wePML-RAR oligomers to recruit multiple N-CoR mole- fused RAR C-terminally to the tetramerization domaincules. present in p53 (Jeffrey et al., 1995; Chen et al., 1998).

To study the binding of PML-RAR oligomers to DNA, SEC analysis showed that p53-RAR (unliganded or RA-we expressed PML-RAR (or RAR) into Xenopus oocytes, bound) formed HMW complexes (Figure 5A), allowingwhich contain low levels of endogenous receptors (Mi- us to analyze RAR oligomers that do not contain PMLnucci et al., 1998). Since RAR and PML-RAR require sequences. Similarly to PML-RAR and CC-RAR, p53-RXR for high-efficiency DNA binding, we coexpressed RAR bound even the lowest amounts of GST-N-CoRRXR in some experiments. Mobility shift assays were tested (Figure 1A) and repressed GAL4-VP16 activity asperformed using the RA responsive element (RARE) strongly (Figure 2A).from the RARb2 promoter as a probe. Coinjection of We then measured the capacity of p53-RAR to blockmRNAs for RAR and RXR caused the formation of a differentiation of primary murine hematopoietic precur-heterodimeric RXR/RAR DNA binding complex (Figure sors transduced with retroviral constructs encoding for4C). RAR did not bind DNA in the absence of RXR, while PML-RAR (or derivatives), and GFP as a marker. GFP-expression of PML-RAR resulted in the formation of a positive cells were sorted and seeded in methylcellulosecomplex (complex I), which migrated more slowly than plates, to allow terminal myeloid differentiation. Pooledthe RXR-RAR complex (Figure 4C). This complex was colonies were analyzed for the expression of differentia-specific, since it was competed by an excess of cold

tion markers (Mac-1 and GR-1). PML-RAR, CC-RAR,RARE (but not by unrelated oligonucleotides: data not

and p53-RAR caused a strong differentiation block (Fig-shown). We performed SEC analysis of the RARE/PML-

ure 5B; data not shown for GR-1). Cells expressing DCC-RAR complex. In control samples, the RARE was found

PML-RAR showed much higher levels of protein com-in fractions corresponding to a predicted MW of 30–60pared to PML-RAR (Figure 5B). High levels of RAR leadkDa, consistent with its length (37 bp). In PML-RAR-to a differentiation block, likely by sequestering of RXRcontaining extracts, a new peak of radioactive RARE(Grignani et al., 1996; Du et al., 1999). We sorted thewas observed, cofractionating with PML-RAR and cor-DCC-PML-RAR GFP1 cells in two populations, ac-responding to an apparent MW .670 kDa (data notcording to their mean fluorescence levels: GFPlow cellsshown). Parallel mobility shift assays revealed the ap-expressed lower levels of DCC-PML-RAR than thepearance of the complex I, showing that this complexGFPhigh cells (Figure 5B). Low levels of DCC-PML-RARrepresents the binding of HMW PML-RAR to DNA (Fig-had no effects, whereas at higher levels it induced aure 4C). Coexpression of PML-RAR and RXR resultedconsistent differentiation block (.30%). These resultsin enhanced binding to DNA and formation of a predomi-show that the capacity of PML-RAR to block myeloidnant complex (complex II), which migrated slightlydifferentiation depends on the CC domain of PML andslower than complex I and represents the oligomericthat addition of an oligomerization domain to RAR isPML-RAR/RXR DNA binding complex (Figure 4C).sufficient to obtain a fusion protein with full transformingMobility shift assays were performed to determinepotential.whether N-CoR might be recruited to DNA by PML-RAR.

Finally, we compared the capacity of PML-RAR, CC-We used agarose as a solid matrix for the electrophoreticRAR, and p53-RAR to respond to RA. The differentiationruns, to allow better resolution. The oligomeric PML-block by PML-RAR and CC-RAR was relieved exclu-RAR/RXR/DNA complex was supershifted by the addi-sively at high concentrations of RA (1 mM, Figure 5B).tion of recombinant GST-N-CoR (but not control GST:In contrast, p53-RAR-expressing cells were insensitiveFigure 4D). RA addition caused the disappearance ofto RA treatment (Figure 5B). Accordingly, RA did notthe supershift and the formation of a complex migratingrelieve transcriptional repression by p53-RAR (Figureslightly faster than in the absence of RA (Figure 4D, lane2B). It appears, therefore, that RAR-fusion protein oligo-2 against lane 5), likely as a result of a conformationalmers exert differential responses to RA on the basis ofchange induced by RA (Chambon, 1996).

These results demonstrate that PML-RAR oligomers the identity of the oligomerization domain fused to RAR.

Molecular Cell816

Figure 6. APL Fusion Proteins Form HMW Complexes Due to thePartners of RAR in the Chromosomal Translocations

(A) SEC analysis of nuclear extracts from COS-1 cells (NPM) orCOS-1 cells transfected with PML or PLZF expression vectors, fol-lowed by SDS-PAGE and Western blotting.(B) Nuclear extracts from COS-1 cells transfected with the indicatedexpression vectors were fractionated and analyzed by Western blot-ting using anti-RAR antibodies.

PML, PLZF, and NPM Form HMW ComplexesOther RAR fusion proteins (such as PLZF-RAR andNPM-RAR) are infrequently associated with APL (Mel-

Figure 5. A Heterologous Oligomerization Domain Activates thenick and Licht, 1999). Both PLZF and NPM containTransforming Potential of RARstrong self-association domains, retained within the fu-

(A) p53-RAR form oligomers. (Upper panel) SEC analysis of RA bind-sion proteins (Chan and Chan, 1995; Ahmad et al., 1998).ing capacity of nuclear extracts from COS-1 cells transfected with

SEC analysis revealed that PML is distributed in HMWp53-RAR or RAR expression vectors, incubated with tritiated RA(10 nM), in the absence (black squares and circles) or in the presence complexes ranging from .670 kDa to the void volume(white squares) of a 100-fold excess of cold RA. (Lower panel) SEC of the column (Figure 6B). PLZF and NPM were alsofractions of in vitro translated, 35S-labeled p53-RAR were analyzed found in HMW complexes: PLZF peaked at an apparentby SDS-PAGE, followed by autoradiography. Two products are gen- MW .440 kDa, whereas NPM was found as a monomererated in the reaction: full-length p53-RAR oligomers and mono-

(30% of total NPM), or in HMW complexes (from 200meric RAR, starting from its internal ATG (maintained in the con-to .400 kDa) consistent with an hexameric state, asstruct).

(B) Differentiation of murine hematopoietic progenitors. Lin2 cells previously described (Figure 6B; Chan and Chan, 1995).were transduced with the indicated retroviral vectors (C: control; NPM-RAR and PLZF-RAR also formed HMW com-P-R: PML-RAR; DCC-P-R: DCC-PML-RAR; CC-R: CC-RAR; GPR: plexes. NPM-RAR peaked with a lower apparent MWGFP-PML-RAR; GDCC: GFP-DCC-PML-RAR; and Gp53R: GFP-p53- (400 kDa), consistent with the lower MW of NPM-RARRAR), and GFP1 cells were sorted by FACS. DCC-PML-RAR, GFP1

(60 kDa; Figure 6B). Therefore, all RAR-fusion proteinscells were sorted in GFPhigh or GFPlow expressors. After sorting, cellsform HMW complexes through their correspondingwere either plated in differentiation medium in the absence or in the

presence of RA (3 nM or 1 mM) (upper panel) or analyzed by Western PML, PLZF, or NPM moieties.blot (lower panels). RA delays myeloid differentiation of control cells,and this effect is counteracted by expression of PML-RAR, CC-RAR, Oligomerization of the AML1/ETO Fusion Proteinand by high levels of DCC-PML-RAR. As described for wild-type ETO forms HMW complexes (Lutterbach et al., 1998) andRAR, expression of high levels of DCC-PML-RAR in the presence

has two putative protein–protein interaction domains,of physiological concentrations of RA relieves the differentiationretained in AML1-ETO: a CC region (P1, residues 444–block observed in the absence of ligand (Du et al., 1999). Note that

p53-RAR was expressed as a GFP-fusion protein: GFP-PML-RAR 492) and an amphipatic a helix (P2, residues 352–378;and GFP-DCC-PML-RAR were also tested in some experiments, Figure 7A). AML1-ETO formed HMW complexes, whilewith identical results to PML-RAR and DCC-PML-RAR, respectively deletion of P1 and P2 shifted the fusion protein to lower(data not shown). molecular weight forms (Figure 7A). AML1a was found

as a monomeric species, confirming the requirement

Oligomerization of Transcription Factors in Leukemias817

despite the fact that DP-AML1-ETO retains the N-CoRbinding site (Figure 7B).

We analyzed whether HMW AML1-ETO complexesare able to bind DNA. AML1-ETO was partially purifiedby DNA affinity (Figure 7C, left panel). Gel filtration ofDNA-eluted material showed that AML1-ETO can berecovered in its oligomeric form after DNA binding (Fig-ure 7C, right panel). These results suggest that the oligo-meric AML1-ETO/DNA complex might recruit N-CoRand repress transcription. Consistently, deletions of ei-ther the N-CoR binding site (DZ) or the oligomerizationregions (DP) impaired the capacity of the fusion proteinto (1) repress transcription from a target promoter—MDR-1—(Figure 7D; Lutterbach et al., 1998) and (2)block differentiation of primary hemopoietic progenitors(Figure 7E). Taken together, these results indicate thatthe efficient recruitment of the N-CoR/HDAC complexby AML1-ETO is required to activate the oncogenic po-tential of AML1 and that this is achieved by the formationof AML1-ETO oligomeric complexes.

Discussion

We propose that oligomerization is the mechanism re-sponsible for the oncogenic activation of RAR upon fu-sion with PML.

Transcription from RA-target genes represents theresult of a “concerted” mode of transcriptional regula-tion, resulting from the cooperation among RARs andother DNA binding transcription factors, with associatedcoregulators (Ptashne and Gann, 1997; Kadonaga, 1998;Minucci and Pelicci, 1999). In this and in an accompa-nying report (Lin and Evans, 2000 [this issue of MolecularCell]), it is shown that oligomerization subverts this regu-latory network by enhancing the capacity of a transcrip-tion factor to recruit coregulators (such as N-CoR, andFigure 7. Oligomerization of AML1-ETOpossibly RXR) and leads to an “a solo” mode of deregu-(A) AML1-ETO forms HMW complexes. A scheme of AML1-ETO andlated transcription. We hypothesize that one criticalthe deletions used (upper panel); Z, zinc fingers (N-CoR interaction

domain). AML1-ETO (A-E) and DP-AML1-ETO were in vitro trans- result of RAR oligomerization is the nonphysiologicallated, fractionated by SEC, and analyzed by SDS PAGE followed recruitment of corepressors, leading to a chromatin con-by autoradiography (lower panel). figuration of target promoters refractory to activating(B–C) Interaction of AML1-ETO with N-CoR and DNA. (B) Pull-down signals from other cis-regulatory elements.assays of in vitro translated AML1-ETO and DP-AML1-ETO incu-

Transcription factors have been shown to oligomerizebated with GST-N-CoR (RDIII), or GST beads (C) as control. Inputphysiologically. Examples are p53, STAT5, Groucho,lanes (I) represent 100% of the total. (C) Nuclear extracts from U937TEL, Sp1 (Jeffrey et al., 1995; Jousset et al., 1997; ChenAML1-ETO cells were incubated with biotinylated oligos containing

a specific AML1 binding site or an unrelated sequence (C) and et al., 1998; John et al., 1999), and, as shown here, PML,then pulled-down with streptavidine-agarose beads (left panel). SEC PLZF, and ETO. Regardless of their implications for leu-analysis of high-salt eluted material (right panel). kemogenesis, our findings provide genetic evidence(D) Transcriptional repression by AML1-ETO. C33A cells were tran- that oligomerization per se has profound effects on thesiently transfected with the MDR1-luc reporter and the indicated

regulatory properties of a transcription factor. In theexpression vectors.case of ETO, the self-association domain is essential(E) Analysis of myeloid differentiation. Lin2 cells transduced within directing efficient recruitment of the N-CoR/HDACthe indicated retroviral vectors were sorted and then treated as

described in the legend to Figure 5. complex and transcriptional repression. Although thenatural targets of ETO are still unknown, our data predictthat oligomerization is crucial for the natural function of

for ETO in the formation of HMW complexes (data not ETO. Increased density of interacting domains due toshown). Bacterially expressed AML1-ETO formed HMW formation of oligomeric complexes may therefore con-complexes, indicating its oligomeric state (data not stitute a general mechanism to generate high local con-shown). We then asked if the loss of the capacity to centrations of coregulators. Notably, a single point mu-form HMW complexes correlated with changes in the tation that prevents STAT5 tetramerization decreasesability of AML1-ETO to recruit N-CoR and to repress levels of STAT5-mediated transcriptional activationtranscription. The P1-P2 motifs do not interact with (John et al., 1999).N-CoR, but their deletion led to a strong decrease in A corollary derives from these considerations: to func-

tion efficiently, the interactions underlying the formationthe amount of fusion protein bound to GST-N-CoR,

Molecular Cell818

(Stratagene). pMAL-PML-RAR was obtained by site-directed muta-of oligomers must be considerably tighter than thosegenesis of the first ATG of the PML-RAR cDNA and insertion of anresponsible for coregulator recruitment. The PML-RAREcoRI site for in-frame cloning in pMAL-C2 (New England BioLabs).HMW complexes, but not the associations of RAR orThe pGEX-CC plasmid was cloned fusing PML residues 224–369

PML-RAR with N-CoR (or RXR), were resistant to strong in-frame at the BamHI site of pGEX 6P1. PSG5-p53-RAR was clonedlysis conditions, such as high salt, detergents, and re- by insertion of a PCR fragment containing the tetramerization do-ducing agents (data not shown). Consistently, in the main of p53 (Chen et al., 1998) carrying an optimal Kozak sequence

at the ATG of pSG5-RAR. pcDNA3-DP-AML1-ETO and pcDNA3-DZ-experimental conditions used for SEC, we did not findAML-ETO were obtained by PCR-mediated deletion mutagenesis.evidence of RAR-associated coregulators in HMW PML-The retroviral vectors were cloned by insertion of the appropriateRAR complexes (RAR itself was found to fractionate ascDNAs into the EcoRI site of PINCO (Grignani et al., 1998a). pRARE-

a monomer). G5-TATA was obtained by inserting five GAL4 binding sites and theOligomerization and strength of self-association are minimal promoter sequence from pG5E1b into the XhoI-HindIII sites

not determined only by CC structures. The p53 tetra- of the pGL2 plasmid (Promega). Oligonucleotides containing theRARE from the RARb2 promoter (Minucci et al., 1998) were insertedmerization domain conferred—upon fusion with RAR—at the MluI site. MDR1-luc was obtained by PCR of the MDR1 pro-altered biochemical, transcriptional, and biologicalmoter region from a genomic clone and subsequent cloning in pGL2.properties, similarly to the CC of PML. PLZF contains

a BTB-POZ domain, which, in the GAGA transcriptionPull-Down Assays and Coimmunoprecipitation Experimentsfactor, mediates the formation of oligomeric complexesGST-N-CoR pull-down assays were performed incubating the indi-(Katsani et al., 1999); NPM contains an amino-terminalcated amounts of GST-N-CoR (attached to a constant amount of

oligomerization domain, which mediates the formation glutathione-agarose beads) in the presence of 35S-labeled, in vitroof hexameric structures (Chan and Chan, 1995). Not translated proteins. Coimmunoprecipitation experiments were per-tested here, NuMA, another protein fused to RAR in APL, formed as described (Gelmetti et al., 1998; Grignani et al., 1998b).also contains a strong oligomerization domain (Harborthet al., 1999). Transactivation Assays

We have shown that oligomerization per se is suffi- Transient transfections of HeLa and C33A cells were performed bycalcium phosphate. PML-RAR (or RAR) expression vector did notcient to activate the oncogenic potential of a transcrip-repress transcription from a RARE-less reporter, and constructstion factor (RAR) and that oligomerization is required forcarrying the AHT mutation, which abrogates N-CoR binding (Horleinbiological activity of both PML-RAR and AML1-ETO.et al., 1995), did not repress activation of the RARE-G5-TATA pro-

Oligomerization might therefore serve as a mechanism moter by GAL4-VP16 (data not shown).of oncogenic activation for transcription factors foundin other leukemia-associated fusion proteins. In the liter- Injection of Xenopus Oocytesature, we found some cases where this mechanism may Ooocyte injection and mobility shift assays were performed as de-be in action. Fusion of a truncated leukemia oncogene, scribed (Minucci et al., 1998).MLL, to bacterial b-galactosidase (a tetrameric protein)caused leukemia in mice (Dobson et al., 2000). The TEL Biochemical Purification of PML-RAR HMW Complexesoligomerization domain is required for transcriptional Nuclear extracts from U937-PML-RAR cells were partially purified

by Heparin-Sepharose and then loaded on a Superose 6 HR 10/repression of TEL-AML1 (Jousset et al., 1997; Uchida30 gel filtration column. The PML-RAR-containing fractions wereet al., 1999). Notably, the portion of AML1 retained indiluted with incubation buffer (Hepes 20 mM [pH 7.4], EDTA 1 mM,this fusion includes a region (lost in AML1-ETO) recentlyDTT 1 mM, aprotinin and leupeptin 10 mg/ml, pepstatin 2 mg/ml, 1shown to recruit corepressors (Levanon et al., 1998;mM PMSF, glycerol 10%, MgCl2 3 mM, NaF 5 mM, NP40 0.1%; final

Uchida et al., 1999). 0.1 M KCl) and incubated for 1 hr at 48C with biotinylated RAREThe oligomerization domain of TEL is also found in other double-stranded oligonucleotide coupled to streptavidine-agarose

leukemia-associated fusion proteins, where it leads to beads (7 mg/mg starting material). Beads were eluted in buffer con-taining 1 M KCl. As controls, we used either streptavidine-agarosethe constitutive activation of the associated tyrosinebeads alone or performed the incubation with RARE-containingkinase (PDGFR-b or JAK2; Lacronique et al., 1997), abeads in the presence of a 100-fold excess RARE competitor infrequent mechanism of oncogene activation in humansolution.tumors. Therefore, oligomerization appears to be a

mechanism of oncogene activation for both tyrosine ki-Expression and Characterization of Recombinant PML-RARnases and transcription factors. Interestingly, oligomer-Bacterial (pMAL-PML-RAR) lysates were incubated with amylose

ization inhibitory peptides are able to revert in vitro the beads for 2 hr at 48C. MBP-PML-RAR was eluted by adding maltosetransforming phenotype of BCR/ABL, a tyrosine kinase (20 mM), loaded onto a MonoQ column (Pharmacia Biotech), andfusion protein found in chronic myelogenous leukemia then subjected to an additional round of amylose affinity chromatog-

raphy. MBP-PML-RAR was eluted and incubated with factor Xa(Guo et al., 1998). In conclusion, with the present workto cleave the MBP moiety and yield purified PML-RAR, that waswe have established the mechanism of altered recruit-subsequently analyzed by gel filtration chromatography (Superosement of the N-CoR/HDAC complex by PML-RAR and6 column).AML1-ETO and presented evidence suggesting that

oligomerization of a transcription factor might representIn Vivo Cross-Linking Experimentsa potentially widespread mechanism of transcriptionalU937-PML-RAR cells were grown for 1 hr in medium devoid of

regulation and oncogenic transformation. As such, cysteine and methionine and then for 8 hr in the presence ofoligomerization might therefore constitute an attractive 35S-labeled cysteine and methionine (Amersham). Before harvest-target for therapeutical intervention. ing, cells were incubated for 30 min at room temperature in PBS

plus 0.1 mM DTBP (Dimethyl 3,39-dithiobisproprionamidate-2HCl,Pierce). Isolated nuclei were extracted in modified RIPA buffer (150Experimental ProceduresmM MaCl, 1% Nonidet P-40, 1% sodyum deoxycholate, 0.2% SDS,2 mM EDTA, 5 mM NaF, aprotinin and leupeptin 10 mg/ml, pepstatinPlasmids2 mg/ml, 1 mM PMSF, and 100 mM Tris-Cl [pH 7.4]). The extractsTo generate PML/RARpSP64UTK for oocyte injections, the human

PMLRAR cDNA was cloned into the NcoI-BamHI sites of PSP64UTK were collected and analyzed by SEC on a Superose 6 HR 10/30

Oligomerization of Transcription Factors in Leukemias819

column (Pharmacia Biotech). HMW PML-RAR complexes were im- Effects on differentiation by the promyelocytic leukemia PML/RARa

protein depend on the fusion of the PML protein dimerization andmunoprecipitated with anti-PML monoclonal antibodies. The immu-RARa DNA binding domains. EMBO J. 15, 4949–4958.noprecipitates were eluted from the beads in 1% SDS, and an aliquot

was further analyzed by SEC. Grignani, F., Kinsella, T., Mencarelli, A., Valtieri, M., Lanfrancone,L., Peschle, C., Nolan, G.P., and Pelicci, P.G. (1998a). High-efficiency

Characterization of the Isolated CC Domain of PML gene transfer and selection of human hematopoietic progenitor cellsThe GST-CC was purified from BL21 cells, incubated with prescission with a hybrid EBV/retroviral vector expressing the green fluores-protease (Pharmacia) to cleave the GST moiety and yield CC, which cence protein. Cancer Res. 58, 14–19.was subsequently purified by Mono-Q, and then analyzed by gel Grignani, F., De Matteis, S., Nervi, C., Gelmetti, V., Cioce, M., Fanelli,filtration chromatography (Superose 12 column, SMART system, M., Ruthardt, M., Ferrara, F.F., Zamir, I., Seiser, C., et al. (1998b).Pharmacia Biotech). In vitro cross-linking was performed in cross- Fusion proteins of the retinoic acid receptor-a recruit histone deace-linking buffer (Hepes 20 mM [pH 7.4], NaCl 150 mM, EDTA 1 mM, tylase in promyelocytic leukaemia. Nature 391, 815–818.and DTT 1 mM), in the presence of cross-linker (BS3 [Pierce] at 0.08,

Grignani, F., Gelmetti, V., Fanelli, M., Ferrara, F.F., Bonci, D., Nervi,0.4, 2 mM). After incubation, the reactions were quenched with Tris

C., and Pelicci, P.G. (1999). Formation of PML/RAR a high molecular(50 mM [pH 6.8]), and then the samples were analyzed by SDS-

weight nuclear complexes through the PML coiled-coil region isPAGE.

essential for the PML/RAR a-mediated retinoic acid response. On-cogene 18, 6313–6321.

In Vitro Differentiation of Murine Hematopoietic ProgenitorsGrisolano, J.L., Wesselschmidt, R.L., Pelicci, P.G., and Ley, T.J.Murine hematopoietic progenitors were purified from the bone mar-(1997). Altered myeloid development and acute leukemia in trans-row of BALB-C mice on the basis of the absence of lineage differenti-genic mice expressing PML-RAR a under control of cathepsin Gation markers (lin-). Purified cells were prestimulated for 2 days inregulatory sequences. Blood 89, 376–387.medium containing IL-3 (20 ng/ml), IL-6 (20 ng/ml), and SCF (100Guo, X.Y., Cuillerot, J.M., Wang, T., Wu, Y., Claxton, D., Bachier, C.,ng/ml), and then attached to Retronectin (Takara Shuzo)-coatedGreenberger, J., and Deisseroth, A.B. (1998). Peptide containing theplates. Cells were incubated with the supernatant from PhoenixBCR oligomerization domain (AA 1–160) reverses the transformedpackaging cells transfected with the indicated retroviral constructs.phenotype of p210bcr-abl positive 32D myeloid leukemia cells. On-GFP1, infected cells were sorted by FACS and seeded in methylcel-cogene 17, 825–833.lulose plates supplemented as above, plus G-CSF (60 ng/ml) andHarborth, J., Wang, J., Gueth-Hallonet, C., Weber, K., and Osborn,GM-CSF (20 ng/ml). After 8–10 days, differentiation was measuredM. (1999). Self assembly of NuMA: multiarm oligomers as structuralby FACS analysis. Uninfected cells and cells infected with the controlunits of a nuclear lattice. EMBO J. 18, 1689–1700.vector behaved identically (data not shown).Horlein, A.J., Naar, A.M., Heinzel, T., Torchia, J., Gloss, B., Kuro-

Acknowledgments kawa, R., Kamei, Y., Soderstrom, M., Glass, C.K., et al. (1995). Li-gand-independent repression by the thyroid hormone receptor me-

We thank F. Grignani, S. Ronzoni, S. Soddu, G. Ferrari, J. Medin, diated by a nuclear receptor co-repressor. Nature 377, 397–404.A. Musacchio, W. Weissenhorn, Y. Nakatani, M. Ruthardt, K. Helin, Hu, X., and Lazar, M.A. (1999). The CoRNR motif controls the recruit-P. P. Di Fiore, and K. Ozato for discussions and reagents. This ment of corepressors by nuclear hormone receptors. Nature 402,research was partially funded by E. U. (P. G. P.), AIRC (P. G. P., 93–96.N. L., and C. N.), FIRC (P. G. P., C. N., and S. M.), and the National Jeffrey, P.D., Gorina, S., and Pavletich, N.P. (1995). Crystal structureInstitutes of Health DK45586 (M. A. L.). of the tetramerization domain of the p53 tumor suppressor at 1.7

angstroms. Science 267, 1498–1502.Received September 28, 1999; revised March 23, 2000.

John, S., Vinkemeier, U., Soldaini, E., Darnell, J.E., Jr., and Leonard,W.J. (1999). The significance of tetramerization in promoter recruit-

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