Proc. Natl. Acad. Sci. USAVol. 91, pp. 10610-10614, October 1994Medical Sciences

11q23 translocations split the "AT-hook" cruciform DNA-bindingregion and the transcriptional repression domain from theactivation domain of the mixed-lineage leukemia (MLL) geneNANCY J. ZELEZNIK-LE*, ALANNA M. HARDEN*, AND JANET D. ROWLEY*t*Departments of *Medicine, Section of Hematology/Oncology and tMolecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637

Contributed by Janet D. Rowley, July 1, 1994

ABSTRACT Translocations involving chromosome band11q23, found in acute lymphoid and myeloid leukemia, dis-rupt theMLL gene. This gene encodes a putative transcriptionfactor with homology to the zinc fingers and other domains ofthe Drosophila trithorax gene product and to the "AT-hook"motif of hig mobility group proteins. To map potentialtranscriptional activation or repression doains of the MLLprotein, yeast GAL4 DNA-binding domain and MLL hybridprotein-expressing plaids were cotransfected with chioram-phenicol acetyltransferase reporter plasmids in a transienttransfection system. We found that MLL contains a strongactivation domain and a repression domain. The former,located telomeric (3') to the breakpoint region, activatedtranscription 18-fold to >200-fold, depending on the promoterand cell line used for transfection. A repression domain thatrepressed transcription 4-fold was located centromeric (5') tothe breakpoint region of MLL. The MLL AT-hook domainprotein was expressed in bacteria and was utilized in a gelmobility shift assay to assess DNA-binding activity. The MLLAT-hook domain could bind cruciform DNA, recognizingstructure rather than sequence of the target DNA. In translo-cations involving MLL, loss of an activation domain withretention of a repression domain and a DNA-binding domainon the der(11) chromosome could alter the expression ofdownstream target genes, suggesting a potential mechanism ofaction for MLL in leukemia.

Chromosome 11 band q23 is involved in many translocationsobserved in acute lymphoblastic leukemia, acute myeloidleukemia, and acute-mixed lineage leukemias, leading to thehypothesis that a gene located in this chromosomal regionplays an important role in a progenitor cell capable of bothlymphoid and myeloid differentiation (1). Interestingly,translocations involving 11q23 are also observed in second-ary leukemias in patients previously treated with DNA to-poisomerase II inhibitors (2, 3). We (4, 5) and others (6-9)have identified and cloned the gene that spans the 11q23breakpoint and is rearranged in patients with these leuke-mias. This gene, MLL, for mixed-lineage leukemia or my-eloid/lymphoid leukemia, has also been called Htrx, ALL-I,and HRX (6-8).The cDNA encoded by the MLL gene shares homology in

size, organization, and with some domains of the Drosophilatrithorax gene (trx), including zinc fingers with potentialDNA-binding capabilities (7-9). The trx gene is thought tocontrol expression of the ultrabithorax complex homeoticgenes by interacting with cis regulatory elements in theirpromoters (10, 11). The MLL protein contains another po-tential DNA-binding motif: "AT-hooks" which, in the highmobility group (HMG)-I(Y) proteins, bind A+T-rich DNA(12). In addition, another family ofHMG proteins, HMG1/2,

binds cruciform DNA (13). Therefore, by homology to do-mains of other proteins, it seems likely that MLL possessesthe capability to bind DNA and to regulate the expression ofdownstream genes. We have used gene fusions to discernwhether MLL contains functional transcriptional activationand/or repression domains. We report here that MLL con-tains an extended activation domain in the C-terminal part ofthe protein and a repression domain in the N-terminal part ofthe protein. Furthermore, the AT-hook region of MLL iscapable ofbinding cruciform DNA, recognizing the structurerather than the sequence of the target DNA.

In many leukemia cases with translocations involvingMLL,both derivative RNAs are expressed (5, 8); however, byanalysis of complex translocations involving more than twochromosomes, the common junction is the one involving theder(11) chromosome (14). This suggests that the transcript andprotein produced by the der(11) chromosome are responsiblefor the leukemogenic phenotype. In this paper, we identify acruciform DNA-binding domain and a repression domain thatwould remain on the der(11) chromosome, as well as a verystrong activation domain that would either be lost (15, 16) ortranslocated to the der(other) chromosome. The splitting ofthese functional domains is likely to contribute to leukemo-genesis in cells containing MLL translocations.

MATERIAL AND METHODSConstrutlon of GAIA-MLL Chimeras. Domains of MLL

were obtained by restriction digestion ofplasmids containingMLL cDNAs or by reverse transcription-PCR from the cellline RCH.ADD (5) (Invitrogen). MLL sequences were clonedinto pSG424 (17), which encodes the DNA-binding domain ofyeast GAL4. The junction of the construct and the MLLsequences were verified by Sequenase dideoxy sequencing(United States Biochemical).

Reporter Pasids. GAL45E1bCAT (18), GAL45tkCAT(19), and GAL45A56fosCAT (20) each contain five copies ofthe GAL4-binding site upstream of various promoter ele-ments.

Transfections and Chloramphenicol Acetyltransferase(CAT) Assays. HeLa cells (1.5 x 106 cells per transfection;from T. McKeithan, University of Chicago) or NIH 3T3 cells(0.75 x 106 cells per transfection; from H. Singh, Universityof Chicago) were transfected by calcium phosphate precipi-tation (Invitrogen). Each precipitate included 5 pg of theinternal reference pCH110, a 3-galactosidase plasmid (Phar-macia), 10 ug (or as noted for titration experiments) ofeffector plasmid, and 10 pg of reporter plasmid. At least twoindependent plasmid preparations for each G4-MLL con-struct were tested. Forty-eight hours after transfection, cellextracts were prepared and assayed for f-galactosidase ac-tivity (20) to normalize for variation in transfection effi-ciency. CAT protein expression was determined by ELISA

Abbreviations: CAT, chloramphenicol acetyltransferase; HMG,high mobility group.MTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(Boehringer Mannheim), analyzed in duplicate. Fold activa-tion or repression was calculated by comparison of theamount ofCAT protein produced (normalized for 3-galacto-sidase activity) in extracts from cells transfected with MLL-containing plasmids relative to extracts from cells transfectedwith the parent pSG424 plasmid.AT-Hook DNA-Binding Assays. DNA encoding MLL

amino acids 142-400 (AT-hook) or, as controls, MLL aminoacids 1101-1400, MLL amino acids 3114-3357, or Egr-1amino acids 281-304 (Egr-1 repression domain) (20) weresubcloned into pGEX-KT (21). The glutathione S-transferasefusion proteins were expressed and purified (22). Artificialcruciform DNAs and control duplex DNAs were prepared asdescribed (13, 23). Cruciforms 1 and 2 consist of oligonucle-otides 1-4 and 7-10, respectively (13). Duplexes 1A and 1Bconsist of oligonucleotides 1 and 6 and oligonucleotides 3 and5, respectively (13). Together, the two duplex moleculescontain the same sequence as contained in cruciform 1 butcannot form a cruciform structure. Gel mobility shift assayswere performed as described (23).

RESULTSGeneral Strategy. MLL cDNA contains regions that code

for potential DNA-binding motifs present in transcriptionfactors: AT-hooks and zinc fingers (Table 1). Therefore,because it is likely that MLL protein is a transcription factor,we wished to determine (1) whether MLL contains eithertranscriptional activation or repression domains and (2)whether MLL AT-hooks are capable of binding to DNA. Toassess transcriptional function, segments ofMLL were fusedto a heterologous DNA-binding domain and assayed fortranscriptional activity. To assess the AT-hook DNA-bindingcapability, we determined whether bacterially expressedMLL AT-hooks can bind DNA in a gel mobility shift assay.GAL4-MLL chimeras (Table 1) were tested for their

ability to transactivate or repress transcription from a CATreporter plasmid containing GAL4 binding sites upstream ofthe herpes simplex virus thymidine kinase promoter (Fig. 1).MLL amino acids 2340-2771 and 2772-3123 activated tran-scription (Fig. 1, constructs 9 and 10), whereas MLL aminoacids 1101-1400 repressed transcription (Fig. 1, construct 5).The rest of the MLL constructs tested did not demonstrateany reproducible effect on transcription; therefore, only theG4-MLL2340, G4-MLL2772, and G4-MLL1101 constructswere studied in further detail.MLL Contains a Potent Activation Domain. The constructs

G4-MLL2340 and G4-MLL2772 activated transcription from

Table 1. MLL constructs used for study of transcriptionallyfunctional domains

No. ofConstruct MLL aa aa Motif/enriched aa

G4-MLL142 142-400 259 AT-hooks/K, R (26%)G4-MLL401 401-778 378 S, T (28%); P (13%)G4-MLL779 779-1100 322 UlsnRNP/S, T (23%);

R (19%6)G4-MLL1101 1101-1400 300 MTase/P (15%);

K (17%)G4-MLL1394 1394-2000* 569 Zinc fingersG4-MLL2001 2001-2213 213 S, T (23%)G4-MLL2214 2214-2339 126 S, T (34%)G4-MLL2340 2340-2771 432 S, T (20%o)G4-MLL2772 2772-3123 352 S, T (24%)G4-MLL3114 3114-3357 244 S, T (31%)G4-MLL3358 3358-3657 300 S, T (24%)G4-MLL3658 3658-3968 311 K, R (16%o)

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FIG. 1. CAT activity ofG4-MLL constructs. CAT activity of cellextracts from HeLa cells cotransfected with 10 ,g of theGAL4stkCAT reporter plasmid, 5 pg of the internal referenceplasmid, and 10 jig of the G4-MLL expression plasmid. CAT proteinproduced was normalized for transfection efficiency and plotted foreach construct. Bar 1, G4 (without any MLL domain); bars 2-13,G4-MLL142, -401, -779, -1101, -1394, -2001, -2214, -2340, -2772,-3114, -3358, and -3658, respectively. The data from one represen-tative experiment are shown.

the GAL45tkCAT plasmid in two different cell lines, HeLaand NIH 3T3. G4-MLL2340 and G4-MLL2772 each activatedtranscription about 30-fold in HeLa cells (Fig. 2A). In NIH3T3 cells, G4-MLL2340 and G4-MLL2772 activated tran-scription an average of 18-fold and 13-fold, respectively (Fig.2A). The GAL45tkCAT reporter plasmid has a high basallevel of CAT activity because it contains a promoter regionwith several transcription elements. We wanted to assess theability of the MLL constructs to activate transcription frompromoters with a low basal level of activity, GAL45E1bCAT(18) and GAL45A56fosCAT (20), that each contain only a

TATA promoter element. In HeLa cells, MLL amino acids2340-2771 and 2772-3123 activate transcription from minimalpromoters an average of 200-fold and 300-fold, respectively(Fig. 2A). The fold activation is much higher for the minimalpromoter constructs than for the GAL45tkCAT construct,mainly due to the difference in the basal level of activity ofthese two different types of promoter constructs. Surpris-ingly, the transactivation activity of the two G4-MLL con-

structs on minimal promoter reporter plasmids was differentin NIH 3T3 cells: G4-MLL2772 activated transcription an

average of 35-fold, whereas G4-MLL2340 did not activatetranscription (Fig. 2A). GAL4-binding sites were requiredbecause reporter constructs lacking GAL4 sites were notactivated (data not shown). An effector construct that con-tained sequences coding for MLL amino acids 2340-3123activated transcription in the same manner as G4-MLL2772alone (data not shown). G4-MLL2772 activation was titrat-able: the amount of CAT protein produced increased withincreasing amounts of effector plasmid transfected, up to a

saturation point (Fig. 2B). Therefore, MLL contains an

extended activation domain (amino acids 2340-3123) that issomewhat redundant in its activity.MLL Contains a Proline-Rich Repression Domain. The

G4-MLL11O1 construct was assayed for its capability torepress transcription from the GAL45tkCAT reporter plas-mid, with a high basal activity suitable for examining repres-sion. The G4-MLL11O1 chimera repressed transcription an

average of 4-fold in HeLa cells and 3-fold in NIH 3T3 cells(Fig. 3A) and was dependent on GAL4-binding sites (data notshown). Titration of the chimeric repressor shows that aslittle as 1 ug of G4-MLL1101 represses transcription 30%6(Fig. 3B), whereas even 15 ug of the GAL4 DNA-bindingdomain does not have this effect (data not shown). Therefore,MLL contains a repression domain that is localized to aminoacids 1101-1400.

aa, Amino acids; snRNP, small nuclear ribonucleoprotein; MTase,methyltransferase.*Excluding MLL exon 8 (amino acids 1407-1444).

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FIG. 2. MLL contains a strong activation domain. Cells weretransfected, and CAT protein expression was determined, as de-scribed in Materials and Methods. (A) HeLa cells or NIH 3T3 (3T3)cells were transiently cotransfected with 10 pg ofGAL45tkCAT (tk),GAL4sElbCAT, or GAL45A56fosCAT; 10 pg of G4-MLL2340 orG4-MLL2772; and 5 pg of internal reference plasmid. Fold activa-tion, calculated relative to the amount of CAT protein produced incells transfected with pSG424, is plotted as the average fold activa-tion plus SD, where n = at least three independent experiments. (B)Titration of transactivation by G4-MLL2772. HeLa cells were tran-siently transfected with 0, 1, 3, 5, or 10 pig of G4-MLL2772, 10 pg ofGALAsElbCAT, 5 pg of internal reference plasmid, and salmonsperm DNA to bring the total amount of G4-MLL2772 plus salmonsperm DNA to 10 pg.

Because not all ofthe G4-MLL fusion constructs possessedtranscriptional activity, we confirmed that each of the con-structs did produce G4-MLL fusion protein capable ofbindingto the target GAL-binding site DNA (data not shown) (20);this demonstrates that the lack of transcriptional activity wasnot simply due to the inability of the constructs to produceprotein capable of binding to the GAL4 binding site.MLL Contains an AT-Hook Domain Capable of Binding

Cruciform DNA. Bacterially expressed proteins correspond-ing to the MLL AT-hook region (amino acids 142-400) or, ascontrols for nonspecific binding, other regions from MLL orthe repression domain of the Egr-1 transcription factor (20)were purified. They were assessed for their ability to bindcruciform DNA with a gel mobility shift system and syntheticcruciform DNA structures as described by Bianchi and co-workers (13, 23) (Fig. 4). The MLL AT-hook domain was ableto bind to two different cruciform DNA structures (Fig. 4A,lanes 1 and 9) that did not contain any obvious sequencehomology to each other. This binding was specific, becauseretarded bands were blocked by competition with an excess ofnonradiolabeled competitor cruciform DNA (Fig. 4A, lanes 2and 10). Surprisingly, the repression domain ofMLL (aminoacids 1101-1400) was also able to bind both cruciform DNAprobes (Fig. 4A, lanes 3 and 11), and this binding could bepartially blocked by competition with a 100-fold molar excess

FIG. 3. MLL contains a repression domain. Cells were trans-fected, and CAT protein expression was determined as described inMaterials and Methods. (A) HeLa cells or NIH 3T3 (3T3) cells weretransiently cotransfected with 10 pg ofGAL4stkCAT, 5 tg ofinternalreference plasmid, and 10 pg of G4-MLL11O1 or 10 pg of pSG424.Fold repression, calculated relative to the amount of CAT proteinproduced in cells transfected~with pSG424, is plotted as the averagefold repression plus SD where n = at least three independentexperiments. (B) Titration ofrepression by G4-MLL11O1. HeLa cellswere transiently transfected with 0, 1, 5, 10, or 15 pg of G4-MLL11O1, 10 pg ofGAL45tkCAT, 5 pig ofpCH11O internal standardplasmid, and salmon sperm DNA to bring the total amount ofG4-MLL11O1 plus salmon sperm DNA to 15 pg.

of nonradiolabeled cruciform DNA (Fig. 4A, lanes 4 and 12).Neither of the other proteins bound cruciform DNA (Fig. 4A,lanes 5-8 and 13-16), even though comparable amounts ofprotein were assayed in each case. To confirm that the MLLAT-hook domain was recognizing structure rather than se-quence ofthe DNA, two duplexDNAs that together containedall ofthe sameDNA sequence as in the cruciform 1 probe wereused as probes. The MLL AT-hook domain was not able tobind either duplex DNA (Fig. 4B, lanes 1 and 8). In contrast,theMLL repression domain was able to bind weakly to duplex1A DNA (Fig. 4B, lane 3) and efficiently to duplex 1B DNA(Fig. 4B, lane 10). However, this binding was not blocked bycompetition with a 100-fold molar excess of nonradiolabeledduplexDNA (Fig. 4B, lanes 4 and 11). Therefore, the AT-hookdomain of MLL can specifically recognize and bind to acruciform DNA structure.

DISCUSSIONTranslocations involving the MLL gene on chromosome 11band q23 have been observed in many cases of acute lym-phoblastic leukemia and acute myeloid leukemia, particularlyin infants and in therapy-related leukemias that occur aftertreatment of a primary neoplasm with topoisomerase IIinhibitors. The MLL gene has been cloned and sequenced byseveral groups (4-9, 24, 25), and regions ofhomology to otherproteins have been identified (8, 9, 24) that include homologyto a DNA methyltransferase (26) and two potential DNA-binding motifs: AT-hooks (12) and the Drosophila trithorax

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FiG. 4. Cruciform DNA binding activity of the MLL AT-hookdomain. Proteins were expressed in bacteria, purified, and tested bygel mobility shift analysis for their ability to bind radiolabeled DNAprobes. Protein-DNA complexes were formed in the presence (+) orabsence (-) of a 100-fold molar excess of cold competitor DNA. Freeprobe is indicated by an arrow. (A) Proteins were incubated withradiolabeled cruciform 1 (lanes 1-8) or cruciform 2 (lanes 9-16)probes. Proteins assayed were MLL(142-400) (lanes 1, 2, 9, and 10),MLL(1101-1400) (lanes 3, 4, 11, and 12), MLL(3114-3357) (lanes 5,

6, 13, and 14), and Egr-1(281-304) (lanes 7, 8, 15, and 16). (B) Proteinswere incubated with either radiolabeled duplex 1A DNA (lanes 1-7)whose sequence corresponds to half of the sequence contained incruciform 1 DNA or with duplex 1B DNA (lanes 8-15) whosesequence corresponds to the other half of the sequence contained incruciform 1 DNA. Proteins assayed were MLL(142-400) (lanes 1, 2,8, and 9), MLL(1101-1400) (lanes 3, 4, 10, and 11), MLL(3114-3357)(lanes 5, 6, 12, and 13), and Egr-1(281-304) (lanes 7, 14, and 15).

(trx) zinc fingers (11) (see Table 1). There is also a similarityin structure and organization to the trx gene (7-9, 11). The trxgene regulates expression of Drosophila genes in the anten-napedia and bithorax complexes during development, andmutations in trx result in homeotic transformations (27). It

has been suggested that trx is a transcription factor that bindsto DNA (10) and that it is likely that MLL is also a tran-scription factor (7-9). However, no experiments have beendone that demonstrate the ability of MLL either to affecttranscription or to bind DNA. Here we present data demon-strating that two domains ofMLL can regulate transcriptionand one domain can recognize and specifically bind DNA.MLL contains a potent activation domain from amino acids

2340 to 3123 that is somewhat redundant in its activity (Figs.1 and 2). Several classes of activation domains have beendescribed that each contain an abundance of certain aminoacids, such as the serine/threonine-rich domains in Pit-1/GHF-1 (28). For other activation domains, however, no motifhas been defined. The activation domain of MLL is serine/threonine-rich: amino acids 2340-2771 and 2772-3123 are20%o and 24% serine/threonine, respectively (see Table 1).However, the whole MLL protein is serine/threonine-rich,containing 20%o of these amino acids. Therefore, the aminoacid composition of this region is not necessarily responsiblefor the activation activity. It has been suggested that serine/threonine-rich domains may be phosphorylated and thusresemble acidic activation domains (29). It is not yet knownif MLL protein is phosphorylated or if the phosphorylationstate of the activation domain determines its activity.We have also identified a repression domain in MLL

(amino acids 1101-1400) that repressed transcription from areporter construct in two different types of cells and in atitratable manner (Fig. 3). This region of MLL containshomology to a DNA methyltransferase (26) and is alsoproline- and lysine-rich (15% and 17%), respectively). Mech-anisms by which transcriptional repression is mediated arenot well defined, but protein-protein interactions do play arole in some cases. It is interesting that the methyltransferasehomologous region within the MLL repression domain isthought to be involved in protein-protein interactions thatactivate enzymatic function, rather than being the catalyticsite (26). Perhaps the MLL repression domain could functionvia protein-protein interactions within this region.The MLL protein seems to be a member of a family of

bifunctional transcription factors that contain both activationand repression domains. This family includes YY1/NF-E1/8(30) and Egr-1 (20). These factors may act as either activatorsor repressors of transcription, depending on their associationwith various other cellular proteins. Because of its large size,we have been unable to express the full-length MLL cDNA ina vector; therefore, we do not know whether the whole proteinhas transcriptional activity. It also remains to be determinedwhetherMLL is capable ofacting as either an activator and/ora repressor of transcription in vivo. The target genes of thistranscriptional regulation are presently unknown.

It had been proposed that the AT-hook region of MLLwould bindDNA because of its homology to the AT-hooks ofthe HMG-I(Y) proteins (12). We found that this region ofMLL could bind to cruciform DNA (Fig. 4A) as has beenshown for the HMG box of the HMG1/2 proteins (13, 23).This binding is specific for DNA structure rather than DNAsequence because binding was not observed in duplex DNAsof the same sequence (Fig. 4B). Two types of natural cruci-form structures occur: Holliday junctions formed duringrecombination (31) and the structures formed when invertedrepeats are extruded from superhelical DNAs. These struc-tures may be associated with initiation of mammalian DNAreplication (32) and are also commonly found in the vicinityof structural genes, so they might play a role in gene expres-sion (23). As has been suggested for HMG1 protein bindingto DNA (33), the AT-hook region ofMLL may also recognizesharp bends in DNA that are not necessarily part of acruciform structure. It is unclear what role the AT-hookbinding to DNA may play in vivo, but it is conceivable that

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FIG. 5. Schematic representation of the normal MLL cDNA on chromosome 11 and an MLL fusion cDNA on the der(11) chromosome aftera translocation event. The figure is drawn to scale, and the positions of pertinent MLL amino acids are indicated by numbers. The AT-hookDNA-binding motif and the potential DNA-binding zinc fingers are identified. The locations of the 0.8-kb breakpoint cluster region (BCR) andthe activation and repression domains are also shown.

it may recognize cruciform and/or bent DNA and therebyaffect gene regulation.

In translocations involving MLL, almost all of the identi-fied breakpoints are clustered within the central region ofMLL (34), which includes amino acids 1250-1630 (Fig. 5). Itis likely that the der(11) protein is responsible for effecting theleukemia in patients with MLL translocations because theder(11) contains the conserved junction in complex three-way translocations (14). Moreover, in about 25% of patientsstudied, the translocated telomeric region ofMLL is deleted(16). Translocations in this region would split the activationdomain from the der(11) protein, which would retain DNA-binding ability and a repression domain. The AT-hook do-main retained on the der(11) could potentially still recognizea DNA target, but probably a different one than is normallyrecognized by the intact MLL protein containing both DNA-binding domains. Dissociating a strong activation domainfrom the rest of the protein could also be involved in theleukemogenic process.

Several translocation partners of MLL have been identi-fied. Two of them, AF9 and ENL (8), share some regions ofhomology (35). Although there is no clear homology to otherfusion partners, they seem to be serine/proline-rich. Whetherthese fusion partners contribute activation domains or otherfunctional domains to the new fusion protein remains to bedetermined.We thank Mark Ptashne, Jim Lillie, Tom Shenk, Vikas Sukhatme,

and Jack Dixon for plasmids. This research was supported in part bygrants from the National Institutes of Health (CA42557 to J.D.R.)and from the Department of Energy (DE-FG02-86ER60408 toJ.D.R.). N.J.Z.-L. is a fellow of the Leukemia Society of America.

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