MLL: How complex does it get?

Journal of Cellular Biochemistry 95:234–242 (2005)

MLL: How Complex Does It Get?

Relja Popovic1 and Nancy J. Zeleznik-Le1,2*1Molecular Biology Program, Loyola University Medical Center, Maywood, Illinois2Department of Medicine, Molecular Oncology Program, Cardinal Bernardin Cancer Center,Loyola University Medical Center, Maywood, Illinois

Abstract The mixed lineage leukemia (MLL) gene encodes a very large nuclear protein homologous toDrosophilatrithorax (trx). MLL is required for the proper maintenance of HOX gene expression during development andhematopoiesis. The exact regulatory mechanism ofHOX gene expression by MLL is poorly understood, but it is believedthat MLL functions at the level of chromatin organization. MLL was identified as a common target of chromosomaltranslocations associatedwith human acute leukemias. About 50 differentMLL fusion partners have been isolated to date,and while similarities exist between groups of partners, there exists no unifying property shared by all the partners.MLLgene rearrangements are found in leukemias with both lymphoid and myeloid phenotypes and are often associated withinfant and secondary leukemias. The immature phenotype of the leukemic blasts suggests an important role forMLL in theearly stages of hematopoietic development.Mll homozygousmutant mice are embryonic lethal and exibit deficiencies inyolk sac hematopoiesis. Recently, two different MLL-containing protein complexes have been isolated. These and othergain- and loss-of-function experiments have provided insight into normal MLL function and altered functions of MLLfusion proteins. This article reviews the progress made toward understanding the function of the wild-type MLL protein.While many advances in understanding this multifaceted protein have been made since its discovery, many challengingquestions remain to be answered. J. Cell. Biochem. 95: 234–242, 2005. � 2005 Wiley-Liss, Inc.

Key words: MLL; leukemia

The mixed lineage leukemia (MLL) gene wasisolated as a common target of chromosomaltranslocations observed in human acute leuke-mias [Ziemin-van der Poel et al., 1991; Gu et al.,1992; Tkachuk et al., 1992; Thirman et al.,1993]. Cytogenetic analyses have identifiedmore than 50 different loci which translocatetoMLL, resulting in either acute lymphoid leu-kemia (ALL) or acute myeloid leukemia (AML)[Rowley, 1998]. Chromosomal translocationsinvolving MLL are predominant in infantleukemias and leukemias occuring in patients

previously treated with DNA topoisomerase IIinhibitors [Tkachuk et al., 1992; Thirman et al.,1993]. In each case, MLL is broken within an8.3 kb breakpoint cluster region (BCR) andexons encoding approximately 1,400 amino-terminal amino acids are fused in-frame to afusion partner gene. One of the interestingaspects of MLL-associated leukemias is thelarge number and the diversity of the fusionpartner genes. In general,MLL rearrangementis associated with a poor prognosis regardless ofthe fusion partner [Ayton and Cleary, 2001].Numerous studies have proposed different rolesfor MLL and fusion partner genes in leukemo-genesis. In this article, we look into some recentadvances in determining the normal MLLfunction and discuss some proposed models ofMLL-related leukemogenesis.

WILD-TYPE MLL FUNCTION

Sequence comparisons and genetic studieshave identified MLL as a functional ortholog ofthe Drosophila trithorax (trx) gene [Tkachuket al., 1992; Yu et al., 1995]. Trx, like MLL,

� 2005 Wiley-Liss, Inc.

Grant sponsor: National Institutes of Health (HIH)/National Cancer Institute (NCI); Grant number:CA40046; Grant sponsor: Dr. Ralph and Marian FalkMedical Research Trust.

*Correspondence to: Nancy J. Zeleznik-Le, Cardinal Ber-nardin Cancer Center, Loyola University Medical Center,2160 South First Avenue, 112-337, Maywood, IL 60153.E-mail: [email protected]

Received 21 December 2004; Accepted 22 December 2004

DOI 10.1002/jcb.20430

belongs to the Trithorax Group (trx-G) ofproteins, which are responsible for positivemaintenance of gene expression during devel-opment. Another evolutionarily conservedfamily of proteins, the Polycomb Group (Pc-G),antagonizes the function of trx-G proteins byacting in a repressive manner on the sametarget genes. Both groups are believed toregulate gene expression through an epigeneticmechanism [Orlando et al., 1998]. The best-studied downstream targets of MLL and trxfunction are the homeobox (HOX) genes, whichcontrol segment specificity and cell fate in thedeveloping embryo [Krumlauf, 1994]. In mam-malian cells, HOX genes are found in fourclusters on different chromosomes. The positionof each HOX gene within the cluster is con-served across species and corresponds to itsanterior-posterior expression pattern duringembryogenensis [Krumlauf, 1994].Targeted homozygous disruption of Mll in

mice is embryonic lethal and, similar to trxmutants in flies,Hox gene expression is initiat-ed but not maintained in these animals [Yuet al., 1998]. Lethality at dayE10.5 is associatedwith numerous defects in segmental identity. Inthese mice, theMll allele was disrupted in exon3, downstream from the AT-hook region (Fig. 1,¥) [Yu et al., 1995]. TargetingMll at exons 12–14 (Fig. 1, #), in the BCR, delayed lethality for acouple of days [Yagi et al., 1998]. Yolk sac andfetal liver hematopoiesis is perturbed in bothtypes of Mll mutants. Colony forming assayswith cells from these tissues show a greatlydecreased number, smaller size, and slowerproliferation in Mll null cells in comparison to

the wild-type [Hess et al., 1997; Yagi et al.,1998]. Surprisingly, embryonic stem (ES) cellshaving Mll disrupted in the third exonupstream from the AT-hook region showed anincreased proliferative capacity in similarassays (Fig. 1, §) [Fidanza et al., 1996]. It is stillunclear why these experiments produce vastlydifferent results, and while there is no evidencefor Mll expression in these cells, it is plausiblethat the differences come from the differenttargeting methods and the portions of Mllprotein expressed. Even animals carrying onlyone normal Mll allele show a phenotype thatdiffers from the wild-type mice. Mllþ/� miceare anemic and show growth retardation withspecific segment transformations [Yu et al.,1995]. The dramatic phenotype of the Mllheterozygous mice suggest that both copies ofthe gene are essential during development thusimplying haploinsuficiency as one of the possi-ble contributing mechanisms of MLL-asso-ciated leukemias.

While it is likely that MLL has many down-stream target genes, HOX genes have beenstudied the most extensively. Embryonic fibro-blasts derived from theMllmutantmice (Fig. 1,¥) show specific downregulation ofmore 50 Hoxaand Hoxc cluster genes (Hoxa5, -a7, -a9, -a10,-c4, -c5, -c6, -c8, -c9) [Hanson et al., 1999]. It isbelieved that Mll controls Hox gene expressionby binding to Hox regulatory elements andassociation with other transcriptional regula-tors. Indeed, MLL binding to the promoters ofHOXA9 and Hoxc8 was described recently[Milne et al., 2002; Nakamura et al., 2002].Overexpression of some Hox genes, includingHoxa9, has been linked to leukemia in mousemodels. Furthermore,HOXA9andHOXA10areoverexpressed in patient leukemia cells withMLL translocations [Ferrando et al., 2003].Together with the studies showing the role ofcertain HOX genes during normal hematopoi-esis, a case can be made that translocation ofMLL leads to the aberrant expression of itsdownstream targets generating the leukemicphenotype [Lawrence et al., 1996]. HOXA9 isnormally downregulated as blood cells differ-entiate from progenitors to more mature stagesof hematopoiesis. Overexpression of these Hoxgenes by some MLL fusion proteins may causean early arrest in hematopoietic differentiationleading to the immature phenotype associatedwith MLL leukemias. The MLL–ENL fusionprotein was shown to require the presence of

Fig. 1. Schematic of the MLL protein and fusion proteins thatresult from chromosomal translocations. The MLL proteinfunctional domains and the proteins they interact with areindicated above and below the diagram, respectively. AT-H, AT-hooks; MT/RD, DNA methyltransferase homology/repressiondomain; BCR, breakpoint cluster region; PHD, plant home-odomain; AD, transcriptional activation domain; SET, histonemethyltransferase domain. Arrow is Taspase 1 cleavage site.Symbols represent regions used for MLL targeting in differentstudies. See text for references on these studies.

MLL: How Complex Does It Get? 235

Hoxa7 and Hoxa9 genes for immortalization ofmouse bonemarrow progenitor cells [Ayton andCleary, 2003]. However, this may not be agenerality for all MLL fusions because MLL–GAS7 is able to transform hematopoietic pro-genitors in the absence ofHoxa7 andHoxa9 [Soet al., 2004]. An alternative explanation forthese discrepant results may be the use of dif-ferent starting populations of progenitor cells.TheMLL–ENL study used 5-fluorouracil-mobi-lized, lineage-depletedprogenitors,whereas theMLL–GAS7 study employed positive selectionwith the c-Kit marker.

MANY PIECES OF THE PUZZLE

MLL translocations lead to the formation offusion proteins where the amino-terminus ofMLL is fused in-frame to the carboxyl-terminusof the translocation partner. Many fusionpartners contain domains that can function astranscriptional activators in reporter geneassays, whereas others contain oligomerizationdomains [Ayton andCleary, 2001]. In both casesit is possible to suggest that a gain-of-functionby the fusion proteins may lead to leukemogen-esis. However, lack of Mll causes defects inembryonic hematopoiesis indicating that thepresence ofnormalMll is absolutely required forproper hematopoietic development. Chromoso-mal translocations cause a loss of several MLLfunctional domains that may be required for itsproper function (Fig. 1).

The most conserved region between trx andMLL is the C-terminal SET domain, alsopresent in numerous chromatin-associated pro-teins. The SET domain was named for Droso-phila founding members containing a region ofhigh sequence similarity: Su-(var)3–9, enhan-cer of zeste, and trx. Many SET-containingproteins have been identified and a number ofthem were shown to possess intrinsic histonemethyltransferase activity [Yeates, 2002].Methylation of the lysine residues on histonetails is part of the proposed histone code inwhich different histone modifications direct therecruitment of transcriptional regulators, ulti-mately exerting an influence on gene expression[Jenuwein and Allis, 2001]. While the initialstudies failed to confirm such enzymatic func-tion in theMLL SET domain, more recently twogroups have shown that mouse and humanMLLSET domains are indeed able tomethylatelysine residues on histone tails [Milne et al.,

2002; Nakamura et al., 2002]. The specificity ofthe MLL SET domain is for lysine 4 (K4) onhistone H3. K4 on histone H3 can be mono-, di-,or tri-methylated and these marks are usuallypresent at transcriptionally active chromatinregions. In yeast, tri-methylation of H3 K4 isassociated with actively expressed genes whiledi-methylation is believed to be present at allaccessible genes [Santos-Rosa et al., 2002]. It ispossible that MLL regulates its target genes byproviding the tri-methylation modification.Since no histone demethylases have beenidentified to date, tri-methylation of lysineresidues could persist over time and present away for regulating gene expression even onceMLL dissociates from the target genes.

Besides binding to the core histones, the SETdomain of MLL interacts with several differentproteins. One of them is the MLL SET domainitself, showing the ability to homo-dimerize[Rozovskaia et al., 2000]. Another protein thatinteracts with the carboxyl-terminus of MLL ishSNF5, a member of the SWI/SNF proteincomplex that functions in chromatin remodel-ing [Rozenblatt-Rosen et al., 1998]. Binding ofMLL to chromatin-associated proteins supportsthe idea that wild-type MLL regulates targetgene expression at the level of chromatinthrough interactions with other transcriptionalregulators.

A region just upstream of the SET domain isalso lost in the MLL fusion proteins caused bychromosomal translocations. Using GAL4–MLL chimeras, it was shown that this regionof the MLL protein contains a transcriptionalactivation domain [Zeleznik-Le et al., 1994].This activation domain binds to theCREB-bind-ing protein (CBP), a known regulator of geneexpression [Ernst et al., 2001]. CBP controlsgene expression by both mediating recruitmentof transcriptional activators, and by its intrinsichistone acetyltransferase (HAT) activity. Thebinding of MLL and CBP is direct and wasshown to be necessary for MLL-mediatedtranscriptional activation. All MLL mutantsunable to bind CBP have a drastic reduction intheir ability to activate expression of a reportergene. Interestingly, CBP is also one of the MLLtranslocation partners, and the MLL–CBPfusion protein retains theHAT activity believedto be important for the transcriptional activa-tion [Sobulo et al., 1997]. There are many otherMLL fusion partners that contain transcrip-tional activation domains which are retained in

236 Popovic and Zeleznik-Le

the chimeric proteins. The presence of theseactivation domains in leukemic cells suggests apossible gain-of-function mechanism for MLL-associated leukemias. The presence of a potenttranscriptional activation domain may lead tooverexpression of MLL downstream targets,causing differentiation arrest and the leukemo-genic phenotype. However, a number of MLLfusion partners are not involved in transcrip-tional regulation suggesting that there is moreto the model than this simple explanation.Recent studies have shown that MLL under-

goes proteolytic processing shortly after trans-lation and that this cleavage divides the proteininto a largerN-terminal portion (320 kDa) and asmaller C-terminal part (180 kDa) [Yokoyamaet al., 2002; Hsieh et al., 2003b]. The enzymeresponsible for this processing is Taspase 1, andtwo cleavage sites are located just upstream ofthe activation domain [Hsieh et al., 2003a](Fig. 1). Enzymatic function of Taspase 1 is re-quired for proper HOX gene expression imply-ing thatMLLprocessing is absolutely necessaryfor its proper function. The two fragmentsgenerated by the cleavage reassociate non-covalently and this interaction confers stabilityto the N-terminal portion and correct localiza-tion to the C-terminal part [Hsieh et al., 2003b].Chromosomal translocations prevent this inter-action from happening, suggesting that onepossible role of the fusion partners may be toprovide stability for the MLL N-terminal frag-ment. However, myc fusion with MLL does notlead to leukemia, indicating that stability of theMLL N-terminal portion is not sufficient forleukemogenesis [Corral et al., 1996].The region of MLL where the breakpoints

occur in patient samples overlaps a group ofspecific zinc fingers called plant homeodomain(PHD). The PHD fingers are the second mostconserved domain between MLL and trx. ThePHD fingers are found in a large number ofchromatin-associated proteins [Aasland et al.,1995]. MLL has four PHD zinc fingers with aregion of homology to bromodomains foundbetween the third and the fourth finger. Pro-teins containing bromodomains have beenimplicated in gene transcription through chro-matin remodeling as they were shown to bindacetylated lysines on histone tails [Dhalluinet al., 1999]. It is possible that the MLL bromo-domain serves a similar function and helpsin targeting the protein to regions of DNAenriched in acetylated histones, although this

function has not been demonstrated. BecauseMLLhas an atypical bromodomain, it could alsohave other, so far unknown, functions. MLLPHD fingers have been shown to function inprotein–protein interactions. The second PHDfinger is required for homodimerization and thethird one was shown to bind the cyclophilinCyp33 [Fair et al., 2001]. Both homodimeriza-tion and Cyp33 interaction are conserved inDrosophila. Cyp33 binds AU-rich RNA se-quence and carries cis/trans prolyl-isomeraseactivity.MLL andCyp33 co-localize, suggestingthat MLL binding may target Cyp33 to specificsubnuclear locations. It is interesting thatoverexpression of Cyp33 results in downregula-tion of some MLL downstream HOX targetgenes. The same effect is not observed in cellsthat do not have the MLL PHD zinc fingerregion intact indicating that Cyp33 affectsMLLtarget genes through its binding to this region.The molecular mechanism of this regulation isnot yet fully understood.

TheN-terminal region ofMLL that is retainedin the fusionproteins encompasses theAT-hooksand transcriptional repression domains. MLLcontains three AT-hook motifs which haveDNA binding activity, specific for AT-rich DNA[Zeleznik-Le et al., 1994]. These domains do notrecognize a specific sequence but rather astructure of cruciform or bent DNA. The MLLAT-hook region was shown to interact with SETprotein (not to be confused with SET domain),protein phosphatase 2A (PP2A), and the pro-apoptotic protein GADD34 [Adler et al., 1997;Adler et al., 1999]. MLL fusion proteins, but notthe wild-type MLL, inhibit GADD34-inducedapoptosis suggesting that this may be animportant factor in MLL-associated leukemias[Adler etal., 1999]. It isunclearwhat role theAT-hooks play in MLL gene regulation, but it ispossible that they aid in the targeting of MLL tospecific genomic region by recognizing theseDNA structures. The MLL repression domaincontains a regionwith homology toDNAmethyl-transferase 1 (DNMT1), also termed the ‘‘CXXC’’domain. The CXXC domain was shown to bindCpG-rich DNA sequences with a preference fornon-methylated DNA [Birke et al., 2002]. Pre-sence of the CXXC domain is required for MLL–ENL immmortalization of myeloid progenitors[Slany et al., 1998]. Recently, the MLL repres-sion domain was shown to interact with histonedeacetylases (HDACs) and to recruit polycombproteins HPC2 and BMI-1, and the co-repressor


protein CtBP [Xia et al., 2003]. Polycombproteins are known functional antagonists ofthe trx-G family.Unlike theactivationdomain intheC-terminus ofMLL, the repression domain isretained in the leukemic fusion proteins. Pre-sence of both of these domains in the wild-typeprotein makes for an attractive hypothesis thatthe regulated balance between the two tran-scriptional functions is important for properMLL function. Thus, loss of the SET domainand the strong activation domain may disturbthis balance leading to aberrant MLL functionsand eventually leukemic transformation.

THE STORY IS TWO COMPLEX

The presence of transcription regulating do-mains within MLL and their interactions withother transcriptional activators and repressorssuggests that many different proteins may beinvolved in mediating the regulatory role ofMLL. Recently two different MLL containingprotein complexes were purified [Nakamuraet al., 2002; Yokoyama et al., 2004]. The firststudy identified a very large complex comprisedof more than 29 proteins associated with MLL[Nakamura et al., 2002]. The complex formationis not mediated by DNA as intercalation ofethidium bromide did not disrupt the proteininteractions. A large number of the 29 proteinsbelong to two HDAC containing complexes,NuRD and SIN3. Both of these complexes havethe same core components (HDAC1/2, RbAp46,and RbAp48) which function in histone bindingand deacetylation of lysine residues on histonetails [Ahringer, 2000]. The NuRD and SIN3complexes diverge in other complex componentsand complex associating proteins. Mi-2, foundin this MLL complex, is another member of theNuRD complex and contains ATP-dependentnucleosome remodeling activity. Other proteinsfound in the MLL complex also have this chro-matin remodeling potential. BRM, BAF170,BAF155, and INI1 associate with MLL and areall components of the SWI/SNFATP-dependentnucleosome remodeling complex [Wang et al.,1996]. A number of proteins found in this MLLcomplex are members of the TFIID complex.TFIID is involved in promoter recognition andfunctions in the recruitment of transcriptionactivators to the RNA polymerase core complex[Lemon and Tjian, 2000]. TFIID is also involvedin the recruitment of proteins necessary forRNA processing, some of which are also found

associated with MLL. Finally, WDR5 andRBBP5, proteins that contain WD40 protein–protein interaction domains are also found intheMLL complex.WDR5andRBBP5 are homo-logs of Swd1 and Swd2 respectively,members oftheyeastSet1protein complex [Yokoyamaetal.,2004]. The Set1 protein contains a SET domain,which is highly homologous to the SET domainof MLL. Interestingly, just like the MLLprotein, the Set1 complex was shown to methy-late lysine 4 on histone H3 [Nagy et al., 2002].This purified MLL complex is able to performchromatin remodeling, histone methylation,acetylation, and deacetylation suggesting thatall the associated proteins retain their enzy-matic activities [Nakamura et al., 2002]. All thecomplex components bind to the promoter ofHOXA9 only whenMLL is present, showing theMLL-dependence of the whole supercomplexbeing targeted to the target gene region. Also,because the complex formation is not DNAdependent, it is possible that the assembledcomplex is recruited to the MLL-dependentregulatory elements as a whole.

A second, much smaller, MLL complex wasisolated by Yokoyama et al. [2004]. It resemblesthe above-mentioned yeast and mammalianSet1 complexes as the majority of the Set1complex components are also found interactingwith MLL. The only unifying members of thetwo different MLL complexes are RBBP5 andWDR5 proteins. RBBP5 and WDR5, along withASH2L, interact with the SET domain of MLL.This binding is also observed in other Set1complexes and is required for the histonemethyltranferase activity [Nagy et al., 2002].It is of interest that a close MLL homolog,MLL2, was also shown to interact with ASH2L,RBBP5, and WDR5. MLL2 also interacts withDPY-30 another component of the yeast Set1complex [Hughes et al., 2004]. HCF, also foundin the human but not the yeast Set1 complex,associates with MLL through its Kelch domain.HCF is known to interact with SET1 and SIN3regulatory complexes, however no recruitmentof SIN3 components to this MLL complex wasdetected [Yokoyama et al., 2004]. The lastcomponent of this MLL complex is menin, alsofound to interact with MLL2 [Hughes et al.,2004]. Menin is a tumor suppressor and muta-tions in this gene have been implicated inmultiple endocrine tumors [Chandrasekhar-appa et al., 1997]. Similar to the AT-hooksof MLL, menin can bind to various DNA


structures in a sequence-independent manner[La et al., 2004]. Menin interacts with the N-terminus of MLL, which suggests that it has apotential to interact with both the wild-typeMLL and the MLL fusion proteins [Yokoyamaet al., 2004]. Indeed, menin does interact withthe MLL–ENL fusion protein. Interestingly,menin is required for efficient HOXA9 expres-sion, and it also binds to theHoxc8 enhancerandpromoter region [Hughes et al., 2004]. Mllbinding to the same Hoxc8 promoter sequencehas been described [Milne et al., 2002]. It is stillunclear what may be the role for menin in MLLtranscriptional regulation or MLL-associatedleukemogenesis andmore studies are needed toanswer this question.

PROSPECTS

Transcriptional regulation byMLL is provingto be a very complex phenomenon. To fullyunderstand the role of wild-type MLL in devel-opment and hematopoiesis, a number of experi-mental systems will need to be analyzed. Whiledisruption of MLL function inhibits growth anddifferentiation of hematopoietic cells in mostsystems, the presence of oncogenic MLL fusionproteins increases their proliferative capacity.BecauseMLL is believed to function at the levelof chromatin organization and its SET domainpossesses intrinsic histone methyltransferaseactivity specific for H3 K4, it is important todetermine if and how different the chromatinstructure of the target genes is in thepresence offusion proteins. The MLL fusion proteins lackthe SETdomainwhich should preventmethyla-tion of H3 K4, a marker associated withactive gene transcription. It is still unclearhow the levels of di- and tri-methylation of thisresidue influence gene expression and if tri-methylation is absolutely required for tran-scription. The majority of HOX genes aredownregulated as hematopoietic cells progressfrom progenitors to a fully differentiated stage.It is possible that, first the increase, and thenthe decrease in gene expression are both me-diated by MLL through interactions with alter-nate complexes. InDrosophila, trxwas shown tobind the regulatory elements of its downstreamtarget genes even when they are not expressed.The same may be true for MLL, and binding oftranscriptional activators and repressors mayfunction in shifting the balance between geneexpression and gene repression. The interaction

of MLL with proteins such as CBP and HDACssuggest an additional potential mechanism bywhich MLL function could be regulated. Anumber of transcription factors are regulatedthroughposttranslationalmodifications suchasphosphorylation or acetylation. It is possiblethat the same type of modifications on specificMLL residues are important determinants of itsfunction. Similarly, Cyp33 may alter MLLstructure/function through its cis/trans prolyl-isomerase enzymatic activity. While these pro-tein interactions may influence normal MLLfunction, it is still not understood how MLL istargeted to the regulatory elements of its HOXgene targets. It is likely that MLL recruitmentinvolves interactions with specific DNA struc-tures as well as interactions with DNA-asso-ciated proteins.One suchproteinmaybemenin,a tumor suppressor which, similar to the AT-hook region of MLL, was shown to bind cruci-form DNA. Menin binds to the N-terminus ofMLL and through this interaction would beable to recruit the wild-type or the oncogenicfusion protein. Interestingly, menin is requiredforHox gene expression adding further supportto the idea that this protein helps in therecruitment of MLL. MLL may also bind tonon-methylated CpG sequences in regulatoryregions of its target genes. The CpG DNAbinding activity of the MLL repression domainmay mediate this in vivo, although only in vitroexperiments have been done to date.

Purification of two different MLL complexesraises a number of interesting questions. Bothcomplexes were isolated from theK562 cell line,therefore differences between the two com-plexes cannot likely be attributed to differentcell types. However, since MLL is expressed inthe majority of tissues, the question arises whe-ther all cell types have both of these complexesor whether different cell types have their ownversions of either complex. Because a number ofproteins in the larger MLL complex eitherbelong to a family of transcriptional repressorsor co-activators, this complex could have bothactivating and repressive functions with onefunctional outcome dominating on a particulartarget gene in a given cell type at a given time.The smaller Set1-like complex would likely beassociated with transcriptional activationbecause in yeast Set1 is found at transcriptio-naly active loci [Ng et al., 2003]. It is unknown ifthe two protein complexes function on the sameor different target genes. In either case, they


may or may not be present in the cells at thesame time. It is possible that the two complexesfunction at two different stages of transcrip-tional regulation. In this model (Fig. 2), thelarge MLL complex would be recruited to thepromoters of MLL downstream target genes bysome as yet unknownmechanism. One outcomecould include MLL recruitment of HDACs andrepressor complexes, causing silencing of thedownstream targets. This would suggest, simi-lar to trx in Drosophila, that MLL is bound tothe regulatory elements of its target genes evenwhen these genes are not expressed. On theother hand, under the appropriate conditions,the SWI/SNF components of the larger complexcould alter the chromatin structure in a waythat allows for binding of transcriptional co-activators to the RNA polymerase core complex,ultimately leading to gene expression. OnceRNA transcription is initiated, the Set1-likeMLL complex could be recruited allowing tri-

methylation of lysine 4 on histone H3 in theopen reading frame (ORF). This is similar to theyeast Set1 complex which is targeted to the 50

coding regions of transcriptionally active geneswhere it catalyzes methylation of the samehistone H3 residue. In yeast, Set1 recruitmentdepends on phosphorylation of the RNA poly-merase II CTD at serine 5. Phosphorylation ofPolII at this residue is a mark for transitionfrom transcriptional initiation to elongation.Comparable to yeast, the MLL Set1-like com-plexmay be required for the elongation phase ofgene expression. If expression of HOX genesdepends on the recruitment of MLL complexesand methylation of histone residues, it is notwell understood how themajority ofMLL fusionproteins, which lack the histone methyltrans-ferase SET domain, accelerate HOX geneexpression. In this case, an active HAT activityrecruited by the fusion may be able to preventsilencing of the target genes. It is also possible

Fig. 2. Model for transcriptional regulation mediated by MLL-containing complexes. Two MLL-containing protein complexeshave been identified. The larger supercomplex contains mem-bers of co-repressor and co-activator protein families. The effecton downstream targets may be silencing (a) or activating (b),depending which members of the supercomplex are present at a

given time. The MLL Set1-like complex (b-bottom diagram) maybe involved in the elongation process and may only be recruitedfollowing phosphorylation of RNA polymerase II. This complexfacilitates methylation of K4 on histone H3 in the ORF, leaving amark of transcriptionally active genes.


that partners provide the ability to recruitelongation function, either via methylationactivity or by some other means.In summary, much work has been done in an

effort to understand transcriptional regulationby MLL. The regulation of HOX genes by MLLpresents a great model for unraveling the com-plexities of how gene expression is controlledthrough changes in chromatin structure.Understanding how normal MLL functions willalso facilitate comprehending MLL-associatedleukemias in order to develop successful MLL-directed therapies.

ACKNOWLEDGMENTS

The authors apologize to colleagues whosework we failed to recognize due to spacelimitations.

REFERENCES

Aasland R, Gibson TJ, Stewart AF. 1995. The PHD finger:Implications for chromatin-mediated transcriptional reg-ulation. Trends Biochem Sci 20:56–59.

Adler HT, Nallaseth FS,Walter G, TkachukDC. 1997. HRXleukemic fusion proteins form a heterocomplex with theleukemia-associated protein SET and protein phospha-tase 2A. J Biol Chem 272:28407–28414.

Adler HT, Chinery R, Wu DY, Kussick SJ, Payne JM,Fornace AJ, Jr., Tkachuk DC. 1999. Leukemic HRXfusion proteins inhibit GADD34-induced apoptosis andassociate with the GADD34 and hSNF5/INI1 proteins.Mol Cell Biol 19:7050–7060.

Ahringer J. 2000. NuRD and SIN3 histone deacetylasecomplexes in development. Trends Genet 16:351–356.

Ayton PM, Cleary ML. 2001. Molecular mechanisms ofleukemogenesis mediated by MLL fusion proteins. Onco-gene 20:5695–5707.

Ayton PM, Cleary ML. 2003. Transformation of myeloidprogenitors by MLL oncoproteins is dependent on Hoxa7and Hoxa9. Genes Dev 17:2298–2307.

Birke M, Schreiner S, Garcia-Cuellar MP, Mahr K,Titgemeyer F, Slany RK. 2002. The MT domain of theproto-oncoprotein MLL binds to CpG-containing DNAand discriminates against methylation. Nucleic AcidsRes 30:958–965.

Chandrasekharappa SC, Guru SC, Manickam P, OlufemiSE, Collins FS, Emmert-Buck MR, Debelenko LV,Zhuang Z, Lubensky IA, Liotta LA, Crabtree JS, WangY, Roe BA, Weisemann J, Boguski MS, Agarwal SK,Kester MB, Kim YS, Heppner C, Dong Q, Spiegel AM,Burns AL, Marx SJ. 1997. Positional cloning of the genefor multiple endocrine neoplasia-type 1. Science 276:404–407.

Corral J, Lavenir I, Impey H,Warren AJ, Forster A, LarsonTA, Bell S, McKenzie AN, King G, Rabbitts TH. 1996. AnMll-AF9 fusion gene made by homologous recombinationcauses acute leukemia in chimeric mice: A method tocreate fusion oncogenes. Cell 85:853–861.

Dhalluin C, Carlson JE, Zeng L, He C, Aggarwal AK, ZhouMM. 1999. Structure and ligand of a histone acetyl-transferase bromodomain. Nature 399:491–496.

Ernst P, Wang J, Huang M, Goodman RH, Korsmeyer SJ.2001. MLL and CREB bind cooperatively to the nuclearcoactivator CREB-binding protein. Mol Cell Biol 21:2249–2258.

Fair K, Anderson M, Bulanova E, Mi H, Tropschug M, DiazMO. 2001. Protein interactions of the MLL PHD fingersmodulate MLL target gene regulation in human cells.Mol Cell Biol 21:3589–3597.

Ferrando AA, Armstrong SA, Neuberg DS, Sallan SE,Silverman LB, Korsmeyer SJ, Look AT. 2003. Geneexpression signatures in MLL-rearranged T-lineage andB-precursor acute leukemias: Dominance of HOX dysre-gulation. Blood 102:262–268.

Fidanza V, Melotti P, Yano T, Nakamura T, Bradley A,Canaani E, Calabretta B, Croce CM. 1996. Doubleknockout of the ALL-1 gene blocks hematopoieticdifferentiation in vitro. Cancer Res 56:1179–1183.

Gu Y, Nakamura T, Alder H, Prasad R, Canaani O, CiminoG, Croce CM, Canaani E. 1992. The t(4;11) chromosometranslocation of human acute leukemias fuses the ALL-1gene, related to Drosophila trithorax, to the AF-4 gene.Cell 71:701–708.

Hanson RD, Hess JL, Yu BD, Ernst P, van Lohuizen M,Berns A, van der Lugt NM, Shashikant CS, Ruddle FH,Seto M, Korsmeyer SJ. 1999. Mammalian trithorax andpolycomb-group homologues are antagonistic regulatorsof homeotic development. Proc Natl Acad Sci USA 96:14372–14377.

Hess JL, Yu BD, Li B, Hanson R, Korsmeyer SJ. 1997.Defects in yolk sac hematopoiesis in Mll-null embryos.Blood 90:1799–1806.

Hsieh JJ, Cheng EH, Korsmeyer SJ. 2003a. Taspase1: Athreonine aspartase required for cleavage of MLL andproper HOX gene expression [see comment]. Cell 115:293–303.

Hsieh JJ, Ernst P, Erdjument-Bromage H, Tempst P,Korsmeyer SJ. 2003b. Proteolytic cleavage of MLLgenerates a complex of N- and C-terminal fragmentsthat confers protein stability and subnuclear localization.Mol Cell Biol 23:186–194.

Hughes CM, Rozenblatt-Rosen O, Milne TA, Copeland TD,Levine SS, Lee JC, Hayes DN, Shanmugam KS,Bhattacharjee A, Biondi CA, Kay GF, Hayward NK,Hess JL, Meyerson M. 2004. Menin associates with atrithorax family histone methyltransferase complex andwith the hoxc8 locus. Mol Cell 13:587–597.

Jenuwein T, Allis CD. 2001. Translating the histone code[see comment]. Science 293:1074–1080.

Krumlauf R. 1994. Hox genes in vertebrate development.Cell 78:191–201.

La P, Silva AC, Hou Z,WangH, Schnepp RW, YanN, Shi Y,Hua X. 2004. Direct binding of DNA by tumor suppressormenin. J Biol Chem 279:49045–49054.

Lawrence HJ, Sauvageau G, Humphries RK, Largman C.1996. The role of HOX homeobox genes in normal andleukemic hematopoiesis. Stem Cells 14:281–291.

Lemon B, Tjian R. 2000. Orchestrated response: Asymphony of transcription factors for gene control. GenesDev 14:2551–2569.

Milne TA, Briggs SD, Brock HW,MartinME, Gibbs D, AllisCD, Hess JL. 2002. MLL targets SET domain methyl-


transferase activity to Hox gene promoters. Mol Cell 10:1107–1117.

Nagy PL, Griesenbeck J, Kornberg RD, Cleary ML. 2002. Atrithorax-group complex purified from Saccharomycescerevisiae is required for methylation of histone H3. ProcNatl Acad Sci USA 99:90–94.

Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia T,Wassell R, Dubois G, Mazo A, Croce CM, Canaani E.2002. ALL-1 is a histone methyltransferase that assem-bles a supercomplex of proteins involved in transcrip-tional regulation. Mol Cell 10:1119–1128.

Ng HH, Robert F, Young RA, Struhl K. 2003. Targetedrecruitment of Set1 histone methylase by elongating PolII provides a localized mark and memory of recenttranscriptional activity. Mol Cell 11:709–719.

Orlando V, Jane EP, Chinwalla V, Harte PJ, Paro R. 1998.Binding of trithorax and Polycomb proteins to thebithorax complex: Dynamic changes during early Droso-phila embryogenesis. Embo J 17:5141–5150.

Rowley JD. 1998. The critical role of chromosome transloca-tions in human leukemias. Annu Rev Genet 32:495–519.

Rozenblatt-Rosen O, Rozovskaia T, Burakov D, Sedkov Y,Tillib S, Blechman J, Nakamura T, Croce CM, Mazo A,Canaani E. 1998. The C-terminal SET domains of ALL-1and TRITHORAX interact with the INI1 and SNR1proteins, components of the SWI/SNF complex. Proc NatlAcad Sci USA 95:4152–4157.

Rozovskaia T, Rozenblatt-Rosen O, Sedkov Y, Burakov D,Yano T, Nakamura T, Petruck S, Ben-Simchon L, CroceCM, Mazo A, Canaani E. 2000. Self-association of theSET domains of human ALL-1 and of DrosophilaTRITHORAX and ASH1 proteins. Oncogene 19:351–357.

Santos-Rosa H, Schneider R, Bannister AJ, Sherriff J,Bernstein BE, Emre NC, Schreiber SL, Mellor J,Kouzarides T. 2002. Active genes are tri-methylated atK4 of histone H3. Nature 419:407–411.

Slany RK, Lavau C, Cleary ML. 1998. The oncogeniccapacity of HRX-ENL requires the transcriptional trans-activation activity of ENL and the DNA binding motifs ofHRX. Mol Cell Biol 18:122–129.

So CW, Karsunky H, Wong P, Weissman IL, Cleary ML.2004. Leukemic transformation of hematopoietic pro-genitors by MLL–GAS7 in the absence of Hoxa7 orHoxa9. Blood 103:3192–3199.

Sobulo OM, Borrow J, Tomek R, Reshmi S, Harden A,Schlegelberger B, Housman D, Doggett NA, Rowley JD,Zeleznik-Le NJ. 1997. MLL is fused to CBP, a histoneacetyltransferase, in therapy-related acute myeloid leu-kemia with a t(11;16)(q23;p13.3). Proc Natl Acad Sci USA94:8732–8737.

Thirman MJ, Gill HJ, Burnett RC, Mbangkollo D, McCabeNR, Kobayashi H, Ziemin-van der Poel S, Kaneko Y,

Morgan R, Sandberg AA, Chaganti RSK, Larson RA,LeBeauMM, DiazMO, Rowley JD. 1993. Rearrangementof the MLL gene in acute lymphoblastic and acutemyeloid leukemias with 11q23 chromosomal transloca-tions. N Engl J Med 329:909–914.

Tkachuk DC, Kohler S, Cleary ML. 1992. Involvement of ahomolog of Drosophila trithorax by 11q23 chromosomaltranslocations in acute leukemias. Cell 71:691–700.

Wang W, Cote J, Xue Y, Zhou S, Khavari PA, Biggar SR,Muchardt C, Kalpana GV, Goff SP, Yaniv M, WorkmanJL, Crabtree GR. 1996. Purification and biochemicalheterogeneity of the mammalian SWI–SNF complex.EMBO J 15:5370–5382.

Xia ZB, Anderson M, Diaz MO, Zeleznik-Le NJ. 2003. MLLrepression domain interacts with histone deacetylases,the polycomb group proteins HPC2 and BMI-1, and thecorepressor C-terminal-binding protein. Proc Natl AcadSci USA 100:8342–8347.

Yagi H, Deguchi K, Aono A, Tani Y, Kishimoto T, Komori T.1998. Growth disturbance in fetal liver hematopoiesis ofMll-mutant mice. Blood 92:108–117.

Yeates TO. 2002. Structures of SET domain proteins:Protein lysine methyltransferases make their mark. Cell111:5–7.

Yokoyama A, Kitabayashi I, Ayton PM, Cleary ML, OhkiM. 2002. Leukemia proto-oncoprotein MLL is proteolyti-cally processed into 2 fragments with opposite transcrip-tional properties. Blood 100:3710–3718.

Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ,Kitabayashi I, Herr W, Cleary ML. 2004. Leukemiaproto-oncoprotein MLL forms a SET1-like histonemethyltransferase complex with menin to regulate Hoxgene expression. Mol Cell Biol 24:5639–5649.

Yu BD, Hess JL, Horning SE, Brown GA, Korsmeyer SJ.1995. Altered Hox expression and segmental identity inMll-mutant mice. Nature 378:505–508.

Yu BD, Hanson RD, Hess JL, Horning SE, Korsmeyer SJ.1998. MLL, a mammalian trithorax-group gene, func-tions as a transcriptional maintenance factor in morpho-genesis. Proc Natl Acad Sci USA 95:10632–10636.

Zeleznik-Le NJ, Harden AM, Rowley JD. 1994. 11q23translocations split the ‘‘AT-hook’’ cruciform DNA-bind-ing region and the transcriptional repression domainfrom the activation domain of the mixed-lineage leuke-mia (MLL) gene. Proc Natl Acad Sci USA 91:10610–10614.

Ziemin-van der Poel S, McCabeNR, Gill HJ, Espinosa R III,Patel Y, Harden A, Rubinelli P, Smith SD, LeBeau MM,Rowley JD, DiazMO. 1991. Identification of a gene,MLL,that spans the breakpoint in 11q23 translocationsassociated with human leukemias. Proc Natl Acad SciUSA 88:10735–10739.


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MLL: How complex does it get?