Proc. Natl. Acad. Sci. USAVol. 91, pp. 6359-6363, July 1994Cell Biology

DNA-binding and trans-activation properties of Drosophila E2F andDP proteinsBRIAN DAVID DYNLACHT, ADAM BROOK, MARLENE DEMBSKI, LYNNE YENUSH, AND NICHOLAS DYSON*Laboratory of Molecular Oncology, Massachusetts General Hospital Cancer Center, Building 149, 13th Street, Charlestown, MA 02129

Communicated by Ed Harlow, January 27, 1994 (received for review December 8, 1993)

ABSTRACT The temporal activation of E2F ansp-tional activity appears to be an important component of themechanisms that prepare m an cells for DNA replica-tion. Regulation ofE2F actvity appears to be a highly complexprocess, and the dissection of the E2F pathway will be greatlyfaclitated by the ability to use genetic approaches. We reportthe isolation of two Drosophila genes that can stimulate E2F-dependent trunscription In Drosophila cells. One ofthese genes,dE2F, contains threedo that are highly conserved in thehuman homologs E2F-1, E2F-2, and E2F-3. Interestingly, oneof these domains Is highly homologous to the retinoblastomaprotein (RB)-binding sequences ofhuman E2F genes. The othergene, dDP, is closely related to the human DP-1 and DP-2genes. We demonstrate that dDP and dE2F interact andcooperate to give sequence-speifc DNA biding and optimaltraactivation. These features suggest that endogenous Dro-sophila E2F, like human E2F, may be composed of het-erodimers and may be regulated by RB-like proteins. Theisolation of these genes will provide important reagents for thegenetic analysis of the E2F pathway.

Several lines ofevidence indicate that the transcription factorE2F coordinates the expression of genes during the progres-sion from the G1 phase of the cell cycle into S phase.E2F-binding sites have been identified in the promoters ofseveral cellular genes whose products are required for cellproliferation (for a review, see refs. 1 and 2). In the c-myc,dihydrofolate reductase, c-myb, thymidine kinase, and cdc2promoters the E2F sites have been shown to be essential forthe transcriptional activation of these genes that occurs asserum-starved cells are stimulated to progress through thecell cycle (3-8). In this experimental system, a short elementcarrying two overlapping E2F sites has been shown to besufficient for the temporal expression of the dihydrofolatereductase gene at the Gj-to-S transition (9).Human E2F is a heterogeneous factor representing the

combined activity ofmany different gene products. Fourgeneshave been demonstrated to encode components of E2F calledE2F-1, E2F-2, E2F-3, and DP-1, and it is likely that additionalgenes exist (10-14). Using partially purified human E2F,Huber et al. (15) found that specific binding to E2F sites wasgreatly stimulated when different E2F fractions were com-bined. E2F-1 and DP-1 associate into stable complexes andactivate transcription in a cooperative manner (16, 17). It is notknown how many different E2F complexes exist or whetherthese multiple forms of E2F are functionally redundant.E2F-dependent transcription of several promoters has been

found to be repressed in cells that lack the retinoblastomaprotein (RB) by the overexpression of either the RB gene orthe related p107 gene (18-24). RB and p107 both associate withE2F in vivo and inhibit E2F by directly binding to a region ofthe protein important for trans-activation (refs. 17 and 24;B.D.D. and E. Harlow, unpublished results). RB, p107, and a

homologous protein, p130 (25), have been detected in DNA-bound E2F complexes at various points in the cell cycle. Thep107/E2F and p130/E2F complexes also contain cyclins (A orE) and Cdk2 and carry a potent kinase activity (25-32). Thetemporal appearance of these E2F complexes suggests thatthey each provide different elements ofE2F regulation, but theprecise roles of these complexes are unknown.To date, studies of E2F have relied primarily on a bio-

chemical approach. However, investigations into the overallrole ofE2F in the control ofproliferation and a determinationofthe functions of the E2F regulatory complexes will requirea combination ofbiochemical and genetic approaches. As thescope of genetic experiments in mammalian cells is limited,we wished to determine whether the E2F-containing growthcontrol pathway could be found in an organism that is moregenetically tractable. Here we report the isolation of twoDrosophila genes encoding E2F-related proteins and dem-onstrate that they share structural and functional character-istics with their human counterparts.t

MATERIALS AND METHODSLibrary Screening and Isolation of cDNAs. One million

plaques from a Drosophila AgtlO cDNA library (33) werescreened with DNA fragments that corresponded to theregions encoding aa 89-214 ofE2F-1, aa 85-200 ofE2F-2, andaa 132-270 of E2F-3. The probes were labeled with[a-32P]dCTP by random primer extension (34) and used inlow-stringency hybridization. Positive clones were plaquepurified and cDNA inserts were subcloned into pBluescriptSK (Stratagene) for sequencing. The inserts were digestedwith exonuclease III and S1 nuclease to generate nested setsof deletions, which were sequenced with Sequenase 2.0(United States Biochemical). To isolate dDP, the same li-brary was screened with a probe to the putative DNA-bindingdomain of DP-1 (17).

Plasmids. pBS-dE2F was made by subcloning a 4.4-kbEcoRI fragment (the entire cDNA) from A phage 16 intopBluescript SK(+). pBS-dE2F.ATG is a modified form ofpBS-dE2F constructed by use of PCR to delete the first 849bp of pBS-dE2F. pBS-dDP was made by subcloning theentire cDNA insert on an EcoRI fragment from A phage 3 intoEcoRI-cut pBluescript SK(+). The expression plasmid Act-PPA, the internal control plasmid copia-lacZ (35), and thechloramphenicol acetyltransferase (CAT) reporter (E2F)4-BCAT (17) used in transfection experiments have beendescribed. Act-dE2F and Act-dDP contain the entire codingregions of the genes.

Cell Culture and Transfections. Schneider line 2 (SL2) cells(36) (generously supplied by Jayne Kassel, MassachusettsGeneral Hospital Cancer Center, Boston) were maintained atroom temperature in Schneider's Drosophila medium and

Abbreviations: GST, glutathione S-transferase; CAT, chloramphen-icol acetyltransferase.*To whom reprint requests should be addressed.the sequences reported in this paper have been deposited in theGenBank data base (accession nos. X78421 and X79708).

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were transfected by a calcium phosphate method (37). Var-ious amounts (0.2-5 ug) of Act-dE2F and Act-dDP expres-sion plasmids were transfected along with 1 pug of copia-lacZand S pug of (E2F)4BCAT. Transfection mixes were supple-mented with pBluescript SK(+) to bring the total amount ofDNA transfected up to 20 pg per 10-cm plate, and the totalamount of expression vector added was kept constant by theaddition of the expression vector Act-PPA, which lacked aninsert. Cell harvesting and extract preparation and treatmentwere as described (37) except that cells were lysed by threefreeze-thaw cycles. CAT assays were performed by theliquid scintillation method of Sleigh (38). (3-Galactosidasewas assayed as described (35). Each transfection was per-formed at least four times independently (twice in duplicate),and representative data are shown.

In Vitro Bindg Assays. dDP and dE2F cRNAs weresynthesized from the linearized cDNAs by in vitro transcrip-tion and translated in rabbit reticulocyte lysate (Promega) inthe presence of [355]methionine (New England Nuclear). Thesynthesized polypeptides were then incubated for 60 min withglutathione-agarose-bound glutathione S-transferase (GST)fusion protein in 200 Al of ELB+ buffer (10). The complexeswere washed four times with ELB+ and then resolved bySDS/10% PAGE.GST fusion proteins were prepared by amplification with

Vent polymerase (New England Biolabs) using the appropri-ate primers with BamHI or EcoRI restriction sites at their 5'ends. The amplified fragments were digested with BamHIand EcoRI and subcloned into pGEX-2T (Pharmacia); theGST fusion proteins were expressed and purified from Esch-erichia coli as described (39). After binding to glutathione-agarose, the size, purity, and concentration ofthe GST fusionproteins were evaluated by Coomassie blue staining of SDS/polyacrylamide gels. Approximately 2 pg of full-length pro-tein was added to each binding reaction mixture.For DNA-binding assays, labeled polypeptides were syn-

thesized by in vitro translation, diluted with 100 pl of ELB+buffer, and incubated with 50 j4 of Sepharose 4B beads thathad been coupled with either wild-type or mutant E2Foligonucleotides. The slurry was rocked for 60 min at 40C toallow binding to occur. The beads were washed four times inELB+, and the bound proteins were analyzed.

RESULTSIsolation of cDNAs E ing dE2F. We prepared probes

that spanned the homologous DNA-binding domains ofE2F-1, E2F-2, and E2F-3 and used this mixture to screen aDrosophila AgtlO cDNA library. The library used had beenconstructed from larval eye imaginal discs (33), a tissuecontaining both growing and quiescent cells. Multiple cloneswere identified, purified, and analyzed. The inserts variedfrom 1.8 to 4.4 kb. The largest had a single long open readingframe that predicted a protein of 805 aa (Fig. 1A). The longestcDNA clone contained a relatively long 5' untranslatedsequence that had multiple in-frame termination codonsupstream of the putative initiator methionine (data notshown). During the course of this work, another laboratoryindependently isolated an E2F-related cDNA from Drosoph-ila (40), and comparison of the two sequences revealed thattheir cDNA and the one reported here were derived from thesame gene and were identical throughout their coding re-gions. A BLAST database search (April 1994) and use ofprotein-aligment programs indicated significant similaritywith the human proteins encoding the E2F family of protein(Fig. 1B). Although the fly and human proteins share threeregions with a high percentage of homology, the Drosophilaprotein is much larger in size, in part due to a 300-aa insertionof unique sequence between the two most C-terminal blocksof homology. The fly gene appears to be equally related to

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MSKFFVNVAP INNSNSSSSH TTTSSNTQRH QQHQQHYGGS GTTGHTMVARRLNYDLHGGT TSINNNNNIV IKNESVDLDY DHVLSSSDSN SNGGVAAHLRDHVYISLDKG HNTGAVATAA AAATAGQTQQ QLQQQHHHQN QQQRKATGKSNDITNYYKVK RRPHAVSDEI HPKKQAKQSA HHQTVYQKHT ASSAPQQLRHSHHQLRHDAD AELDEDVVER VAKPASHHPF SLSTPQQQLA ASVASSSSSGDRNRADTSLG ILTKKFVDLL QESPDGVVDL NEASNRLHVQ KRRIYDITNVLEGINILEKK SKNNIQWRCG QSMVSQERSR HIEADSLRLE QQENELNKAIDLMRENLAEI SQEVENSGGM AYVTQNDLLN VDLFKDQIVI VIKAPPEAKLVLPNTKLPRE IYVKAENSGE INVFLCHDTS PENSPIAPGA GYVGAPGAGCVRTATSTRLH PLTNQRLNDP LFNNIDAMST KGLFQTPYRS ARNLSKSIEEAAKQSQPEYN NICDIAMGQH HNLNQQQQQQ QQQLLQQPEE DDVDVELNQLVPTLTNPVVR THQFQQHQQP SIQELFSSLT ESSPPTPTKR RREAAAAAIAAGSSTTATTT LNSHNNRNHS NHSNHSNHSS SNNSKSQPPT IGYGSSQRRSDVPMYNCAME GATTTSATAD TTAATSRSAA ASSLQMQFAA VAESNNGSSSGGGGGGGGYG SIAGAGANAD PHQPYSHDRN SLPPGVADCD ANSNSSSVTLQGLDALFNDI GSDYFSNDIA PVSINPPDDN DYPYALNANE GIDRLFDFGSDAYGP*

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FIG. 1. (A) Predicted amino acid sequence of dE2F. Star indi-cates termination codon. (B) An alignment of the three regions mostconserved between dE2F and three members of the human E2Ffamily of proteins (10-13). Numbering above the amino acid se-quence corresponds to the Drosophila E2F sequence, and dotsindicate gaps introduced for sequence alignment. Boxed residuesindicate amino acids that are similar in fly E2F and all three humanproteins, and shaded boxes indicate residues that are identical in allfour proteins. Stringent similarity rules were used: Q. N; V, I, L; S,T; K, R; and D, E. The bar indicates the previously defined minimalRB-binding domain in human E2F-1, and the star denotes the singleresidue within this domain that is identical in all three human proteinsbut differs in dE2F.

each of the human E2F family members (13). To reflect thisfact, we have termed this protein dE2F.aa 249-318 ofdE2F share striking homology (Fig. 1B) with

DNA-binding domains of human E2F genes. In addition, aregion termed the "marked box" (13) that is highly conservedbetween the human proteins is also conserved within dE2F.The function of this domain is uncertain but may be involvedin protein dimerization (17). The RB-binding domain has alsobeen largely conserved in dE2F, sharing 56% similarity overthis region (Fig. 1B). dE2F contains numerous repetitiveamino acid stretches including poly(glutamine) segmentsreminiscent of those found in several mammalian and Dro-sophila transcription factors, such as Spl (37) and NTF-1(41). Similar repetitive sequences are encoded in a largenumber of Drosophila genes. Consistent with this, DNAprobes prepared from the full-length dE2F cDNA hybridizedto multiple DNA fragments on Southern blots of genomic

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80* 60

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FiG. 2. Trans-activation by dE2F expressed in insect cells. From0.2 to 5 Ag of dE2F expression vector was transfected into Dro-sophila SL2 cells along with an E2F-responsive CAT reporter gene[(E2F)4BCAT]. Fold activation is the ratio of CAT activity toP-galactosidase activity expressed from the internal control plasmidcopia-lacZ. The activity of the internal control plasmid was notaffected by the expression vectors used here or in Fig. 5.

DNA even at high stringency (data not shown). Thesefragments do not appear to represent E2F-related genes,since a probe prepared from the putative DNA-bindingdomain ofdE2F, containing sequences that are most likely tobe conserved in a homologous gene, produced a single stronghybridization signal even at low stringency (data not shown).We conclude that the dE2F gene is a single-copy gene and isnot a member of a large family of genes that can be readilydetected by cross-hybridization.TracripWal Activatio Properties of dE2F. We tested

whether the expression ofdE2F in insect cells could promotetranscriptional activation of an E2F-dependent promoter,since this functional assay represents the most rigorous testofa putative E2F gene. For this assay, dE2F expression wasdriven from the Drosophila actin 5C promoter. This vectorwas cotransfected into the Drosophila SL2 cells (36) togetherwith aCAT reporter gene [(E2F)4BCAT (17)] containing fourconsensus E2F binding sites (Fig. 2). Neither the parentreporter plasmid [BCAT, which contains only a TATA box(42)] nor the internal control plasmid [copia-lacZ (35)] wasaffected by the expression of dE2F (data not shown and Fig.2). Expression of dE2F led to a significant increase in

transcriptional activity of the reporter, with optimal activa-tion levels approaching 100-fold, indicating that dE2F is abona fide homolog of the human E2F genes.

Isolatin of eDNAsE g dDP. In light of the transcrip-tional activation properties ofdE2F we were surprised to findthat recombinant dE2F synthesized by in vitro translation wasunable to bind to oligonucleotides containing E2F sites (datanot shown; see also Fig. 5A). As the DNA-binding andtrans-activation properties ofhuman E2F proteins are greatlyenhanced by a heterodimeric partner (15-17), we hypothe-sized that the SL2 cells contained such apartnerfordE2F. Themost likely candidate for such an activity would be the productof a homolog of the mammalian DP-1 genes (14), and wetherefore screened the Drosophila cDNA library with a la-beled fragment corresponding to the putative DNA-bindingdomain ofmouse DP-1. DNA sequencing ofcDNAs from fourindependent positive phage revealed that the inserts containedoverlapping sequences. The longest cDNA clone (2.1 kb) wascompletely sequenced and found to contain a long openreading frame of 377 aa (Fig. 3). The Drosophila and humanDP-1 proteins share 61% identity over a region betweenresidues 91 and 315 that includes the putative DNA-bindingdomain (Fig. 3). The Drosophila gene appears to be equallyrelated to both human DP-1 and a highly related gene, humanDP-2, that has been isolated recently by hybridization with aDP-1 probe (C.-L. Wu, personal communication). It is unclearwhether the Drosophila gene is the homolog of either one orboth ofthe human genes; thus, we have termed the Drosophilagene dDP. Genomic Southern blots ofDrosophila DNA wereprobed under low and hih stringency with the entire dDPcDNA. Under both sets of conditions, only a single stronglyhybridizing fiagment was seen (data not shown). Although itappears that dDP is encoded by a single gene in Drosophila,we cannot rule out the possibility that other, more distantlyrelated genes exist in the fly.dE2F and dDP Associate in Vibt). dDP and dE2F were

expressed as GST fusion proteins in bacteria and tested forbinding to [35S]methionine-labeled proteins produced by invitro transcription and translation of the cDNA clones. La-beled dE2F bound strongly to GST-dDP but failed to bind toGST-dE2F controls (Fig. 4A). In the converse experiment,labeled dDP bound to GST-dE2F, and deletion mutants of

dDP _TXIi.AIFM..C.....'T. .I. . I 2K......- T .1T'S -:

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MDP-1 Alsits G1 A LIA TS| 4 .:. YS;MX--Gc R-n'E T P-. Y sV.:... )DDE A;, 1_XhDP-1 '-'TRT

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FIG. 3. Predicted amino acidsequence ofdDP aligned with hu-man (h) and mouse (m) homologs(14, 17). Numbering correspondsto the mouse and human aminoacid sequence, and dots representgaps introduced to allow the bestalignment. Residues are boxedand shaded as described in thelegend of Fig. 1. Similarity rulesare described in Fig. 1.

dDPmDP-lhDP-1

dDPmDP-1hDP-1

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in vitro. Binding assays were per-formed usingGST fusion proteins and

-46 dE2F or dDP synthesized by in vitrotranscription of the cDNA clone andtranslation using [35S]methionine.GST fusion proteins containing fNll-length dDP, dE2F, and deletion mu-

-30 tants were tested for binding. Theamino acids present in the dE2F fu-sion proteins are indicated. (A) Invitro translated dE2F binds to GST-dDP. Two GST-dE2F deletion mu-tants were included as negative con-

trols. (B)In vitro translatd dDPbindsto GST-dE2F and to several dE2Fdeletion mutants.

dE2F were constructed to identify the regions important forthis interaction (Fig. 4B). dDP bound to dE2F fusion proteinscontaining aa 225-805 or 1-432 but failed to bind to a fusionprotein that included only aa 47-343. These data suggest thata region of dE2F between aa 225 and 432 may be required forinteraction with dDP. Interestingly, this region of the proteinis highly conserved between Drosophila E2F and humanE2F-1, -2, and -3 (Fig. 1B), and an overlapping portion ofhuman E2F-1 has been implicated in bindinghuman DP-1 (17).DNA-Biding nd sAcdvatir i ofdDP. Next,

we tested the ability of recombinant dt)P or dE2F to bindDNA, either alone or in combination. Labeled proteins weregenerated by in vitro translation and incubated with oligonu-cleotides bearing either wild-type or mutant E2F binding sites(Fig. SA). In these experiments, dDP appeared to have a weak

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nonspecific DNA-binding activity; dE2F alone was unable tobind Wild-type or mutant E2F DNA sequences. Interestingly,when dDP and dE2F were mixed, dE2F acquired DNA-binding activity, and the affinity of dDP for DNA was dra-

matically enhanced. The enhanced binding by these proteinswas site-specific and was observed with wild-type but notmutant E2F sites. These data, together with the fact that dDPand dE2F can associate, suggest that dDP promotes thesequence-specific DNA-binding ofdE2F and that this bindingis achieved through the interaction of these two proteins.Given that dDP was able to bind DNA weakly and stim-

ulate the DNA-binding ability of dE2F, we investigated the

effect of dDP expression on the transcriptional activity of anE2F-responsive promoter. Hence, we performed transienttransfection experiments in SL2 cells, using eitherdDP alone

FIG. 5. dDP potentiates spe-cific DNA binding by dE2F andenhances trans-activation bydE2F. (A) In vitro translated dDPand dE2F were incubated eitherindividually or in combinationwith Sepharose beads bearing ei-ther wild-type or mutant E2FDNA binding sites. (B)DrosophilaSL2 cells were transfected with

_DP 1111 a_ either dE2F (1 pg) or dDP (1dDP dE2Fe vector alone or a combination of

both (1 pg of dE2F vector witheither 0.2 or 1 jg ofdDP vector).

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or in combination with dE2F as described earlier. Interest-ingly, overexpression ofdDP alone had no effect on the levelof transcription from the E2F-containing promoter (Fig. 5B).However, when dDP and dE2F were coexpressed, the com-bination of proteins resulted in a level of activation consid-erably greater than that seen with dE2F alone (Fig. 5B). Themagnitude of the effect ofdDP enhancement depended on theamount of dE2F plasmid used. At subsaturating amounts ofdE2F plasmid (1 ,ug in Fig. 5B), coexpression of dDP in-creased activation from 25- to 90-fold. A greater level ofenhancement was observed when less dE2F plasmid wasused or when dE2F and dDP were expressed in these insectcells using a weaker promoter (data not shown). We concludefrom these experiments that dE2F and dDP interact produc-tively to form a heteromeric complex capable of site-specificDNA binding and potent transcriptional activation.

DISCUSSIONThe two Drosophila genes reported here, dE2F and dDP,encode proteins with many of the characteristics of thehuman E2F genes. The dDP amino acid sequence is highlyhomologous to human DP-1 and DP-2 genes, and dE2Fcontains three regions that are conserved in human E2Fgenes. dDP and dE2F bind together and act cooperatively tostimulate sequence-specific DNA binding and site-specifictrans-activation. Thus the dE2F and dDP genes are bona fidehomologs of their maimalian counterparts, since they con-serve both the structural and the functional properties of thehuman genes. As such, they will serve as the focal point fora genetic study aimed at examining the regulators and targetsof E2F activity.

Southern blot analysis of Drosophila DNA, however,failed to detect genes closely related to dE2F or dDP. Thissuggests that flies have bypassed the need for a large familyof E2F-1-related polypeptides. Further studies will be neededto determine whether there are related genes that are toodivergent to be found by cross-hybridization. If no additionalgenes are found this may limit the parallels that can be drawnbetween the regulation of E2F activity in human and Dro-sophila cells. It appears, however, that at least one potentialtarget ofdE2F and dDP has been conserved between flies andhumans: the Drosophila DNA polymerase a gene, like itshuman counterpart, contains multiple E2F-binding siteswhich can confer dE2F responsiveness on a heterologousreporter gene (40).The question of the existence of an E2F pathway in

Drosophila was of particular interest for several reasons. (i)In mammalian cells, E2F activity has been shown to bedownregulated during differentiation and implicated as aregulator of genes necessary for cellular proliferation (1-8,43). (ii) E2F appears to provide a direct connection betweencyclin-dependent kinases and the regulation of specific tran-scriptional events. (iii) E2F-dependent transcription is ifihib-ited by RB, the product of a known tumor-suppressor gene.In some settings, RB itself has cell cycle-regulatory proper-ties and may provide a direct link to cell cycle-regulatorykinases. Although, it would be useful to study RB in Dro-sophila, no homolog has been found by direct screeningprocedures. Within the minimal RB-binding domain ofE2F-l, 9 of the 10 aa conserved among all three human E2Fsare also identical in the dE2F sequeiqce (Fig. 1B). Thisstriking similarity suggests that the regulatory interactionbetween RB-related proteins and E2F activity may be con-served in Drosophila, and a genetic analysis of E2F activityin Drosophila might permit the isolation of homologs for RBor RB-related proteins.

B.D.D. and A.B. contributed equally to this work. We thank J.

Colgan and J. Manley for the copia-lacZ and Act-PPA plasmids. Weacknowledge the valuable advice and assistance of I. Hariharan andE. Harlow and their colleagues at the Massachusetts General Hos-pital Cancer Center. We thank J. Lees, M. Vidal, C.-L. Wu, and K.Helin for reagents. We thank K. Ohtani and J. Nevins for exchangingdE2F sequences; C.-L. Wu for'sharing information on DP-2 prior topublication; and E. Harlow, K. Helin, and I. Sanchez for criticalreading of the manuscript. This work was supported by NationalInstitutes ofHealth funds. B.D.D. is supported by a Damon Runyon-Walter Winchell Cancer Fund Fellowship.

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