The LMO2 oncogene regulates DNA replication inhematopoietic cellsMarie-Claude Sincennesa,b,1, Magali Humberta,1, Benoît Grondina, Véronique Lisia,b, Diogo F. T. Veigaa, André Hamana,Christophe Cazauxc,2, Nazar Mashtalird, EL Bachir Affard, Alain Verreaulta,b,e, and Trang Hoanga,b,e,3

aInstitute of Research in Immunology and Cancer, University of Montreal, Montreal, QC, Canada H3C 3J7; bMolecular Biology Program, University ofMontreal, Montreal, QC, Canada H1T 2M4; cCancer Research Center of Toulouse, Toulouse 31024, France; dMaisonneuve-Rosemont Hospital ResearchCenter, Department of Medicine, University of Montreal, Montreal, QC, Canada H1T 2M4; and eDepartments of Pharmacology and Biochemistry, Universityof Montreal, Montreal, QC, Canada H3T 1J2

Edited by Mark Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved December 2, 2015 (received for review July 30, 2015)

Oncogenic transcription factors are commonly activated in acuteleukemias and subvert normal gene expression networks to reprogramhematopoietic progenitors into preleukemic stem cells, as exemplifiedby LIM-only 2 (LMO2) in T-cell acute lymphoblastic leukemia (T-ALL).Whether or not these oncoproteins interferewith other DNA-dependentprocesses is largely unexplored. Here, we show that LMO2 is recruitedto DNA replication origins by interaction with three essential replicationenzymes: DNA polymerase delta (POLD1), DNA primase (PRIM1), andminichromosome 6 (MCM6). Furthermore, tethering LMO2 to syntheticDNA sequences is sufficient to transform these sequences into origins ofreplication.We next addressed the importance of LMO2 in erythroid andthymocyte development, two lineages in which cell cycle and differen-tiation are tightly coordinated. Lowering LMO2 levels in erythroidprogenitors delays G1-S progression and arrests erythropoietin-depen-dent cell growth while favoring terminal differentiation. Conversely,ectopic expression in thymocytes induces DNA replication and drivesthese cells into cell cycle, causing differentiation blockade. Our resultsdefine a novel role for LMO2 in directly promoting DNA synthesis andG1-S progression.

LMO2 | cell cycle | DNA replication | hematopoietic cells | T-cell acutelymphoblastic leukemia

More than 70% of recurring chromosomal translocations inT-cell acute lymphoblastic leukemia (T-ALL) involve tran-

scription factors that are master regulators of cell fate. These on-cogenic transcription factors determine the gene signature andleukemic cell types (1). Whether these DNA-bound factors mayhave additional roles beyond modulating gene expression remainsunknown. LMO2, a 17-kDa protein defined by tandem zinc fingerdomains, is an essential nucleation factor that assembles a multi-partite transcriptional regulatory complex on gene regulatory re-gions via direct interaction with the TAL1/SCL transcription factor,LDB1, and other DNA binding transcription factors (2–4, reviewedin refs. 5, 6). These complexes drive gene expression programs thatdetermine hematopoietic cell fates at critical branchpoints bothduring embryonic development (7) and in adult hematopoietic stemcells (8, 9). Lmo2 function is essential in highly proliferative ery-throid progenitors (10, reviewed in refs. 5, 6). Interestingly, Lmo2down-regulation is required for terminal erythroid differentiation(11, 12). Because commitment to terminal differentiation iscoordinated with growth arrest (13), Lmo2 may have additionalmolecular functions that impede this critical step marked bygrowth cessation.In mouse models of T-ALL, LMO1 or LMO2 collaborates with

SCL to inhibit the activity of two basic helix–loop–helix (bHLH)transcription factors that control thymocyte differentiation, E2A/TCF3 and HEB/TCF12, causing differentiation arrest (reviewed inref. 14). However, this inhibition is not sufficient, per se, for leu-kemogenesis, because both TAL1 and LYL1 inhibit E proteins butrequire interaction with LMO1/2 to activate the transcription ofa self-renewal gene network in thymocytes (15, 16) and to in-duce T-ALL (17, 18). Of note, downstream target genes cannot

substitute for LMO1/2 to induce T-ALL, suggesting additionalfunctions for LMO1/2.Together, these studies underscore the dominant oncogenic

properties of LMO2, as revealed by recurring retroviral integrationsupstream of LMO2 in the gene therapy trial (19, 20) or by recurrentchromosomal rearrangements in T-ALL (21). As a consequence,LMO2 is misexpressed in the T lineage, where it is normally absent.In addition, LMO proteins are frequently deregulated in breastcancers (22) and neuroblastomas (23), pointing to their importancein cell transformation. In particular, in patients who eventuallydeveloped T-ALL associated with LMO2 activation after genetherapy, T-cell hyperproliferation was observed early during thepreleukemic stage (19). How LMO2 affects erythroid progenitor orT-cell proliferation cannot be inferred from its downstream targetgenes (12, 24–28).To understand LMO2 functions, we performed an unbiased

screen for LMO2 interaction partners. We show that LMO2 asso-ciates with three replication proteins, minichromosome 6 (MCM6),DNA primase (PRIM1), and DNA polymerase delta (POLD1),and that LMO2 influences cell cycle progression and DNA repli-cation in hematopoietic cells, indicating an unexpected functionfor LMO2.

Significance

Understanding how cell cycle and cell differentiation are coordinatedduring normal hematopoiesis will reveal molecular insights in leu-kemogenesis. LIM-only 2 (LMO2) is a transcriptional regulator thatcontrols the erythroid lineage via activation of an erythroid-specificgene expression program. Here, we uncover an unexpected functionfor LMO2 in controlling DNA replication via protein–protein inter-actions with essential DNA replication enzymes. To our knowledge,this work provides the first evidence for a nontranscriptional func-tion of LMO2 that drives the cell cycle at the expense of differenti-ation in the erythroid lineage and in thymocyteswhenmisexpressedfollowing genetic alterations. We propose that the nontranscrip-tional control of DNA replication uncovered here for LMO2may be amore common function of oncogenic transcription factors thanpreviously appreciated.

Author contributions: M.-C.S., M.H., B.G., and T.H. designed research; M.-C.S., M.H., B.G.,V.L., D.F.T.V., A.H., and N.M. performed research; A.V. contributed new reagents/analytictools; M.-C.S., M.H., B.G., V.L., D.F.T.V., A.H., C.C., N.M., E.B.A., and T.H. analyzed data;and M.-C.S., M.H., B.G., E.B.A., A.V., and T.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1M.-C.S. and M.H. contributed equally to this work.2Deceased August 3, 2015.3To whom correspondence should be addressed. Email: trang.hoang@umontreal.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1515071113/-/DCSupplemental.

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ResultsIdentification of New LMO2 Protein–Protein Interactions in HematopoieticProgenitors. Lmo2 is expressed in c-Kit+ hematopoietic stem andprogenitor cells (HSPCs) and in immature prothymocytes, but not atlater stages of T-cell differentiation (29). To identify new LMO2binding proteins in HSPCs, we constructed a cDNA library frompurified murine Kit+Lin− hematopoietic progenitors for a yeast two-hybrid screen and used LMO2 as bait. In addition to known LMO2-interacting proteins, such as LDB1, and to proteins associated withtranscription, we unexpectedly identified interactions with three es-sential components of prereplication complexes (pre-RCs),namely, MCM6, POLD1, and PRIM1 (30) (Fig. 1A and Table S1).In comparison, a screen performed using GAL4-SCL identified onlyknown interactions (Table S1). LMO2 interaction was specific tothese three replication proteins, as confirmed by independent yeasttwo-hybrid assays with full-length cDNAs (Fig. 1 B and C). In ad-dition, we identified BAZ1A/ACF1, required for DNA replicationthrough heterochromatin (31); SetD8, for replication licensing (32);MYST2/HBO1, controlling MCM loading via ORC1 binding (33);and CCNA2-CDK1, regulating origin firing (34) (Fig. 1A). The top-ranking pathways by gene set enrichment analysis were cell cycle,DNA synthesis, and DNA replication (Fig. 1D), concurring with theview that LMO2 controls these processes via its protein partners.Finally, LMO2 coimmunoprecipitated with PRIM1, MCM6, andMYST2 in mammalian cells, and both LIM domains contributed tothis interaction (Fig. 1E), whereas LDB1 binding required mostlyLIM1, as expected (4). We conclude that in addition to its associa-tion with transcription regulators, LMO2 engages in protein–proteininteractions with DNA replication proteins.

LMO2 Associates with Replication Complexes. We next addressedthe question of whether LMO2 associates with the pre-RC inhematopoietic cells. Because pre-RC formation often requires aDNA template (35), we prepared DNA-containing chromatinextracts from CD34+Kit+ progenitors (TF-1 cells) (3, 13, 36, 37)to assess the interaction of LMO2 with endogenous replicationproteins by immunoprecipitation. We found that POLD1, PRIM1,and MCM6, but not lamin B or beta-actin (negative controls),reproducibly and specifically coimmunoprecipitate with LMO2,but not with control Ig, in TF-1 chromatin extracts (Fig. 1F). We

also detected the other members of the MCM hexameric com-plex (MCM2–MCM7), as well as ORC2, CDC6, CDT1, andPCNA, albeit with weaker efficiency for the latter three (Fig.1F). In contrast, MCM6, POLD1, and PRIM1 did not coim-munoprecipitate with SCL (Fig. 1G), whereas LMO2 and LDB1were found with SCL in this assay, as expected. Therefore, replica-tion proteins do not stably associate with the SCL transcriptioncomplex but more specifically with LMO2.

LMO2 Binds to Origins of Replication. LMO2 shares importanttranscriptional targets with SCL (15, 16, 25, 36). Nonetheless,LMO2 is rarely found at gene promoters (3%) by ChIP sequencingin multipotent progenitors, whereas SCL is more frequently ob-served within transcriptional start sites (28%) (24), suggesting thatLMO2 has SCL-independent functions. We assessed whetherLMO2 associates with DNA replication origins, using ChIP of TF-1cells with anti-LMO2, or anti-MCM5 antibodies. We first studiedseven well-characterized human DNA replication origins (38) andfound that both MCM5 and LMO2 occupied four of these repli-cation origins, c-MYC, G6PD, TOP1, and MCM4 (Fig. 2A), allmapping to early replicating G1 (ERG1) segments (39, 40) (Fig.S1A). In contrast, MCM5 did not bind the GYPA promoter, a well-defined SCL-LMO2 transcriptional target (36), whereas LMO2occupancy was confirmed, together with SCL and GATA1, twoLMO2 transcription factor partners. SCL was detected at two of theseven tested origins, although binding was 10- to 20-fold lowercompared with GYPA, whereas GATA1 binding was below thedetection limit (Fig. 2A). Therefore, LMO2 is recruited to well-characterized DNA replication origins with MCM5, in the absenceof GATA1 and frequently of SCL.These results led us to assess LMO2 occupancy of replication

initiation zones identified in two independent tiling array-basedstudies in HeLa cells, using Lambda-exonuclease digestion (38, 41)and anti-BrdU immunoprecipitation to purify origin-centered na-scent DNA strands. We focused on a subset of 45 replication ini-tiation zones that overlapped within a distance of 1 kb between thetwo studies (Fig. 2B and Fig. S1 B and C). In TF-1 cells, MCM5 andLMO2 were each detected on 20 and 19 replication initiation zones,respectively, nearly half of which were positive for both (Fig. 2B).We aligned these 45 replication initiation zones with ERG1 seg-ments identified in mammalian cells (39, 42) and found that 68% of

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Fig. 1. LMO2 interacts with DNA replication proteins.(A) New LMO2 protein–protein interactions in hema-topoietic progenitors revealed by yeast two-hybridscreens using LMO2 as bait (*). (B and C) LMO2 spe-cifically interacts with MCM6, PRIM1, and POLD1 byyeast two-hybrid assay. (D) Gene set enrichment anal-ysis for LMO2 interaction partners. Shown are the topseven Reactome pathways significantly enriched in theLMO2 Y2H screen [false discovery rate (FDR): q < 0.001]according to the Molecular Signatures database v5.0(PubMed identifier 16199517). (E) Both LIM domainsof LMO2 are required for interaction with PRIM1,MCM6, andMYST2. GAL4-LMO2WT or LIM1/2 mutantswere cotransfected with Flag-tagged PRIM1, MCM6, orMYST2. Protein extracts were immunoprecipitated withanti-FLAG Ab, followed by Western blotting. (F) DNAreplication proteins coimmunoprecipitate with LMO2(**). Immunoprecipitation of TF-1 chromatin extractswith Abs against LMO2. Inputs (4%) and immune pel-lets (IP) were analyzed by IB. (G) SCL does not stablyassociate with DNA replication proteins. SCL or isotypecontrol Abs were analyzed as in F (*). IB, immunoblot-ting; IP, immune pellet; SN, supernatant. Data shownare typical of at least two (*) or three (**) independentexperiments.

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LMO2-bound zones mapped to ERG1 segments, which is almosttwice the percentage of ERG1 segments found in the absence ofLMO2 (Fig. 2B). Therefore, LMO2 is recruited to a significantproportion of initiation zones in TF-1 cells, corresponding, in mostpart, to ERG1 segments.

Tethering LMO2 to DNA Recruits Replication Proteins and Induces DNAReplication. To determine whether LMO2 tethering to DNA wassufficient to stimulate DNA replication, we optimized a syntheticDNA replication assay in mammalian cells described by Takedaet al. (43). We assessed whether LMO2 fused to the DNA bindingdomain of GAL4 can drive DNA replication from GAL4 bindingsites (5XUAS) in transfected 293 cells (Fig. 2C). Newly replicatedDNA is hemimethylated or unmethylated and can be distinguishedfrom transfected, bacterially derived DNA by its resistance to Dpn1digestion (43). As expected, GAL4-ORC2 induced an approx-imately fivefold increase in newly synthesized DNA comparedwith control GAL4-VP16 (Fig. 2D). In this assay, GAL4-LMO2reproductively directed a dose-dependent increase of newly repli-cated DNA, reaching a maximum of eightfold (Fig. 2D), whereasGAL4-SCL did not produce the same results. This activity wasabrogated when GAL4-binding UAS sequences were mutated (Fig.2E) or absent (pBluescript vector). We therefore conclude thatLMO2 anchoring to DNA was sufficient to direct DNA replication.LMO2-driven DNA replication occurred most likely in the absenceof transcription because 293T cells lack hematopoietic transcrip-tion factors that recruit LMO2 to DNA, namely SCL and GATA1/2. To determine if DNA-bound GAL4-LMO2 could recruit DNAreplication proteins to the UAS template, we performed DNAcapture as previously described (44), using UAS sequencesimmobilized on beads. LMO2 recruited POLD1, MCM5, and, to alesser extent, CDT1 and PCNA to DNA, and this recruitmentrequired the integrity of the GAL4 binding site (Fig. 2F). Together,our results indicate that LMO2 tethering to DNA was sufficient tonucleate the assembly of prereplication/preinitiation complexesspecifically at the site of binding and to direct DNA replication.

LMO2 Expression Levels Control the Rate of DNA Synthesis andCellular Outcome in the Erythroid Lineage. Cell cycle is highly reg-ulated in the erythroid lineage because ∼80% of primary pro-erythroblasts (E1 stage; Fig. 3A) are in S phase, exactly at theonset of erythropoietin (Epo) dependence (45), and this pro-portion sharply drops at the E3 and E4 stages of terminal dif-ferentiation. We observed that the E1 population segregatedinto cells with high and low endogenous LMO2 protein levels(Fig. 3B), corresponding to high and low proportions of cells inS/G2/M (Fig. 3C). Strikingly, LMO2 protein levels decrease fromthe E1hi to E4 stage, directly correlating with a decrease in theproportion of cells in S phase (Fig. 3C), whereas GATA1 andSCL protein levels increase (Fig. 3D). This process is highlycoordinated because most LMO2 partners identified here, in-cluding CCNA2, are synchronously down-regulated with Lmo2during differentiation from proerythroblast to orthochromaticerythroblasts (Fig. S2).We addressed the functional importance of LMO2 by decreasing

LMO2 protein levels in erythroid progenitors via RNAi (shRNALmo2) (Fig. S3A). Lmo2 depletion in Ter119− fetal liver erythroidprogenitors decreased by twofold the proportion of cells in S phase asdetermined by DAPI staining and flow cytometry analysis, comparedwith control cells (Fig. 3E). In addition, Lmo2 depletion almost ab-rogated the proliferation of primary erythroid progenitors at a keycommitment step marked by Epo responsiveness (Fig. 3 F and G),while enhancing the generation of mature E4 cells (CD71−Ter119+)(Fig. 3H). Therefore, high LMO2 levels are required for the Epo-dependent proliferation of CD71+ erythroid precursors, whereaslowering LMO2 accelerates terminal erythroid differentiation.Decreased LMO2 levels caused a twofold decrease in the rate of

DNA synthesis monitored by the kinetics of 32P orthophosphateincorporation into synchronized mouse erythroleukemia (MEL) cells(Fig. 3I), without causing apoptosis (Fig. S3B). Consequently, thesecells failed to proliferate in culture (Fig. S3C). Decreased DNAsynthesis was unlikely due to decreased expression levels for repli-cation genes assessed by RT-quantitative PCR (Fig. S3D), consistentwith transcriptome analysis of erythroid cells and lymphoid cells

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Fig. 2. LMO2 and MCM5 occupy DNA replication origins. (A) ChIP of well-characterized DNA replication origins in TF-1 cells with anti-LMO2, anti-MCM5,anti-SCL, and anti-GATA1 Abs. Shown are the ratio of enrichment over controlIg and background (c-Kit, −20 kb). GYPA promoter sequences were amplified asa control (36). (Left) Maps depicting the location of well-characterized DNAreplication origins and PCR probes. Gene exons are illustrated by black boxes.PCR primers are shown as red bars. E, E47 consensus sites; G, GATA1/2 consensussites. n.d., not detectable. (B) LMO2-bound replication initiation zones (gray,first column) are preferentially enriched within ERG1 segments. Forty-five rep-lication initiation zones found in common in two studies (38, 41) were analyzedby ChIP for LMO2 binding. The replication timing of these origins according totwo datasets is illustrated: study 1 [Gilbert and coworkers (42)] in leukemicpatients and lymphoblastoid cell lines and study 2 [Stamatoyannopoulos andcoworkers (39)] in B cells and ES cells. A higher proportion of early replicatingsegments (gray, last column) is found in LMO2-bound origins (P = 0.02). Mix:Some samples from study 1 showed early replication, whereas others showedlate replication. (C) Diagram illustrating the synthetic DNA replication assay.Plasmid DNA was transfected into HEK293 cells and detected by PCR (primerpairs as shown). (Left) Dpn1 sensitivity distinguishes unreplicated methylatedplasmid DNA (sensitive) from replicated hemi- or unmethylated DNA (resistant).(D) LMO2 fused to GAL4 stimulates the replication of a GAL4-responsive plas-mid in mammalian cells. HEK293 cells were transfected with the indicated GAL-fusion genes. After Dpn1 digestion, Dpn1-resistant extrachromosomalDNA was quantified by real-time PCR (n = 2). P < 0.05. (E) LMO2-induced DNAreplication depends on LMO2 tethering to DNA via GAL4 binding sites (UAS).The experiment was performed as in D. (F) LMO2 binding to artificial origins ofreplication is sufficient to recruit DNA replication proteins specifically. DNAcapture was performed using immobilized WT or mutant UAS sequences onbeads. Bound proteins were revealed byWestern blotting. Data are the mean ±SD of at least two independent experiments performed in duplicate.

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in which Lmo2 was knocked down or overexpressed, respectively(12, 46). Furthermore, replication genes were not occupied by theSCL–LMO2 complex in leukemic T cells (25). Together, these ob-servations indicate a critical role for LMO2 in erythroid cell fate, viathe control of DNA replication and the cell cycle that drives Epo-dependent expansion of erythroid progenitors while impeding theircommitment to terminal maturation.

LMO2 Expression Regulates S-Phase Entry. We next addressed theimportance of LMO2 levels on the kinetics of cell cycle pro-gression (Fig. 3J). Briefly, we found that viable MEL cells withlow side and forward scatter properties are mostly in G0/G1 (Fig.S3E). These G0/G1 cells were purified and released in culturemedium to analyze their DNA content by Hoechst staining (Fig.S3E). Decreased LMO2 (shRNA Lmo2) reproducibly caused a

2-h delay in G1/S transition, occurring at 3 h after releasecompared with 1 h for control cells (Vector), as well as delayedS-phase progression (Fig. 3J and Fig. S3E), indicating thatLMO2 levels control S-phase entry in erythroblasts.To mimic retroviral integration upstream of the LMO2 locus in

the X-SCID gene therapy trial (19, 20, 46) and define the conse-quences of ectopic LMO2 expression in vivo, we delivered LMO2 inhematopoietic stem cells by retroviral infection, (Fig. 4 A–F). Upontransplantation, LMO2-transduced bone marrow cells inducedT-ALL in 60% of recipient mice, with a median of 270 days (46) (Fig.4B and Fig. S4). During the preleukemic stage (30 days), LMO2overexpression mostly affected thymocytes and led to an accumula-tion of CD44+CD25−CD4−D8− double-negative thymocyte (DN)cells and a decrease in CD4+CD8+ double-positive thymocyte(DP) cells in the thymus, reproducing the differentiation blockadeat the DN stage reported in LMO2 transgenic mice (Fig. 4C),despite the fact that the retroviral vector allowed for transgeneexpression in all cells. Elevating LMO2 in thymocytes modified thecell cycle status of thymocyte progenitors (Fig. 4D) without af-fecting bone marrow stem cells (Fig. S4B), consistent with the roleof LMO2 as a T-cell specific oncogene (46). Interestingly, elevatingLMO2 enhanced the cell cycle status of both DN1 and DP cells,suggesting that differentiation blockade (lower number of DP cells)could be due to increased cell cycle. Furthermore, when DN1thymocytes were synchronized in G0/G1 and released in culture,LMO2-expressing cells entered more rapidly into S phase com-pared with control cells (vector) (Fig. 4E), indicating that LMO2facilitates the G1-S transition and S phase progression.The above results indicate that LMO2 overexpression in hema-

topoietic cells reproduced the T-cell proliferation phenotypereported for preleukemic patients in the X-SCID gene therapy trial.Actively dividing cells are more sensitive to 5-fluorouracil (5-FU), anantimetabolite that inhibits thymidylate synthase and therefore de-pletes the pool of dTTP. Consistent with increased DNA replication,LMO2-expressing thymocytes cocultured with MS5 stromal cellsexpressing DL4 were 100-fold more sensitive thanWT thymocytes to5-FU (EC50 of 5 nM vs. 500 nM) (Fig. 4F).

DiscussionLMO2 has a well-established function in transcriptional regulationvia direct interaction with transcription factors, mostly of the bHLHfamily, SCL/TAL1, TAL2, and LYL1 or GATA proteins (2–4). Inthis study, we revealed unexpected new functions of LMO2 in he-matopoiesis and leukemogenesis through a yeast two-hybrid screenof LMO2 interaction partners in hematopoietic progenitors. Ourobservations indicate that LMO2 controls cell fate by directly pro-moting DNA replication, a hitherto unrecognized mechanism thatmight also account for its oncogenic properties.Our study unravels unexpected interactions between LMO2 and

three essential replication proteins, MCM6, PRIM1, and POLD1(30), as well as chromatin-modifying enzymes that have well-characterized roles in DNA replication, BAZ1A, SETD8, MYST2,and UHRF1 (31–33, 47). More importantly, tethering LMO2 toDNA via the GAL4-DNA binding domain was sufficient to recruitMCM5 and POLD1 to DNA and to transform UAS into origins ofreplication, indicating that LMO2 directly controls DNA replication.Unlike other transcription complexes that have a dual role in DNAreplication (48), LMO2 interaction with the RC is distinct from thewell-known recruitment of LMO2 to the SCL transcription complex.Our data are in line with the observations that there is only a partialoverlap between SCL and LMO2 chromatin occupancy in hema-topoietic progenitors (24).LMO2 binding to replication initiation zones reported here

overlaps with early G1 replication segments described in lymphoidcells (39, 42), a possibility that may be favored by its interaction withMLL2 (Fig. 1A). Indeed, the H3K4me3 histone mark found in earlyreplicating domains (40) can be controlled by MLL2. In eukaryotes,origins are licensed in excess, and not all licensed origins are active

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Fig. 3. LMO2 levels determine the proliferation of erythroid progenitors.(A) Diagram of erythroid differentiation according to flow cytometry profiles.(B) LMO2 levels in bone marrow erythroid progenitors (Lin−CD71+Ter119−) cor-relate with their cell cycle status by Hoechst staining: E1low and E1high (P ≤ 0.05).The mean fluorescence intensity (MFI) for LMO2 per cell was assessed by flowcytometry. (C) Correlation between LMO2 levels and the proportion of cells inS/G2/M during erythroid differentiation (E1 to E4). (D) Expression levels of LMO2,SCL, and GATA1 during erythroid differentiation. (E) Decreased LMO2 in ery-throid progenitors reduced the fraction of cells in S phase. The shRNA Lmo2 wasdelivered in Ter119− fetal liver cells, which were then stimulated with Epo for 2 d.The cell cycle in erythroid progenitors (E1, CD71+Ter119−) was analyzed by DAPIstaining. (F) Lmo2 is required for the response of proerythroblasts to Epo. NT,nontarget (control). (G) LMO2 levels control the proliferation of erythroid progen-itors in culture. (H) Decreased LMO2 abolishes the growth of erythroid progenitors(E1) in response to Epo but favors terminally differentiating erythroid cells (E4).(I) Decreased DNA synthesis induced by shRNA-mediated Lmo2 depletion. MEL cellswere purified in G0/G1 and released in culture for different times in the presence ofα32P-dCTP. After electrophoresis, total DNAwas quantified by autoradiography (n=2). The slopes of the two curves were 0.48 ± 0.02 (Vector) and 0.29 ± 0.01 (shLmo2).*P < 0.0001. (J) Decreased proportion of cells in S phase after Lmo2 depletion. MELcells expressing shLmo2 or control (Vector) were purified in G0/G1 and analyzed forcell cycle progression at different time points in culture by Hoechst staining (n = 3).All data shown are typical of at least two independent experiments.

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during a given cell cycle, which requires recruitment of DNA poly-merases to initiate DNA synthesis (30). Accordingly, we show thatLMO2 levels influence the rate of DNA replication in MEL cells.Although we focused on the basic components of the pre-RC

in the present study, several other proteins identified in thescreen could have a regulatory function. For example, cyclinA2-CDK2 favors the onset of DNA replication at the G1-Stransition and prevents rereplication during S phase (49).These possibilities would be consistent with the effects ofLMO2 levels on the kinetics of G1-S transition that we ob-served in two cell types, delayed when Lmo2 is knocked downin murine erythroleukemic cells and, conversely, accelerated inLMO2-overexpressing DN1 thymocytes. In addition, LMO2associates with two cell cycle checkpoint proteins, CDK9 andBUB1B (Table S1). CDK9 associates with cyclin K in replica-tion stress response and prevents DNA damage in replicatingcells (50). BUB1B is an essential component of the mitoticcheckpoint. The importance of these cell cycle proteins forLMO2-induced cell proliferation remains to be addressed.Cell cycle is a highly regulated process in the erythroid lineage.

CD71+Ter119− proerythroblasts represent a critical transitionalstage marked by Epo dependency (51) and elevated DNA repli-cation (45), both shown here to require high LMO2 protein ex-pression. Conversely, terminal erythroid differentiation to theCD71−Ter119+ stage is necessarily coordinated with growth ar-rest (13, 51), which we now show to be favored by Lmo2 down-

regulation, in agreement with previous work indicating that LMO2overexpression prevents terminal erythroid differentiation (11). Weconclude that LMO2 down-regulation is required for the switch toterminal erythroid differentiation due to the implication of LMO2in DNA replication/cell cycle (Fig. 4G).It is well established that transcription factor gene networks

drive hematopoietic cell development and lineage outcome, viasynergistic or antagonistic interactions (reviewed in refs. 52 and 53).It is unclear how LMO2 inhibits cell differentiation in both theerythroid (11) and T-lymphoid lineages (reviewed in ref. 53).We now propose that LMO2-dependent DNA replication inboth lineages governs the switch between a proliferative state inprogenitors and commitment to terminal differentiation (Fig.4G). Failure to regulate this proliferative switch caused by ec-topic LMO2 expression (19) may lead to T-ALL.Oncogene-induced DNA replication stress (54) could lead to

replication errors and ultimately cause genetic lesions, such as acti-vating NOTCH1 mutations (55, 56), that convert preleukemic stemcells (15, 16) into leukemia initiating cells (15). Two other LIM-onlyproteins, LMO1 and LMO4, are also important determinants of cellcycle progression in neuroblastoma (23) and in breast cancer asso-ciated with genomic instability (22), respectively, suggesting that themechanism(s) described here may be extended to these proteins.Emerging evidence indicates that oncogenes, such as c-MYC orHOXD13, can be part of nontranscriptional complexes involvedin DNA replication (54, 57). Taken together, we propose that on-cogenic transcription factors in acute leukemias and possibly othertumor types transform cells by at least two DNA-dependent mech-anisms, the control of gene expression programs, as initially pro-posed (1), but also by deregulating DNA replication (42), with bothprocesses being important determinants of cell fate.

Materials and MethodsYeast Two-Hybrid. LMO2 full-length protein was subcloned in the pGBKT7vector and transformed in the Y187 yeast strain. The cDNA library fromc-Kit+Lin− murine bone marrow cells was constructed in the pGADT7 vectorwith the Matchmaker Library Construction and Screening Kit (BD Biosci-ences) and transformed in the AH109 yeast strain. The screen (selection ofAde+His+Leu+Trp+ colonies) was performed by yeast mating. Positive cloneswere isolated and sequenced. To confirm the interactions and check forspecificity, full-length cDNAs for Polδ1, Prim1, Mcm2-5-6-10, Cdt1, Pcna, andCaf1 were subcloned into pGADT7 and transformed in AH109 cells.

Retroviral Gene Transfer and RNAi. LMO2 retroviral gene transfer and mousebone marrow transplantation were performed as described (8). For RNAi ex-periments, vesicular stomatitis virus (VSV-G)–producing cells were transfectedwith plasmids encoding shRNAs against LMO2, with a nontarget shRNA or withthe empty pLKO.1 vector (Sigma). MEL cells or Lin− fetal liver cells were in-cubated with VSV-G supernatant for 48 h and then selected with puromycin asdescribed (8). All mice were kept under pathogen-free conditions according toinstitutional animal care and use guidelines. The protocols for gene transferand transplantation in mice were approved by the Committee of Ethics andAnimal Deontology of the University of Montreal.

Flow Cytometry, Cell Cycle Analysis, and Cell Sorting. Flow cytometry, cell cycleanalysis, and cell sorting are described in Supporting Information.

32P Orthophosphate Labeling and Quantification of de Novo DNA Synthesis.MEL cells synchronized in G0/G1 were incubated with α32P-dCTP (100 μCi/mL;PerkinElmer) in DMEM [10 mMHepes (pH 7.4), 10% (vol/vol) FCS] at 8 × 105 cellsper milliliter for the indicated times. Cells were lysed in DNA extraction buffer[80 mM Tris·HCl (pH 8.0), 8 mM EDTA, 100 mM NaCl, 0.5% (g/100 mL) SDS].After proteinase K digestion and phenol/chloroform extraction, DNA was re-solved on an alkaline (NaOH) agarose gel. The gel was dried on a Biodynemembrane (Pall Corporation). 32P incorporation was visualized by autoradi-ography and quantitated using ImageQuant software (GE Healthcare).

Protein Extraction, Immunoprecipitation, Immunoblot, Abs, RNA, and ChIP Analysis.Protein extraction, immunoprecipitation, immunoblot (IB), Abs, RNA, and ChIPanalysis methods are fully described in Supporting Information. Primer sequences

T

T

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A B

C D

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Fig. 4. Ectopic LMO2 expression in thymocytes triggers G1/S transition, T-cellhyperproliferation, and T-ALL development. (A) Experimental strategy to deliverLMO2 in hematopoietic cells using the murine stem cell virus (MSCV) retroviralvector. BM, bone marrow. (B) LMO2 overexpression induces T-ALL in mice.Kaplan–Meier curves showing the time of leukemia onset in mice transplantedwithMSCV-LMO2 or MSCV transduced cells. (C and D) LMO2 overexpression leadsto an increase of the DN1 thymocyte subset but a decrease in DP cells in vivo,despite higher proportions of cycling cells in both populations. (E) LMO2 over-expression induces an increase in the percentage of DN1 cells in S phase. DN1 cellswere purified in G0/G1 and analyzed for cell cycle progression by DAPI staining atdifferent time points after coculture with MS5-DL4 stromal cells. (F) LMO2-expressing thymocytes are more sensitive to 5-FU treatment in vitro. Cells werecocultured onMS5-DL4 stromal cells and treated with 5-FU at the indicated doses.Viable Thy1+ cells were scored by flow cytometry. (G) Proposed model: LMO2controls DNA replication, favors progenitor cell proliferation, and inhibits com-mitment to terminal differentiation in the erythroid and T-lymphoid lineages.Data shown are typical of at least two independent experiments.

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used are shown in Tables S2 and S3, and Abs used for ChIP, IB, and immuno-fluorescence are listed in Table S4.

Transient Replication Assay in Mammalian Cells. The transient replicationassay is adapted from Takeda et al. (43) and described in SupportingInformation.

ACKNOWLEDGMENTS. We thank Danièle Gagné [Institute of Research in Immu-nology and Cancer (IRIC)] for her assistance with flow cytometry, VéroniqueLitalien for mouse handling, Geneviève Boucher for bioinformatic analyses,Francis Migneault and David Flaschner for assistance with the yeast two-hybridconfirmation assays, Drs. Jalila Chagraoui and Richard Martin for help with the

retroviral gene transfer and the yeast two-hybrid, and Dr. Jana Krosl for criticalcomments on the manuscript. This work was funded by the Cancer ResearchSociety, Inc. (2012–2014); the Canadian Institutes for Health Research (CIHR; GrantMOP111050, 2011–2016); Canadian Cancer Society Research Institute Grant019222 (to T.H.); the Leukemia & Lymphoma Society (2013–2015) (T.H. andE.B.A.); CIHR Grant 89928 (to A.V.); a CIHR multiuser grant to support the flowcytometry and imaging service; and a group grant from the Fonds de Recherchedu Québec-Santé to support, in part, IRIC infrastructure. M.-C.S. was supported bya Canada Graduate Scholarship Doctoral Award (CIHR) and a doctoral award fromthe Cole Foundation. M.H. was supported by a postdoctoral fellowship awardof the Swiss National Foundation (PBBEP3 144798), by Swiss Foundationfor Fellowships in Medicine and Biology, and by Novartis (P3SMP3 151720).V.L. and D.F.T.V. were supported by Cole Foundation awards.

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