The LMO2 Oncogene Regulates DNA Replication in Hematopoietic Cells

Home , LDB1, LMO2, MCM6, ORC1, POLD1, PRIM1

The LMO2 oncogene regulates DNA replication in hematopoietic cells

Marie-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 of Montreal, Montreal, QC, Canada H1T 2M4; cCancer Research Center of Toulouse, Toulouse 31024, France; dMaisonneuve-Rosemont Hospital Research Center, Department of Medicine, University of Montreal, Montreal, QC, Canada H1T 2M4; and eDepartments of Pharmacology and Biochemistry, University of 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 acute substitute for LMO1/2 to induce T-ALL, suggesting additional leukemias and subvert normal gene expression networks to reprogram functions for LMO1/2. hematopoietic progenitors into preleukemic stem cells, as exemplified Together, these studies underscore the dominant oncogenic by LIM-only 2 (LMO2) in T-cell acute lymphoblastic leukemia (T-ALL). properties of LMO2, as revealed by recurring retroviral integrations Whether or not these oncoproteins interfere with other DNA-dependent upstream of LMO2 in the gene therapy trial (19, 20) or by recurrent processes is largely unexplored. Here, we show that LMO2 is recruited chromosomal rearrangements in T-ALL (21). As a consequence, to DNA replication origins by interaction with three essential replication LMO2 is misexpressed in the T lineage, where it is normally absent. enzymes: DNA polymerase delta (POLD1), DNA primase (PRIM1), and minichromosome 6 (MCM6). Furthermore, tethering LMO2 to synthetic In addition, LMO proteins are frequently deregulated in breast DNA sequences is sufficient to transform these sequences into origins of cancers (22) and neuroblastomas (23), pointing to their importance replication. We next addressed the importance of LMO2 in erythroid and in cell transformation. In particular, in patients who eventually thymocyte development, two lineages in which cell cycle and differen- developed T-ALL associated with LMO2 activation after gene tiation are tightly coordinated. Lowering LMO2 levels in erythroid therapy, T-cell hyperproliferation was observed early during the progenitors delays G1-S progression and arrests erythropoietin-depen- preleukemic stage (19). How LMO2 affects erythroid progenitor or dent cell growth while favoring terminal differentiation. Conversely, T-cell proliferation cannot be inferred from its downstream target MEDICAL SCIENCES ectopic expression in thymocytes induces DNA replication and drives genes (12, 24–28). these cells into cell cycle, causing differentiation blockade. Our results To understand LMO2 functions, we performed an unbiased define a novel role for LMO2 in directly promoting DNA synthesis and screen for LMO2 interaction partners. We show that LMO2 asso- G1-S progression. ciates with three replication proteins, minichromosome 6 (MCM6), DNA primase (PRIM1), and DNA polymerase delta (POLD1), LMO2 | cell cycle | DNA replication | hematopoietic cells | T-cell acute lymphoblastic leukemia and that LMO2 influences cell cycle progression and DNA replication in hematopoietic cells, indicating an unexpected function ore than 70% of recurring chromosomal translocations in for LMO2. MT-cell acute lymphoblastic leukemia (T-ALL) involve transcription factors that are master regulators of cell fate. These on- Significance cogenic transcription factors determine the gene signature and leukemic cell types (1). Whether these DNA-bound factors may Understanding how cell cycle and cell differentiation are coordinated have additional roles beyond modulating gene expression remains during normal hematopoiesis will reveal molecular insights in leu- unknown. LMO2, a 17-kDa protein defined by tandem zinc finger kemogenesis. LIM-only 2 (LMO2) is a transcriptional regulator that domains, is an essential nucleation factor that assembles a multi- controls the erythroid lineage via activation of an erythroid-specific partite transcriptional regulatory complex on gene regulatory re- gene expression program. Here, we uncover an unexpected function gions via direct interaction with the TAL1/SCL transcription factor, for LMO2 in controlling DNA replication via protein–protein inter- LDB1, and other DNA binding transcription factors (2–4, reviewed actions with essential DNA replication enzymes. To our knowledge, in refs. 5, 6). These complexes drive gene expression programs that this work provides the first evidence for a nontranscriptional func- determine hematopoietic cell fates at critical branchpoints both tion of LMO2 that drives the cell cycle at the expense of differenti- during embryonic development (7) and in adult hematopoietic stem ation in the erythroid lineage and in thymocytes when misexpressed cells (8, 9). Lmo2 function is essential in highly proliferative ery- following genetic alterations. We propose that the nontranscrip- throid progenitors (10, reviewed in refs. 5, 6). Interestingly, Lmo2 tional control of DNA replication uncovered here for LMO2 may be a down-regulation is required for terminal erythroid differentiation more common function of oncogenic transcription factors than (11, 12). Because commitment to terminal differentiation is previously appreciated. coordinated with growth arrest (13), Lmo2 may have additional Author contributions: M.-C.S., M.H., B.G., and T.H. designed research; M.-C.S., M.H., B.G., molecular functions that impede this critical step marked by V.L., D.F.T.V., A.H., and N.M. performed research; A.V. contributed new reagents/analytic growth cessation. tools; 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; In mouse models of T-ALL, LMO1 or LMO2 collaborates with and M.-C.S., M.H., B.G., E.B.A., A.V., and T.H. wrote the paper. SCL to inhibit the activity of two basic helix–loop–helix (bHLH) The authors declare no conflict of interest. transcription factors that control thymocyte differentiation, E2A/ This article is a PNAS Direct Submission. TCF3 and HEB/TCF12, causing differentiation arrest (reviewed in Freely available online through the PNAS open access option. ref. 14). However, this inhibition is not sufficient, per se, for leu- 1M.-C.S. and M.H. contributed equally to this work. kemogenesis, because both TAL1 and LYL1 inhibit E proteins but 2Deceased August 3, 2015. require interaction with LMO1/2 to activate the transcription of 3To whom correspondence should be addressed. Email: [email protected]. a self-renewal gene network in thymocytes (15, 16) and to in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. duce T-ALL (17, 18). Of note, downstream target genes cannot 1073/pnas.1515071113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1515071113 PNAS | February 2, 2016 | vol. 113 | no. 5 | 1393–1398 Downloaded by guest on September 26, 2021 Results also detected the other members of the MCM hexameric com- Identification of New LMO2 Protein–Protein Interactions in Hematopoietic plex (MCM2–MCM7), as well as ORC2, CDC6, CDT1, and + Progenitors. Lmo2 is expressed in c-Kit hematopoietic stem and PCNA, albeit with weaker efficiency for the latter three (Fig. F progenitor cells (HSPCs) and in immature prothymocytes, but not at 1 ). In contrast, MCM6, POLD1, and PRIM1 did not coim- G later stages of T-cell differentiation (29). To identify new LMO2 munoprecipitate with SCL (Fig. 1 ), whereas LMO2 and LDB1 binding proteins in HSPCs, we constructed a cDNA library from were found with SCL in this assay, as expected. Therefore, replica- + − purified murine Kit Lin hematopoietic progenitors for a yeast two- tion proteins do not stably associate with the SCL transcription hybrid screen and used LMO2 as bait. In addition to known LMO2- complex but more specifically with LMO2. interacting proteins, such as LDB1, and to proteins associated with LMO2 Binds to Origins of Replication. LMO2 shares important transcription, we unexpectedly identified interactions with three es- transcriptional targets with SCL (15, 16, 25, 36). Nonetheless, sential components of prereplication complexes (pre-RCs), LMO2 is rarely found at gene promoters (3%) by ChIP sequencing namely, MCM6, POLD1, and PRIM1 (30) (Fig. 1A and Table S1). in multipotent progenitors, whereas SCL is more frequently ob- In comparison, a screen performed using GAL4-SCL identified only served within transcriptional start sites (28%) (24), suggesting that known interactions (Table S1). LMO2 interaction was specific to LMO2 has SCL-independent functions. We assessed whether these three replication proteins, as confirmed by independent yeast B C LMO2 associates with DNA replication origins, using ChIP of TF-1 two-hybrid assays with full-length cDNAs (Fig. 1 and ). In ad- cells with anti-LMO2, or anti-MCM5 antibodies. We first studied dition, we identified BAZ1A/ACF1, required for DNA replication seven well-characterized human DNA replication origins (38) and through heterochromatin (31); SetD8, for replication licensing (32); found that both MCM5 and LMO2 occupied four of these repli- MYST2/HBO1, controlling MCM loading via ORC1 binding (33); cation origins, c-MYC, G6PD, TOP1,andMCM4 (Fig. 2A), all A and CCNA2-CDK1, regulating origin firing (34) (Fig. 1 ). The top- mapping to early replicating G1 (ERG1) segments (39, 40) (Fig. ranking pathways by gene set enrichment analysis were cell cycle, S1A). In contrast, MCM5 did not bind the GYPA promoter, a well- D DNA synthesis, and DNA replication (Fig. 1 ), concurring with the defined SCL-LMO2 transcriptional target (36), whereas LMO2 view that LMO2 controls these processes via its protein partners. occupancy was confirmed, together with SCL and GATA1, two Finally, LMO2 coimmunoprecipitated with PRIM1, MCM6, and LMO2 transcription factor partners. SCL was detected at two of the MYST2 in mammalian cells, and both LIM domains contributed to seven tested origins, although binding was 10- to 20-fold lower this interaction (Fig. 1E), whereas LDB1 binding required mostly compared with GYPA, whereas GATA1 binding was below the LIM1, as expected (4). We conclude that in addition to its associa- detection limit (Fig. 2A). Therefore, LMO2 is recruited to well- tion with transcription regulators, LMO2 engages in protein–protein characterized DNA replication origins with MCM5, in the absence interactions with DNA replication proteins. of GATA1 and frequently of SCL. These results led us to assess LMO2 occupancy of replication LMO2 Associates with Replication Complexes. We next addressed initiation zones identified in two independent tiling array-based the question of whether LMO2 associates with the pre-RC in studies in HeLa cells, using Lambda-exonuclease digestion (38, 41) hematopoietic cells. Because pre-RC formation often requires a and anti-BrdU immunoprecipitation to purify origin-centered na- DNA template (35), we prepared DNA-containing chromatin + + scent DNA strands. We focused on a subset of 45 replication ini- extracts from CD34 Kit progenitors (TF-1 cells) (3, 13, 36, 37) tiation zones that overlapped within a distance of 1 kb between the to assess the interaction of LMO2 with endogenous replication two studies (Fig. 2B and Fig. S1 B and C). In TF-1 cells, MCM5 and proteins by immunoprecipitation. We found that POLD1, PRIM1, LMO2 were each detected on 20 and 19 replication initiation zones, and MCM6, but not lamin B or beta-actin (negative controls), respectively, nearly half of which were positive for both (Fig. 2B). reproducibly and specifically coimmunoprecipitate with LMO2, We aligned these 45 replication initiation zones with ERG1 seg- but not with control Ig, in TF-1 chromatin extracts (Fig. 1F). We ments identified in mammalian cells (39, 42) and found that 68% of

LMO2 A B 4 C

3 MCM6 PRIM1 POLD1 LDB1 Transcription (total 2 LMO2 39 16 12 26 Fig. 1. LMO2 interacts with DNA replication proteins. =52) Lamin 0000 LMO2 1 (A)NewLMO2protein–protein interactions in hema- TAF6LTTA 66LL Empty 0000

FLIILIIL interactions 0 LDBLDLLDB1DBB Tp53 0000 topoietic progenitors revealed by yeast two-hybrid GTGTFGTF3C2TF3C2F3C2 MLMMLLMLL2L

6 screens using LMO2 as bait (*). (B and C)LMO2spe- LDB1 CDT1 PCNA D R MCM2 MCM5 MCM6

PSME3PS PRIM1 RPA32

Chromatin MYST2MYYST22 POLD1 MCM10 cifically interacts with MCM6, PRIM1, and POLD1 by modification SETD8ETETD8D8 PSMC1PS 1 FD 4 POPPOLDPOLD1OLD yeast two-hybrid assay. (D) Gene set enrichment anal- ACF1A F CCNA2CCC 2 HIPK2 IP 2 ysis for LMO2 interaction partners. Shown are the top MCM6MCM UHRF1 -log(10) BUB1BU F 0 seven Reactome pathways significantly enriched in the

G1-S input LMO2 IgG N PRIM1P M CDK9 LMO2 Y2H screen [false discovery rate (FDR): q < 0.001] POLD1 DNA according to the Molecular Signatures database v5.0 DNA PRIM1 S PHASE

replication Cell cycle MCM2 E MITOTIC (PubMed identifier 16199517). (E) Both LIM domains CELL CYCLE of LMO2 are required for interaction with PRIM1, MCM3 YCL S TRANSITION MCM6, and MYST2. GAL4-LMO2 WT or LIM1/2 mutants

L C MCM5 G1 DNA REPLICATIO

SYNTHESIS OF E EL were cotransfected with Flag-tagged PRIM1, MCM6, or

PRIM1 MCM6 MYST2 C pcDNA3 LDB1 MCM6 DNA STRAND ELONG. MYST2. Protein extracts were immunoprecipitated with GAL4-LMO2 + + + + + MCM7 IP SN Δ anti-FLAG Ab, followed by Western blotting. (F) DNA GAL4- LIM1 + + + + + ORC2 G GAL4-ΔLIM2 + + + + + replication proteins coimmunoprecipitate with LMO2 CDC6 inputIgG SCLIgG SCL (**). Immunoprecipitation of TF-1 chromatin extracts IB: GAL4 CDT1 SCLp42 * with Abs against LMO2. Inputs (4%) and immune pel- PCNA SCLp22 * lets (IP) were analyzed by IB. (G) SCL does not stably

IP FLAG IP SCL FLAG MCM6 associate with DNA replication proteins. SCL or isotype LDB1 LMO2 control Abs were analyzed as in F (*). IB, immunoblot- LMO2 POLD1 ting; IP, immune pellet; SN, supernatant. Data shown LaminB GAL4 PRIM1 are typical of at least two (*) or three (**) independent

Input β -actin experiments.

1394 | www.pnas.org/cgi/doi/10.1073/pnas.1515071113 Sincennes et al. Downloaded by guest on September 26, 2021 ABImmunoprecipitated Tethering LMO2 to DNA Recruits Replication Proteins and Induces DNA chromatin (fold enrichment) ChIP ERG1* Replication. To determine whether LMO2 tethering to DNA was 1 2 5 10 20 50 100 sufficient to stimulate DNA replication, we optimized a synthetic GYPA promoter: DNA replication assay in mammalian cells described by Takeda LMO2 MCM5 1 Study 2 Study G Annot_Ori (3-4) et al. (43). We assessed whether LMO2 fused to the DNA binding MYC: exon1 24 mix 264 mix 272 mix domain of GAL4 can drive DNA replication from GAL4 binding e2 G e1 G 193 mix 50 sites (5XUAS) in transfected 293 cells (Fig. 2C). Newly replicated G6PD: 93 n.d. 130 155 DNA is hemimethylated or unmethylated and can be distinguished e1 e2 54 165 TOP1: 194 mix from transfected, bacterially derived DNA by its resistance to Dpn1 29 n.d. 220 K *Early replicating G1 segment e1 e1 221 K digestion (43). As expected, GAL4-ORC2 induced an approx- MCM4: 23 217 K imately fivefold increase in newly synthesized DNA compared (PRDKC) (MCM4) 87 n.d. 88 D e1 G GG 212 with control GAL4-VP16 (Fig. 2 ). In this assay, GAL4-LMO2 HSP70: 162 n.d. 117 126 reproductively directed a dose-dependent increase of newly repli- 163 e2 G LMO2 167 cated DNA, reaching a maximum of eightfold (Fig. 2D), whereas DNMT1: 190 mix MCM5 259 mix n.d. 188 mix GAL4-SCL did not produce the same results. This activity was e1 e12 G 53 K SCL 225 K LMNB2: 105 abrogated when GAL4-binding UAS sequences were mutated (Fig. (TIMM13) (LMNB2) n.d. GATA-1 151 0.5 kb 218 K E 219 K 2 ) or absent (pBluescript vector). We therefore conclude that 228 K 230 K LMO2 anchoring to DNA was sufficient to direct DNA replication. C 237 DNA replication assay DNA capture assay (F) 20 202 LMO2-driven DNA replication occurred most likely in the absence (D, E) 206 GAL4-LMO2 + UAS WTO 207 208 of transcription because 293T cells lack hematopoietic transcrip- UAS WT nuc. extract or UAS mutO 209 NdeI DpnI DpnI 210 233 tion factors that recruit LMO2 to DNA, namely SCL and GATA1/ Bound proteins 241 UAS mut XXXXX 2. To determine if DNA-bound GAL4-LMO2 could recruit DNA NdeI DpnI DpnI Western blotting mix: early or late K: early in K562 replication proteins to the UAS template, we performed DNA capture as previously described (44), using UAS sequences D 10 E 400 immobilized on beads. LMO2 recruited POLD1, MCM5, and, to a F 150 8 POLD1 lesser extent, CDT1 and PCNA to DNA, and this recruitment 300 6 MCM5 100 required the integrity of the GAL4 binding site (Fig. 2F). Together, (% control) 200 4 CDT1 75 our results indicate that LMO2 tethering to DNA was sufficient to MEDICAL SCIENCES 2 100 GAL4 50 nucleate the assembly of prereplication/preinitiation complexes plasmid Replicated DNA specifically at the site of binding and to direct DNA replication.

0 Extra chromosomal PCNA (Fold over control) 0 37 Gal4-VP16 + ------VP16 LMO2 UAS Wt Mut Gal4-ORC2 --+ --- -- Gal4- LMO2 Expression Levels Control the Rate of DNA Synthesis and Gal4-LMO2 -- - - - UAS Cellular Outcome in the Erythroid Lineage. Cell cycle is highly reg- Gal4-SCL ---- - Wt mut pBS ulated in the erythroid lineage because ∼80% of primary proerythroblasts (E1 stage; Fig. 3A) are in S phase, exactly at the 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, onset of erythropoietin (Epo) dependence (45), and this pro- anti-SCL, and anti-GATA1 Abs. Shown are the ratio of enrichment over control portion sharply drops at the E3 and E4 stages of terminal dif- Ig and background (c-Kit, −20 kb). GYPA promoter sequences were amplified as ferentiation. We observed that the E1 population segregated a control (36). (Left) Maps depicting the location of well-characterized DNA into cells with high and low endogenous LMO2 protein levels replication origins and PCR probes. Gene exons are illustrated by black boxes. (Fig. 3B), corresponding to high and low proportions of cells in PCR primers are shown as red bars. E, E47 consensus sites; G, GATA1/2 consensus S/G2/M (Fig. 3C). Strikingly, LMO2 protein levels decrease from sites. n.d., not detectable. (B) LMO2-bound replication initiation zones (gray, the E1hi to E4 stage, directly correlating with a decrease in the first column) are preferentially enriched within ERG1 segments. Forty-five rep- proportion of cells in S phase (Fig. 3C), whereas GATA1 and lication initiation zones found in common in two studies (38, 41) were analyzed D by ChIP for LMO2 binding. The replication timing of these origins according to SCL protein levels increase (Fig. 3 ). This process is highly two datasets is illustrated: study 1 [Gilbert and coworkers (42)] in leukemic coordinated because most LMO2 partners identified here, in- patients and lymphoblastoid cell lines and study 2 [Stamatoyannopoulos and cluding CCNA2, are synchronously down-regulated with Lmo2 coworkers (39)] in B cells and ES cells. A higher proportion of early replicating during differentiation from proerythroblast to orthochromatic segments (gray, last column) is found in LMO2-bound origins (P = 0.02). Mix: erythroblasts (Fig. S2). Some samples from study 1 showed early replication, whereas others showed We addressed the functional importance of LMO2 by decreasing late replication. (C) Diagram illustrating the synthetic DNA replication assay. LMO2 protein levels in erythroid progenitors via RNAi (shRNA − Plasmid DNA was transfected into HEK293 cells and detected by PCR (primer Lmo2)(Fig. S3A). Lmo2 depletion in Ter119 fetal liver erythroid pairs as shown). (Left) Dpn1 sensitivity distinguishes unreplicated methylated progenitors decreased by twofold the proportion of cells in S phase as plasmid DNA (sensitive) from replicated hemi- or unmethylated DNA (resistant). determined by DAPI staining and flow cytometry analysis, compared (D) LMO2 fused to GAL4 stimulates the replication of a GAL4-responsive plas- E Lmo2 mid in mammalian cells. HEK293 cells were transfected with the indicated GAL- with control cells (Fig. 3 ). In addition, depletion almost ab- fusion genes. After Dpn1 digestion, Dpn1-resistant extrachromosomal rogated the proliferation of primary erythroid progenitors at a key DNA was quantified by real-time PCR (n = 2). P < 0.05. (E) LMO2-induced DNA commitment step marked by Epo responsiveness (Fig. 3 F and G), − + replication depends on LMO2 tethering to DNA via GAL4 binding sites (UAS). while enhancing the generation of mature E4 cells (CD71 Ter119 ) The experiment was performed as in D.(F) LMO2 binding to artificial origins of (Fig. 3H). Therefore, high LMO2 levels are required for the Epo- + replication is sufficient to recruit DNA replication proteins specifically. DNA dependent proliferation of CD71 erythroid precursors, whereas capture was performed using immobilized WT or mutant UAS sequences on ± lowering LMO2 accelerates terminal erythroid differentiation. beads. Bound proteins were revealed by Western blotting. Data are the mean Decreased LMO2 levels caused a twofold decrease in the rate of SD of at least two independent experiments performed in duplicate. DNA synthesis monitored by the kinetics of 32P orthophosphate incorporation into synchronized mouse erythroleukemia (MEL) cells LMO2-bound zones mapped to ERG1 segments, which is almost (Fig. 3I), without causing apoptosis (Fig. S3B). Consequently, these twice the percentage of ERG1 segments found in the absence of cells failed to proliferate in culture (Fig. S3C). Decreased DNA LMO2 (Fig. 2B). Therefore, LMO2 is recruited to a significant synthesis was unlikely due to decreased expression levels for repli- proportion of initiation zones in TF-1 cells, corresponding, in most cation genes assessed by RT-quantitative PCR (Fig. S3D), consistent part, to ERG1 segments. with transcriptome analysis of erythroid cells and lymphoid cells

Sincennes et al. PNAS | February 2, 2016 | vol. 113 | no. 5 | 1395 Downloaded by guest on September 26, 2021 A B high E1 2-h delay in G1/S transition, occurring at 3 h after release TF1 MEL E1 E2 compared with 1 h for control cells (Vector), as well as delayed HSPC E1 E2 E3 E4 J E E3 low S-phase progression (Fig. 3 and Fig. S3 ), indicating that Kit+ Lin- Ter119 CD71 IgG LMO2 LMO2 levels control S-phase entry in erythroblasts. E4 To mimic retroviral integration upstream of the LMO2 locus in CD71 C Ter119 LMO2 the X-SCID gene therapy trial (19, 20, 46) and define the conse- shRNA LMO2 LMO2 E CD71+Ter119- F NT quences of ectopic expression in vivo, we delivered in Lmo2 A–F vector hematopoietic stem cells by retroviral infection, (Fig. 4 ). Upon transplantation, LMO2-transduced bone marrow cells induced

LMO2 (MFI) 44.5%

(%) T-ALL in 60% of recipient mice, with a median of 270 days (46) (Fig. 4B and Fig. S4). During the preleukemic stage (30 days), LMO2 NT E1 overexpression mostly affected thymocytes and led to an accumula- + − − − tion of CD44 CD25 CD4 D8 double-negative thymocyte (DN) 52.2% Ctrl Epo + + %S/G2/M + - cells and a decrease in CD4 CD8 double-positive thymocyte lo hi 2 3 4 CD71 Ter119 1 1 E E E G (DP) cells in the thymus, reproducing the differentiation blockade E E 3 shRNA Lmo2 NT at the DN stage reported in LMO2 transgenic mice (Fig. 4C), MFI E1 E2 E3 E4 D 2 despite the fact that the retroviral vector allowed for transgene LMO2 4.5 3.1 25.4% LMO2 Cell count expression in all cells. Elevating in thymocytes modified the shRNA GATA1 1 Lmo2 cell cycle status of thymocyte progenitors (Fig. 4D) without af- 7.41820 DAPI SCL fecting bone marrow stem cells (Fig. S4B), consistent with the role Fold expansion 0 01234 of LMO2 as a T-cell specific oncogene (46). Interestingly, elevating H Abundance ratio of E1-4 I Time (d) LMO2 Lmo2 enhanced the cell cycle status of both DN1 and DP cells, subsets in MEL J shRNA/control suggesting that differentiation blockade (lower number of DP cells) MEL could be due to increased cell cycle. Furthermore, when DN1 thymocytes were synchronized in G0/G1 and released in culture, time(h) E1 E2 E3 E4 Vector Vector shRNA LMO2-expressing cells entered more rapidly into S phase com- 0 3.1 0.5 0.5 3.4 Lmo2 E shRNA pared with control cells (vector) (Fig. 4 ), indicating that LMO2 24 0.5 0.9 3.5 2.5 Lmo2 facilitates the G1-S transition and S phase progression. 48 0.1 1.1 5.4 96.9 The above results indicate that LMO2 overexpression in hema- 72 0.1 2.6 4.2 94.2 Time (h) S phase (% total) Time (h) Newly synthesized DNA synthesized Newly topoietic cells reproduced the T-cell proliferation phenotype reported for preleukemic patients in the X-SCID gene therapy trial. Fig. 3. LMO2 levels determine the proliferation of erythroid progenitors. (A) Diagram of erythroid differentiation according to flow cytometry profiles. Actively dividing cells are more sensitive to 5-fluorouracil (5-FU), an − + − (B) LMO2 levels in bone marrow erythroid progenitors (Lin CD71 Ter119 )cor- antimetabolite that inhibits thymidylate synthase and therefore de- relate with their cell cycle status by Hoechst staining: E1low and E1high (P ≤ 0.05). pletes the pool of dTTP. Consistent with increased DNA replication, The mean fluorescence intensity (MFI) for LMO2 per cell was assessed by flow LMO2-expressing thymocytes cocultured with MS5 stromal cells cytometry. (C) Correlation between LMO2 levels and the proportion of cells in expressing DL4 were 100-fold more sensitive than WT thymocytes to S/G2/M during erythroid differentiation (E1 to E4). (D) Expression levels of LMO2, 5-FU (EC50 of5nMvs.500nM)(Fig.4F). SCL, and GATA1 during erythroid differentiation. (E) Decreased LMO2 in erythroid progenitors reduced the fraction of cells in S phase. The shRNA Lmo2 was Discussion − delivered in Ter119 fetal liver cells, which were then stimulated with Epo for 2 d. + − LMO2 has a well-established function in transcriptional regulation The cell cycle in erythroid progenitors (E1, CD71 Ter119 ) was analyzed by DAPI via direct interaction with transcription factors, mostly of the bHLH staining. (F) Lmo2 is required for the response of proerythroblasts to Epo. NT, – nontarget (control). (G) LMO2 levels control the proliferation of erythroid progen- family, SCL/TAL1, TAL2, and LYL1 or GATA proteins (2 4). In itors in culture. (H) Decreased LMO2 abolishes the growth of erythroid progenitors this study, we revealed unexpected new functions of LMO2 in he- (E1) in response to Epo but favors terminally differentiating erythroid cells (E4). matopoiesis and leukemogenesis through a yeast two-hybrid screen (I) Decreased DNA synthesis induced by shRNA-mediated Lmo2 depletion. MEL cells of LMO2 interaction partners in hematopoietic progenitors. Our were purified in G0/G1 and released in culture for different times in the presence of observations indicate that LMO2 controls cell fate by directly pro- α32P-dCTP. After electrophoresis, total DNA was quantified by autoradiography (n = moting DNA replication, a hitherto unrecognized mechanism that 2). The slopes of the two curves were 0.48 ± 0.02 (Vector) and 0.29 ± 0.01 (shLmo2). might also account for its oncogenic properties. < *P 0.0001. (J) Decreased proportion of cells in S phase after Lmo2 depletion. MEL Our study unravels unexpected interactions between LMO2 and cells expressing shLmo2 or control (Vector) were purified in G0/G1 and analyzed for cell cycle progression at different time points in culture by Hoechst staining (n = 3). three essential replication proteins, MCM6, PRIM1, and POLD1 All data shown are typical of at least two independent experiments. (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 to in which Lmo2 was knocked down or overexpressed, respectively DNAviatheGAL4-DNAbindingdomain was sufficient to recruit (12, 46). Furthermore, replication genes were not occupied by the MCM5 and POLD1 to DNA and to transform UAS into origins of SCL–LMO2 complex in leukemic T cells (25). Together, these ob- replication, indicating that LMO2 directly controls DNA replication. servations indicate a critical role for LMO2 in erythroid cell fate, via Unlike other transcription complexes that have a dual role in DNA the control of DNA replication and the cell cycle that drives Epo- replication (48), LMO2 interaction with the RC is distinct from the dependent expansion of erythroid progenitors while impeding their well-known recruitment of LMO2 to the SCL transcription complex. commitment to terminal maturation. Our data are in line with the observations that there is only a partial overlap between SCL and LMO2 chromatin occupancy in hema- LMO2 Expression Regulates S-Phase Entry. We next addressed the topoietic progenitors (24). importance of LMO2 levels on the kinetics of cell cycle pro- LMO2 binding to replication initiation zones reported here gression (Fig. 3J). Briefly, we found that viable MEL cells with overlaps with early G1 replication segments described in lymphoid low side and forward scatter properties are mostly in G0/G1 (Fig. cells (39, 42), a possibility that may be favored by its interaction with S3E). These G0/G1 cells were purified and released in culture MLL2 (Fig. 1A). Indeed, the H3K4me3 histone mark found in early medium to analyze their DNA content by Hoechst staining (Fig. replicating domains (40) can be controlled by MLL2. In eukaryotes, S3E). Decreased LMO2 (shRNA Lmo2) reproducibly caused a origins are licensed in excess, and not all licensed origins are active

1396 | www.pnas.org/cgi/doi/10.1073/pnas.1515071113 Sincennes et al. Downloaded by guest on September 26, 2021 A B regulation, in agreement with previous work indicating that LMO2 overexpression prevents terminal erythroid differentiation (11). We conclude that LMO2 down-regulation is required for the switch to terminal erythroid differentiation due to the implication of LMO2 in DNA replication/cell cycle (Fig. 4G). It is well established that transcription factor gene networks drive hematopoietic cell development and lineage outcome, via synergistic or antagonistic interactions (reviewed in refs. 52 and 53). C D It is unclear how LMO2 inhibits cell differentiation in both the * erythroid (11) and T-lymphoid lineages (reviewed in ref. 53). * * We now propose that LMO2-dependent DNA replication in both lineages governs the switch between a proliferative state in * progenitors and commitment to terminal differentiation (Fig. 4G). Failure to regulate this proliferative switch caused by ectopic LMO2 expression (19) may lead to T-ALL. Oncogene-induced DNA replication stress (54) could lead to E F G replication errors and ultimately cause genetic lesions, such as activating NOTCH1 mutations (55, 56), that convert preleukemic stem T cells (15, 16) into leukemia initiating cells (15). Two other LIM-only proteins, LMO1 and LMO4, are also important determinants of cell

T cycle progression in neuroblastoma (23) and in breast cancer associated with genomic instability (22), respectively, suggesting that the mechanism(s) described here may be extended to these proteins. Emerging evidence indicates that oncogenes, such as c-MYC or Fig. 4. Ectopic LMO2 expression in thymocytes triggers G1/S transition, T-cell HOXD13, can be part of nontranscriptional complexes involved hyperproliferation, and T-ALL development. (A) Experimental strategy to deliver in DNA replication (54, 57). Taken together, we propose that on- LMO2 in hematopoietic cells using the murine stem cell virus (MSCV) retroviral cogenic transcription factors in acute leukemias and possibly other

vector. BM, bone marrow. (B) LMO2 overexpression induces T-ALL in mice. tumor types transform cells by at least two DNA-dependent mech- MEDICAL SCIENCES Kaplan–Meiercurvesshowingthetimeofleukemia onset in mice transplanted anisms, the control of gene expression programs, as initially pro- with MSCV-LMO2 or MSCV transduced cells. (C and D) LMO2 overexpression leads posed (1), but also by deregulating DNA replication (42), with both to 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)LMO2over- processes being important determinants of cell fate. expression induces an increase in the percentage of DN1 cells in S phase. DN1 cells were purified in G0/G1 and analyzed for cell cycle progression by DAPI staining at Materials and Methods different time points after coculture with MS5-DL4 stromal cells. (F)LMO2- Yeast Two-Hybrid. LMO2 full-length protein was subcloned in the pGBKT7 expressing thymocytes are more sensitive to 5-FU treatment in vitro. Cells were vector and transformed in the Y187 yeast strain. The cDNA library from + − cocultured on MS5-DL4 stromal cells and treated with 5-FU at the indicated doses. c-Kit Lin murine bone marrow cells was constructed in the pGADT7 vector + Viable Thy1 cells were scored by flow cytometry. (G) Proposed model: LMO2 with the Matchmaker Library Construction and Screening Kit (BD Biosci- controls DNA replication, favors progenitor cell proliferation, and inhibits com- ences) and transformed in the AH109 yeast strain. The screen (selection of + + + + mitment to terminal differentiation in the erythroid and T-lymphoid lineages. Ade His Leu Trp colonies) was performed by yeast mating. Positive clones Data shown are typical of at least two independent experiments. were isolated and sequenced. To confirm the interactions and check for specificity, full-length cDNAs for Polδ1, Prim1, Mcm2-5-6-10, Cdt1, Pcna,and Caf1 were subcloned into pGADT7 and transformed in AH109 cells. during a given cell cycle, which requires recruitment of DNA poly- merases to initiate DNA synthesis (30). Accordingly, we show that Retroviral Gene Transfer and RNAi. LMO2 retroviral gene transfer and mouse LMO2 levels influence the rate of DNA replication in MEL cells. bone marrow transplantation were performed as described (8). For RNAi ex- Although we focused on the basic components of the pre-RC periments, vesicular stomatitis virus (VSV-G)–producing cells were transfected with plasmids encoding shRNAs against LMO2, with a nontarget shRNA or with in the present study, several other proteins identified in the − screen could have a regulatory function. For example, cyclin the empty pLKO.1 vector (Sigma). MEL cells or Lin fetal liver cells were incubated with VSV-G supernatant for 48 h and then selected with puromycin as A2-CDK2 favors the onset of DNA replication at the G1-S described (8). All mice were kept under pathogen-free conditions according to transition and prevents rereplication during S phase (49). institutional animal care and use guidelines. The protocols for gene transfer These possibilities would be consistent with the effects of and transplantation in mice were approved by the Committee of Ethics and LMO2 levels on the kinetics of G1-S transition that we ob- Animal Deontology of the University of Montreal. served in two cell types, delayed when Lmo2 is knocked down in murine erythroleukemic cells and, conversely, accelerated in Flow Cytometry, Cell Cycle Analysis, and Cell Sorting. Flow cytometry, cell cycle LMO2-overexpressing DN1 thymocytes. In addition, LMO2 analysis, and cell sorting are described in Supporting Information. associates with two cell cycle checkpoint proteins, CDK9 and BUB1B (Table S1). CDK9 associates with cyclin K in replica- 32P Orthophosphate Labeling and Quantification of de Novo DNA Synthesis. tion stress response and prevents DNA damage in replicating MEL cells synchronized in G0/G1 were incubated with α32P-dCTP (100 μCi/mL; 5 cells (50). BUB1B is an essential component of the mitotic PerkinElmer) in DMEM [10 mM Hepes (pH 7.4), 10% (vol/vol) FCS] at 8 × 10 cells checkpoint. The importance of these cell cycle proteins for per milliliter for the indicated times. Cells were lysed in DNA extraction buffer · LMO2-induced cell proliferation remains to be addressed. [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- Cell cycle is a highly regulated process in the erythroid lineage. + − solved on an alkaline (NaOH) agarose gel. The gel was dried on a Biodyne CD71 Ter119 proerythroblasts represent a critical transitional membrane (Pall Corporation). 32P incorporation was visualized by autoradi- stage marked by Epo dependency (51) and elevated DNA repli- ography and quantitated using ImageQuant software (GE Healthcare). cation (45), both shown here to require high LMO2 protein expression. Conversely, terminal erythroid differentiation to the − + Protein Extraction, Immunoprecipitation, Immunoblot, Abs, RNA, and ChIP Analysis. CD71 Ter119 stage is necessarily coordinated with growth ar- Protein extraction, immunoprecipitation, immunoblot (IB), Abs, RNA, and ChIP rest (13, 51), which we now show to be favored by Lmo2 down- analysis methods are fully described in Supporting Information. Primer sequences

Sincennes et al. PNAS | February 2, 2016 | vol. 113 | no. 5 | 1397 Downloaded by guest on September 26, 2021 used are shown in Tables S2 and S3, and Abs used for ChIP, IB, and immuno- retroviral gene transfer and the yeast two-hybrid, and Dr. Jana Krosl for critical fluorescence are listed in Table S4. comments on the manuscript. This work was funded by the Cancer Research Society, Inc. (2012–2014); the Canadian Institutes for Health Research (CIHR; Grant MOP111050, 2011–2016); Canadian Cancer Society Research Institute Grant Transient Replication Assay in Mammalian Cells. The transient replication 019222 (to T.H.); the Leukemia & Lymphoma Society (2013–2015) (T.H. and assay is adapted from Takeda et al. (43) and described in Supporting E.B.A.); CIHR Grant 89928 (to A.V.); a CIHR multiuser grant to support the flow Information. cytometry and imaging service; and a group grant from the Fonds de Recherche du Québec-Santé to support, in part, IRIC infrastructure. M.-C.S. was supported by ACKNOWLEDGMENTS. We thank Danièle Gagné [Institute of Research in Immu- a Canada Graduate Scholarship Doctoral Award (CIHR) and a doctoral award from nology and Cancer (IRIC)] for her assistance with flow cytometry, Véronique the Cole Foundation. M.H. was supported by a postdoctoral fellowship award Litalien for mouse handling, Geneviève Boucher for bioinformatic analyses, of the Swiss National Foundation (PBBEP3 144798), by Swiss Foundation Francis Migneault and David Flaschner for assistance with the yeast two-hybrid for Fellowships in Medicine and Biology, and by Novartis (P3SMP3 151720). confirmation assays, Drs. Jalila Chagraoui and Richard Martin for help with the V.L. and D.F.T.V. were supported by Cole Foundation awards.

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