The LMO2 oncogene regulates DNA replication in hematopoietic cells

Marie Sincennes

Magali Humbert

Benoît Grondin

Véronique Lisi

El Affar+2 authors

^{Institute of Research in Immunology and Cancer, University of Montreal, Montreal, QC, Canada, H3C 3J7;}

^{Molecular Biology Program, University of Montreal, Montreal, QC, Canada, H1T 2M4;}

^{Cancer Research Center of Toulouse, Toulouse 31024, France;}

^{Maisonneuve-Rosemont Hospital Research Center, Department of Medicine, University of Montreal, Montreal, QC, Canada, H1T 2M4;}

^{Departments of Pharmacology and Biochemistry, University of Montreal, Montreal, QC, Canada, H3T 1J2}

^{To whom correspondence should be addressed. Email:}ac.laertnomu@gnaoh.gnart.

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

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/analytic 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; and M.-C.S., M.H., B.G., E.B.A., A.V., and T.H. wrote the paper.

^{M.-C.S. and M.H. contributed equally to this work.}

^{Deceased August 3, 2015.}

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

Freely available online through the PNAS open access option.

Significance

Understanding how cell cycle and cell differentiation are coordinated during normal hematopoiesis will reveal molecular insights in leukemogenesis. LIM-only 2 (LMO2) is a transcriptional regulator that controls the erythroid lineage via activation of an erythroid-specific gene expression program. Here, we uncover an unexpected function for LMO2 in controlling DNA replication via protein–protein interactions with essential DNA replication enzymes. To our knowledge, this work provides the first evidence for a nontranscriptional function of LMO2 that drives the cell cycle at the expense of differentiation in the erythroid lineage and in thymocytes when misexpressed following genetic alterations. We propose that the nontranscriptional control of DNA replication uncovered here for LMO2 may be a more common function of oncogenic transcription factors than previously appreciated.

Keywords: LMO2, cell cycle, DNA replication, hematopoietic cells, T-cell acute lymphoblastic leukemia

Significance

Abstract

Oncogenic transcription factors are commonly activated in acute leukemias and subvert normal gene expression networks to reprogram hematopoietic progenitors into preleukemic stem cells, as exemplified by LIM-only 2 (LMO2) in T-cell acute lymphoblastic leukemia (T-ALL). Whether or not these oncoproteins interfere with other DNA-dependent processes is largely unexplored. Here, we show that LMO2 is recruited to DNA replication origins by interaction with three essential replication enzymes: DNA polymerase delta (POLD1), DNA primase (PRIM1), and minichromosome 6 (MCM6). Furthermore, tethering LMO2 to synthetic DNA sequences is sufficient to transform these sequences into origins of replication. We next addressed the importance of LMO2 in erythroid and thymocyte development, two lineages in which cell cycle and differentiation are tightly coordinated. Lowering LMO2 levels in erythroid progenitors delays G1-S progression and arrests erythropoietin-dependent cell growth while favoring terminal differentiation. Conversely, ectopic expression in thymocytes induces DNA replication and drives these cells into cell cycle, causing differentiation blockade. Our results define a novel role for LMO2 in directly promoting DNA synthesis and G1-S progression.

Abstract

More than 70% of recurring chromosomal translocations in T-cell acute lymphoblastic leukemia (T-ALL) involve transcription factors that are master regulators of cell fate. These oncogenic transcription factors determine the gene signature and leukemic cell types (1). Whether these DNA-bound factors may have additional roles beyond modulating gene expression remains unknown. LMO2, a 17-kDa protein defined by tandem zinc finger domains, is an essential nucleation factor that assembles a multipartite transcriptional regulatory complex on gene regulatory regions via direct interaction with the TAL1/SCL transcription factor, LDB1, and other DNA binding transcription factors (2 –4, reviewed in refs. 5, 6). These complexes drive gene expression programs that determine hematopoietic cell fates at critical branchpoints both during embryonic development (7) and in adult hematopoietic stem cells (8, 9). Lmo2 function is essential in highly proliferative erythroid progenitors (10, reviewed in refs. 5, 6). Interestingly, Lmo2 down-regulation is required for terminal erythroid differentiation (11, 12). Because commitment to terminal differentiation is coordinated with growth arrest (13), Lmo2 may have additional molecular functions that impede this critical step marked by growth 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 in ref. 14). However, this inhibition is not sufficient, per se, for leukemogenesis, because both TAL1 and LYL1 inhibit E proteins but require interaction with LMO1/2 to activate the transcription of a self-renewal gene network in thymocytes (15, 16) and to induce T-ALL (17, 18). Of note, downstream target genes cannot substitute for LMO1/2 to induce T-ALL, suggesting additional functions for LMO1/2.

Together, these studies underscore the dominant oncogenic properties of LMO2, as revealed by recurring retroviral integrations upstream of LMO2 in the gene therapy trial (19, 20) or by recurrent chromosomal 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 breast cancers (22) and neuroblastomas (23), pointing to their importance in cell transformation. In particular, in patients who eventually developed T-ALL associated with LMO2 activation after gene therapy, T-cell hyperproliferation was observed early during the preleukemic stage (19). How LMO2 affects erythroid progenitor or T-cell proliferation cannot be inferred from its downstream target genes (12, 24 –28).

To understand LMO2 functions, we performed an unbiased screen for LMO2 interaction partners. We show that LMO2 associates with three replication proteins, minichromosome 6 (MCM6), DNA primase (PRIM1), and DNA polymerase delta (POLD1), and that LMO2 influences cell cycle progression and DNA replication in hematopoietic cells, indicating an unexpected function for LMO2.

Illustrated are proteins for which there were at least two independent clones per screen (analysis of 6 × 10^{clones from a cDNA library of Kit}^LIN^{hematopoietic cells). LMO2 interacts with 52 proteins, including two Lys methyltransferases, MLL2 and SetD8, both controlling erythroid gene expression (}61, 62). In contrast, TAL1 interacts with six proteins only, mostly E protein transcription factors, DRG1 as well as ERG and FLI1. IP, immunoprecipitation; N/A, not available.

Fw, forward; Rv, reverse.

The BTL-73 mouse anti-SCL/Tal1 antiserum was generously provided by D. Mathieu (Institut de Génétique Moléculaire, Montpellier, France).

Acknowledgments

We thank Danièle Gagné [Institute of Research in Immunology and Cancer (IRIC)] for her assistance with flow cytometry, Véronique Litalien for mouse handling, Geneviève Boucher for bioinformatic analyses, Francis Migneault and David Flaschner for assistance with the yeast two-hybrid confirmation assays, Drs. Jalila Chagraoui and Richard Martin for help with the retroviral gene transfer and the yeast two-hybrid, and Dr. Jana Krosl for critical 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 019222 (to T.H.); the Leukemia & Lymphoma Society (2013–2015) (T.H. and E.B.A.); CIHR Grant 89928 (to A.V.); a CIHR multiuser grant to support the flow 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 a Canada Graduate Scholarship Doctoral Award (CIHR) and a doctoral award from the Cole Foundation. M.H. was supported by a postdoctoral fellowship award of the Swiss National Foundation (PBBEP3 144798), by Swiss Foundation for Fellowships in Medicine and Biology, and by Novartis (P3SMP3 151720). V.L. and D.F.T.V. were supported by Cole Foundation awards.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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

Footnotes

References

1. Ferrando AA, et al Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell. 2002;1(1):75–87.[PubMed][Google Scholar]
2. Wadman I, et al Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukemia. EMBO J. 1994;13(20):4831–4839.[Google Scholar]
3. Lécuyer E, et al Protein stability and transcription factor complex assembly determined by the SCL-LMO2 interaction. J Biol Chem. 2007;282(46):33649–33658.[PubMed][Google Scholar]
4. El Omari K, et al Structural basis for LMO2-driven recruitment of the SCL:E47bHLH heterodimer to hematopoietic-specific transcriptional targets. Cell Reports. 2013;4(1):135–147.[Google Scholar]
5. Lécuyer E, Hoang TSCL: From the origin of hematopoiesis to stem cells and leukemia. Exp Hematol. 2004;32(1):11–24.[PubMed][Google Scholar]
6. Love PE, Warzecha C, Li LLdb1 complexes: The new master regulators of erythroid gene transcription. Trends Genet. 2014;30(1):1–9.[Google Scholar]
7. Org T, et al Scl binds to primed enhancers in mesoderm to regulate hematopoietic and cardiac fate divergence. EMBO J. 2015;34(6):759–777.[Google Scholar]
8. Lacombe J, et al Scl regulates the quiescence and the long-term competence of hematopoietic stem cells. Blood. 2010;115(4):792–803.[PubMed][Google Scholar]
9. Li L, et al Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells. Nat Immunol. 2011;12(2):129–136.[Google Scholar]
10. Warren AJ, et al The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell. 1994;78(1):45–57.[PubMed][Google Scholar]
11. Visvader JE, Mao X, Fujiwara Y, Hahm K, Orkin SHThe LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation. Proc Natl Acad Sci USA. 1997;94(25):13707–13712.[Google Scholar]
12. Fujiwara T, Lee HY, Sanalkumar R, Bresnick EHBuilding multifunctionality into a complex containing master regulators of hematopoiesis. Proc Natl Acad Sci USA. 2010;107(47):20429–20434.[Google Scholar]
13. Goardon N, et al ETO2 coordinates cellular proliferation and differentiation during erythropoiesis. EMBO J. 2006;25(2):357–366.[Google Scholar]
14. Anderson MKAt the crossroads: Diverse roles of early thymocyte transcriptional regulators. Immunol Rev. 2006;209:191–211.[PubMed][Google Scholar]
15. Gerby B, et al SCL, LMO1 and Notch1 reprogram thymocytes into self-renewing cells. PLoS Genet. 2014;10(12):e1004768.[Google Scholar]
16. McCormack MP, et al The Lmo2 oncogene initiates leukemia in mice by inducing thymocyte self-renewal. Science. 2010;327(5967):879–883.[PubMed][Google Scholar]
17. Chervinsky DS, et al Disordered T-cell development and T-cell malignancies in SCL LMO1 double-transgenic mice: Parallels with E2A-deficient mice. Mol Cell Biol. 1999;19(7):5025–5035.[Google Scholar]
18. Homminga I, et al Characterization of a pediatric T-cell acute lymphoblastic leukemia patient with simultaneous LYL1 and LMO2 rearrangements. Haematologica. 2012;97(2):258–261.[Google Scholar]
19. Hacein-Bey-Abina S, et al LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302(5644):415–419.[PubMed][Google Scholar]
20. Howe SJ, et al Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest. 2008;118(9):3143–3150.[Google Scholar]
21. Boehm T, Foroni L, Kaneko Y, Perutz MF, Rabbitts THThe rhombotin family of cysteine-rich LIM-domain oncogenes: Distinct members are involved in T-cell translocations to human chromosomes 11p15 and 11p13. Proc Natl Acad Sci USA. 1991;88(10):4367–4371.[Google Scholar]
22. Montañez-Wiscovich ME, et al LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression. Oncogene. 2009;28(41):3608–3618.[Google Scholar]
23. Wang K, et al Integrative genomics identifies LMO1 as a neuroblastoma oncogene. Nature. 2011;469(7329):216–220.[Google Scholar]
24. Wilson NK, et al Combinatorial transcriptional control in blood stem/progenitor cells: Genome-wide analysis of ten major transcriptional regulators. Cell Stem Cell. 2010;7(4):532–544.[PubMed][Google Scholar]
25. Sanda T, et al Core transcriptional regulatory circuit controlled by the TAL1 complex in human T cell acute lymphoblastic leukemia. Cancer Cell. 2012;22(2):209–221.[Google Scholar]
26. Palii CG, et al Differential genomic targeting of the transcription factor TAL1 in alternate haematopoietic lineages. EMBO J. 2011;30(3):494–509.[Google Scholar]
27. Kassouf MT, et al Genome-wide identification of TAL1's functional targets: Insights into its mechanisms of action in primary erythroid cells. Genome Res. 2010;20(8):1064–1083.[Google Scholar]
28. Palomero T, et al Transcriptional regulatory networks downstream of TAL1/SCL in T-cell acute lymphoblastic leukemia. Blood. 2006;108(3):986–992.[Google Scholar]
29. Herblot S, Steff AM, Hugo P, Aplan PD, Hoang TSCL and LMO1 alter thymocyte differentiation: Inhibition of E2A-HEB function and pre-T alpha chain expression. Nat Immunol. 2000;1(2):138–144.[PubMed][Google Scholar]
30. Remus D, Diffley JFEukaryotic DNA replication control: Lock and load, then fire. Curr Opin Cell Biol. 2009;21(6):771–777.[PubMed][Google Scholar]
31. Collins N, et al An ACF1-ISWI chromatin-remodeling complex is required for DNA replication through heterochromatin. Nat Genet. 2002;32(4):627–632.[PubMed][Google Scholar]
32. Beck DB, et al The role of PR-Set7 in replication licensing depends on Suv4-20h. Genes Dev. 2012;26(23):2580–2589.[Google Scholar]
33. Wu ZQ, Liu XRole for Plk1 phosphorylation of Hbo1 in regulation of replication licensing. Proc Natl Acad Sci USA. 2008;105(6):1919–1924.[Google Scholar]
34. Katsuno Y, et al Cyclin A-Cdk1 regulates the origin firing program in mammalian cells. Proc Natl Acad Sci USA. 2009;106(9):3184–3189.[Google Scholar]
35. Méndez J, Stillman BChromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: Assembly of prereplication complexes in late mitosis. Mol Cell Biol. 2000;20(22):8602–8612.[Google Scholar]
36. Lahlil R, Lécuyer E, Herblot S, Hoang TSCL assembles a multifactorial complex that determines glycophorin A expression. Mol Cell Biol. 2004;24(4):1439–1452.[Google Scholar]
37. Kitamura T, et al Efficient screening of retroviral cDNA expression libraries. Proc Natl Acad Sci USA. 1995;92(20):9146–9150.[Google Scholar]
38. Cadoret JC, et al Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc Natl Acad Sci USA. 2008;105(41):15837–15842.[Google Scholar]
39. Hansen RS, et al Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc Natl Acad Sci USA. 2010;107(1):139–144.[Google Scholar]
40. Ryba T, et al Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types. Genome Res. 2010;20(6):761–770.[Google Scholar]
41. Karnani N, Taylor CM, Malhotra A, Dutta AGenomic study of replication initiation in human chromosomes reveals the influence of transcription regulation and chromatin structure on origin selection. Mol Biol Cell. 2010;21(3):393–404.[Google Scholar]
42. Ryba T, et al Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia. Genome Res. 2012;22(10):1833–1844.[Google Scholar]
43. Takeda DY, Shibata Y, Parvin JD, Dutta ARecruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells. Genes Dev. 2005;19(23):2827–2836.[Google Scholar]
44. Grondin B, et al c-Jun homodimers can function as a context-specific coactivator. Mol Cell Biol. 2007;27(8):2919–2933.[Google Scholar]
45. Pop R, et al A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression. PLoS Biol. 2010;8(9):e1000484.[Google Scholar]
46. Treanor LM, et al Functional interactions between Lmo2, the Arf tumor suppressor, and Notch1 in murine T-cell malignancies. Blood. 2011;117(20):5453–5462.[Google Scholar]
47. Nishiyama A, et al Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication. Nature. 2013;502(7470):249–253.[PubMed][Google Scholar]
48. Karmakar S, Mahajan MC, Schulz V, Boyapaty G, Weissman SMA multiprotein complex necessary for both transcription and DNA replication at the β-globin locus. EMBO J. 2010;29(19):3260–3271.[Google Scholar]
49. Girard F, Strausfeld U, Fernandez A, Lamb NJCyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell. 1991;67(6):1169–1179.[PubMed][Google Scholar]
50. Yu DS, et al Cyclin-dependent kinase 9-cyclin K functions in the replication stress response. EMBO Rep. 2010;11(11):876–882.[Google Scholar]
51. Zhang J, Socolovsky M, Gross AW, Lodish HFRole of Ras signaling in erythroid differentiation of mouse fetal liver cells: Functional analysis by a flow cytometry-based novel culture system. Blood. 2003;102(12):3938–3946.[PubMed][Google Scholar]
52. Graf T, Enver TForcing cells to change lineages. Nature. 2009;462(7273):587–594.[PubMed][Google Scholar]
53. Yui MA, Rothenberg EVDevelopmental gene networks: A triathlon on the course to T cell identity. Nat Rev Immunol. 2014;14(8):529–545.[Google Scholar]
54. Srinivasan SV, Dominguez-Sola D, Wang LC, Hyrien O, Gautier JCdc45 is a critical effector of myc-dependent DNA replication stress. Cell Reports. 2013;3(5):1629–1639.[Google Scholar]
55. Tremblay M, et al Modeling T-cell acute lymphoblastic leukemia induced by the SCL and LMO1 oncogenes. Genes Dev. 2010;24(11):1093–1105.[Google Scholar]
56. Weng AP, et al Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science. 2004;306(5694):269–271.[PubMed][Google Scholar]
57. Salsi V, et al HOXD13 binds DNA replication origins to promote origin licensing and is inhibited by geminin. Mol Cell Biol. 2009;29(21):5775–5788.[Google Scholar]
58. Mashtalir N, et al Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O. Mol Cell. 2014;54(3):392–406.[PubMed][Google Scholar]
59. An X, et al Global transcriptome analyses of human and murine terminal erythroid differentiation. Blood. 2014;123(22):3466–3477.[Google Scholar]
60. Wong P, et al Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes. Blood. 2011;118(16):e128–e138.[Google Scholar]
61. Demers C, et al Activator-mediated recruitment of the MLL2 methyltransferase complex to the beta-globin locus. Mol Cell. 2007;27(4):573–584.[Google Scholar]
62. DeVilbiss AW, et al Epigenetic Determinants of Erythropoiesis: Role of the Histone Methyltransferase SetD8 in Promoting Erythroid Cell Maturation and Survival. Mol Cell Biol. 2015;35(12):2073–2087.[Google Scholar]