The Ets transcription factor PU.1 is essential for inducing the differentiation of monocytes, macrophages, and B cells in fetal liver and adult bone marrow. PU.1 controls hematopoietic differentiation through physical interactions with other transcription factors, such as C/EBPα and the AP-1 family member c-Jun. We found that PU.1 recruits c-Jun to promoters without the AP-1 binding sites. To address the functional importance of this interaction, we generated PU.1 point mutants that do not bind c-Jun while maintaining normal DNA binding affinity. These mutants lost the ability to transactivate a target reporter that requires a physical PU.1-c-Jun interaction, and did not induce monocyte/macrophage differentiation of PU.1-deficient cells. Knock-in mice carrying these point mutations displayed an almost complete block in hematopoiesis and perinatal lethality. While the PU.1 mutants were expressed in hematopoietic stem and early progenitor cells, myeloid differentiation was severely blocked, leading to an almost complete loss of mature hematopoietic cells. Differentiation into mature macrophages could be restored by expressing PU.1 mutant fused to c-Jun, demonstrating that a physical PU.1-c-Jun interaction is crucial for the transactivation of PU.1 target genes required for myeloid commitment and normal PU.1 function in vivo during macrophage differentiation.

Albert Einstein College of Medicine New York NY USA

Cancer Science Institute of Singapore Singapore Singapore

Childhood Leukemia Investigation Prague Department of Pediatric Haematology and Oncology 2nd Faculty of Medicine Charles University Prague University Hospital Motol Videnska Czech Republic

Department of Biomedical Sciences College of Life and Health Sciences Chubu University Kasugai Aichi Japan

Department of Hematology oncology Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská Prague Czech Republic

Department of Medicine 1 Division of Hematology and Hemostaseology Medical University of Vienna Vienna Austria

Department of Medicine Northwestern University Chicago IL USA

Division of BioMedical Sciences Faculty of Medicine Memorial University of Newfoundland St John's NL Canada

Harvard Stem Cell Institute Harvard Medical School Boston MA 02115 USA

Institute for Research in Immunology and Cancer Department of Pharmacology and Physiology Université de Montréal Montréal QC H3C 3J7 Canada

MOMA Therapeutics Cambridge MA USA

Union Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Hubei China

Zobrazit více v PubMed

Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 2000;404:193–197. doi: 10.1038/35004599. PubMed DOI

Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell. 1990;61:113–124. doi: 10.1016/0092-8674(90)90219-5. PubMed DOI

Reddy MA, et al. Opposing actions of c-ets/PU.1 and c-myb protooncogene products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes. J. Exp. Med. 1994;180:2309–2319. doi: 10.1084/jem.180.6.2309. PubMed DOI PMC

Zhang DE, Hetherington CJ, Chen HM, Tenen DG. The macrophage transcription factor PU. 1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol. Cell. Biol. 1994;14:373–373. PubMed PMC

Hohaus S, et al. PU. 1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene. Mol. Cell. Biol. 1995;15:5830–5830. doi: 10.1128/MCB.15.10.5830. PubMed DOI PMC

Smith LT, Hohaus S, Gonzalez DA, Dziennis SE, Tenen DG. PU. 1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood. 1996;88:1234–1234. doi: 10.1182/blood.V88.4.1234.bloodjournal8841234. PubMed DOI

Iwasaki H, et al. Distinctive and indispensable roles of PU. 1 in maintenance of hematopoietic stem cells and their differentiation. Blood. 2005;106:1590–1590. doi: 10.1182/blood-2005-03-0860. PubMed DOI PMC

Staber PB, et al. Sustained PU.1 levels balance cell-cycle regulators to prevent exhaustion of adult hematopoietic stem cells. Mol. Cell. 2013;49:934–946. doi: 10.1016/j.molcel.2013.01.007. PubMed DOI PMC

Staber PB, et al. The Runx-PU.1 pathway preserves normal and AML/ETO9a leukemic stem cells. Blood. 2014;124:2391–2399. doi: 10.1182/blood-2014-01-550855. PubMed DOI PMC

Rosenbauer F, et al. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. Nat. Genet. 2004;36:624–630. doi: 10.1038/ng1361. PubMed DOI

Metcalf D, et al. Inactivation of PU. 1 in adult mice leads to the development of myeloid leukemia. Proc. Natl Acad. Sci. USA. 2006;103:1486–1486. doi: 10.1073/pnas.0510616103. PubMed DOI PMC

Burda P, et al. PU. 1 activation relieves GATA-1–mediated repression of Cebpa and Cbfb during leukemia differentiation. Mol. Cancer Res. 2009;7:1693–1693. doi: 10.1158/1541-7786.MCR-09-0031. PubMed DOI PMC

Liew CW. Molecular analysis of the interaction between the hematopoietic master transcription factors GATA-1 and PU.1. J. Biol. Chem. 2006;281:28296–28306. doi: 10.1074/jbc.M602830200. PubMed DOI

Nerlov C, Querfurth E, Kulessa H, Graf T. GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood. 2000;95:2543–2551. doi: 10.1182/blood.V95.8.2543. PubMed DOI

Stopka T, Amanatullah DF, Papetti M, Skoultchi AI. PU. 1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure. EMBO J. 2005;24:3712–3712. doi: 10.1038/sj.emboj.7600834. PubMed DOI PMC

Takemoto, C. M. et al. PU.1 positively regulates GATA-1 expression in mast cells. J. Immunol.184 4349–4361 (2010). PubMed

Zhang P, et al. Negative cross-talk between hematopoietic regulators: GATA proteins repress PU. 1. Proc. Natl Acad. Sci. USA. 1999;96:8705–8705. doi: 10.1073/pnas.96.15.8705. PubMed DOI PMC

Zhang P, et al. PU. 1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding. Blood. 2000;96:2641–2641. doi: 10.1182/blood.V96.8.2641. PubMed DOI

Walsh JC, et al. Cooperative and antagonistic interplay between PU. 1 and GATA-2 in the specification of myeloid cell fates. Immunity. 2002;17:665–676. doi: 10.1016/S1074-7613(02)00452-1. PubMed DOI

Chang HC, et al. PU. 1 regulates TCR expression by modulating GATA-3 activity. J. Immunol. 2009;183:4887–4887. doi: 10.4049/jimmunol.0900363. PubMed DOI PMC

Chang H-C, et al. PU.1 expression delineates heterogeneity in primary Th2 cells. Immunity. 2005;22:693–703. doi: 10.1016/j.immuni.2005.03.016. PubMed DOI

Petrovick MS, et al. Multiple functional domains of AML1: PU. 1 and C/EBPalpha synergize with different regions of AML1. Mol. Cell. Biol. 1998;18:3915–3915. doi: 10.1128/MCB.18.7.3915. PubMed DOI PMC

Reddy VA, et al. Granulocyte inducer C/EBPalpha inactivates the myeloid master regulator PU.1: possible role in lineage commitment decisions. Blood. 2002;100:483–490. doi: 10.1182/blood.V100.2.483. PubMed DOI

Yang Z, Wara-Aswapati N, Chen C, Tsukada J, Auron PE. NF-IL6 (C/EBPbeta) vigorously activates il1b gene expression via a Spi-1 (PU.1) protein-protein tether. J. Biol. Chem. 2000;275:21272–21277. doi: 10.1074/jbc.M000145200. PubMed DOI

Ueki N, Zhang L, Haymann MJ. Ski can negatively regulates macrophage differentiation through its interaction with PU.1. Oncogene. 2007;27:300–307. doi: 10.1038/sj.onc.1210654. PubMed DOI PMC

Laricchia-Robbio L, Premanand K, Rinaldi CR, Nucifora G. EVI1 impairs myelopoiesis by deregulation of PU. 1 function. Cancer Res. 2009;69:1633–1633. doi: 10.1158/0008-5472.CAN-08-2562. PubMed DOI

Rekhtman N, et al. PU. 1 and pRB interact and cooperate to repress GATA-1 and block erythroid differentiation. Mol. Cell. Biol. 2003;23:7460–7460. doi: 10.1128/MCB.23.21.7460-7474.2003. PubMed DOI PMC

Wei F, Zaprazna K, Wang J, Atchison ML. PU.1 can recruit BCL6 to DNA to repress gene expression in germinal center B cells. Mol. Cell. Biol. 2009;29:4612–4622. doi: 10.1128/MCB.00234-09. PubMed DOI PMC

Pongubala JM, et al. PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3’ enhancer activity. Mol. Cell. Biol. 1992;12:368–378. PubMed PMC

Meraro D, et al. Protein-protein and DNA-protein interactions affect the activity of lymphoid-specific IFN regulatory factors. J. Immunol. 1999;163:6468–6478. PubMed

Akashi K. Lineage promiscuity and plasticity in hematopoietic development. Ann. N. Y. Acad. Sci. 2005;1044:125–131. doi: 10.1196/annals.1349.016. PubMed DOI

Rekhtman N, Radparvar F, Evans T, Skoultchi AI. Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells. Genes Dev. 1999;13:1398–1411. doi: 10.1101/gad.13.11.1398. PubMed DOI PMC

Lord KA, Abdollahi A, Hoffman-Liebermann B, Liebermann DA. Proto-oncogenes of the fos/jun family of transcription factors are positive regulators of myeloid differentiation. Mol. Cell. Biol. 1993;13:841–851. PubMed PMC

Mechta-Grigoriou F, Gerald D, Yaniv M. The mammalian Jun proteins: redundancy and specificity. Oncogene. 2001;20:2378–2389. doi: 10.1038/sj.onc.1204381. PubMed DOI

Jochum W, Passegué E, Wagner EF. AP-1 in mouse development and tumorigenesis. Oncogene. 2001;20:2401–2412. doi: 10.1038/sj.onc.1204389. PubMed DOI

Passegué E, Wagner EF, Weissman IL. JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells. Cell. 2004;119:431–443. doi: 10.1016/j.cell.2004.10.010. PubMed DOI

Gaynor R, Simon K, Koeffler P. Expression of c-jun during macrophage differentiation of HL-60 cells. Blood. 1991;77:2618–2623. doi: 10.1182/blood.V77.12.2618.2618. PubMed DOI

Hass R, Prudovsky I, Kruhøffer M. Differential effects of phorbol ester on signaling and gene expression in human leukemia cells. Leuk. Res. 1997;21:589–594. doi: 10.1016/S0145-2126(97)00010-6. PubMed DOI

Sherman ML, Stone RM, Datta R, Bernstein SH, Kufe DW. Transcriptional and post-transcriptional regulation of c-jun expression during monocytic differentiation of human myeloid leukemic cells. J. Biol. Chem. 1990;265:3320–3323. doi: 10.1016/S0021-9258(19)39769-8. PubMed DOI

Grant S, et al. Effect of 1-beta-D-arabinofuranosylcytosine on apoptosis and differentiation in human monocytic leukemia cells (U937) expressing a c-Jun dominant-negative mutant protein (TAM67) Cell Growth Differ. 1996;7:603–613. PubMed

Behre G, et al. c-Jun is a JNK-independent coactivator of the PU. 1 transcription factor. J. Biol. Chem. 1999;274:4939–4939. doi: 10.1074/jbc.274.8.4939. PubMed DOI

Grondin B, et al. c-Jun homodimers can function as a context-specific coactivator. Mol. Cell. Biol. 2007;27:2919–2933. doi: 10.1128/MCB.00936-06. PubMed DOI PMC

Jia X, Lee LK, Light J, Palmer AG, Assa-Munt N. Backbone dynamics of a short PU.1 ETS domain. J. Mol. Biol. 1999;292:1083–1093. doi: 10.1006/jmbi.1999.3123. PubMed DOI

Kodandapani R, et al. A new pattern for helix-turn-helix recognition revealed by the PU.1 ETS-domain-DNA complex. Nature. 1996;380:456–460. doi: 10.1038/380456a0. PubMed DOI

Bassuk AG, Leiden JM. A direct physical association between ETS and AP-1 transcription factors in normal human T cells. Immunity. 1995;3:223–237. doi: 10.1016/1074-7613(95)90092-6. PubMed DOI

Vangala RK, et al. The myeloid master regulator transcription factor PU. 1 is inactivated by AML1-ETO in t (8; 21) myeloid leukemia. Blood. 2003;101:270–270. doi: 10.1182/blood-2002-04-1288. PubMed DOI

Uppaluri R, Towle HC. Genetic dissection of thyroid hormone receptor beta: identification of mutations that separate hormone binding and transcriptional activation. Mol. Cell. Biol. 1995;15:1499–1512. doi: 10.1128/MCB.15.3.1499. PubMed DOI PMC

Shih HM, et al. A positive genetic selection for disrupting protein-protein interactions: identification of CREB mutations that prevent association with the coactivator CBP. Proc. Natl Acad. Sci. USA. 1996;93:13896–13901. doi: 10.1073/pnas.93.24.13896. PubMed DOI PMC

Escalante CR, et al. Crystallization and characterization of PU.1/IRF-4/DNA ternary complex. J. Struct. Biol. 2002;139:55–59. doi: 10.1016/S1047-8477(02)00514-2. PubMed DOI

McKercher SR, Lombardo CR, Bobkov A, Jia X, Assa-Munt N. Identification of a PU. 1–IRF4 protein interaction surface predicted by chemical exchange line broadening. Proc. Natl Acad. Sci. USA. 2003;100:511–511. doi: 10.1073/pnas.0136910100. PubMed DOI PMC

Szabo E, Preis LH, Birrer MJ. Constitutive cJun expression induces partial macrophage differentiation in U-937 cells. Cell Growth Differ. 1994;5:439–446. PubMed

Li J, King I, Sartorelli AC. Differentiation of WEHI-3B D+ myelomonocytic leukemia cells induced by ectopic expression of the protooncogene c-jun. Cell Growth Differ. 1994;5:743–751. PubMed

Wang X, Studzinski GP. The requirement for and changing composition of the activating protein-1 transcription factor during differentiation of human leukemia HL60 cells induced by 1,25-dihydroxyvitamin D3. Cancer Res. 2006;66:4402–4409. doi: 10.1158/0008-5472.CAN-05-3109. PubMed DOI PMC

Anderson KL, Smith KA, Pio F, Torbett BE, Maki RA. Neutrophils deficient in PU. 1 do not terminally differentiate or become functionally competent. Blood. 1998;92:1576–1576. doi: 10.1182/blood.V92.5.1576. PubMed DOI

Pear WS, et al. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood. 1998;92:3780–3792. doi: 10.1182/blood.V92.10.3780. PubMed DOI

Weigelt K, Lichtinger M, Rehli M, Langmann T. Transcriptomic profiling identifies a PU.1 regulatory network in macrophages. Biochem. Biophys. Res. Commun. 2009;380:308–312. doi: 10.1016/j.bbrc.2009.01.067. PubMed DOI

Otero K, et al. Macrophage colony-stimulating factor induces the proliferation and survival of macrophages via a pathway involving DAP12 and beta-catenin. Nat. Immunol. 2009;10:734–743. doi: 10.1038/ni.1744. PubMed DOI PMC

Heinz S, et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell. 2010;38:576–589. doi: 10.1016/j.molcel.2010.05.004. PubMed DOI PMC

Zambelli F, Pesole G, Pavesi G. Pscan: finding over-represented transcription factor binding site motifs in sequences from co-regulated or co-expressed genes. Nucleic Acids Res. 2009;37:W247–W252. doi: 10.1093/nar/gkp464. PubMed DOI PMC

Portales-Casamar E, et al. JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles. Nucleic Acids Res. 2010;38:D105–D110. doi: 10.1093/nar/gkp950. PubMed DOI PMC

Badis G, et al. Diversity and complexity in DNA recognition by transcription factors. Science. 2009;324:1720–1723. doi: 10.1126/science.1162327. PubMed DOI PMC

Gangenahalli GU, et al. Stem cell fate specification: role of master regulatory switch transcription factor PU. 1 in differential hematopoiesis. Stem Cells Dev. 2005;14:140–152. doi: 10.1089/scd.2005.14.140. PubMed DOI

Tenen DG, Hromas R, Licht JD, Zhang DE. Transcription factors, normal myeloid development, and leukemia. Blood. 1997;90:489–519. doi: 10.1182/blood.V90.2.489. PubMed DOI

Spooner CJ, Cheng JX, Pujadas E, Laslo P, Singh H. A recurrent network involving the transcription factors PU.1 and Gfi1 orchestrates innate and adaptive immune cell fates. Immunity. 2009;31:576–586. doi: 10.1016/j.immuni.2009.07.011. PubMed DOI PMC

Steidl U, et al. Essential role of Jun family transcription factors in PU.1 knockdown–induced leukemic stem cells. Nat. Genet. 2006;38:1269–1277. doi: 10.1038/ng1898. PubMed DOI

Adolfsson J, et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potentiala revised road map for adult blood lineage commitment. Cell. 2005;121:295–306. doi: 10.1016/j.cell.2005.02.013. PubMed DOI

Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO J. 1997;16:4226–4237. doi: 10.1093/emboj/16.14.4226. PubMed DOI PMC

Higuchi M, et al. Expression of a conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia. Cancer Cell. 2002;1:63–74. doi: 10.1016/S1535-6108(02)00016-8. PubMed DOI

Huntly BJ, et al. MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors. Cancer Cell. 2004;6:587–596. doi: 10.1016/j.ccr.2004.10.015. PubMed DOI

Pahl HL, et al. The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter. J. Biol. Chem. 1993;268:5014–5020. doi: 10.1016/S0021-9258(18)53496-7. PubMed DOI

Behre G, Smith LT, Tenen DG. Use of a promoterless Renilla luciferase vector as an internal control plasmid for transient co-transfection assays of Ras-mediated transcription activation. BioTechniques. 1999;26:24–26. doi: 10.2144/99261bm03. PubMed DOI

DeKoter, R. P., Lee, H.-J. & Singh, H. PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity16, 297–309 (2002). PubMed

Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA. 2001;98:5116–5121. doi: 10.1073/pnas.091062498. PubMed DOI PMC

Saeed AI, et al. TM4: a free, open-source system for microarray data management and analysis. BioTechniques. 2003;34:374–378. doi: 10.2144/03342mt01. PubMed DOI

Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.8 (2015).