ABSTRACT The Ets family transcription factor PU.1 and the interferon regulatory factor (IRF)4 and IRF8 regulate gene expression by binding to composite DNA sequences known as Ets/interferon

consensus elements. Although all three factors are expressed from the onset of B-cell development, single deficiency of these factors in B-cell progenitors only mildly impacts on bone marrow

B lymphopoiesis. Here we tested whether PU.1 cooperates with IRF factors in regulating early B-cell development. Lack of PU.1 and IRF4 resulted in a partial block in development the

pre-B-cell stage. The combined deletion of PU.1 and IRF8 reduced recirculating B-cell numbers. Strikingly, all PU.1/IRF4 and ~50% of PU.1/IRF8 double deficient mice developed pre-B-cell

development and to prevent pre-B-ALL formation. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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IKAROS AND SYK Article 23 February 2021 INHIBITION OF INFLAMMATORY SIGNALING IN _PAX5_ MUTANT CELLS MITIGATES B-CELL LEUKEMOGENESIS Article Open access 05 November 2020 UPF1 PLAYS CRITICAL

ROLES IN EARLY B CELL DEVELOPMENT Article Open access 09 July 2024 REFERENCES * Pongubala JM, Nagulapalli S, Klemsz MJ, McKercher SR, Maki RA, Atchison ML . PU1 recruits a second nuclear

factor to a site important for immunoglobulin kappa 3' enhancer activity. _Mol Cell Biol_ 1992; 12: 368–378. Article  CAS  PubMed  PubMed Central  Google Scholar  * Kanno Y, Levi B-Z,

Tamura T, Ozato K . Immune cell-specific amplification of interferon signaling by the IRF-4/8-PU.1 complex. _J Interferon Cytokine Res_ 2005; 25: 770–779. Article  CAS  PubMed  Google

Scholar  * Eisenbeis CF, Singh H, Storb U . Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. _Genes Dev_ 1995; 9: 1377–1387. Article  CAS 

PubMed  Google Scholar  * Eisenbeis CF, Singh H, Storb U . PU.1 is a component of a multiprotein complex which binds an essential site in the murine immunoglobulin lambda 2-4 enhancer. _Mol

Cell Biol_ 1993; 13: 6452–6461. Article  CAS  PubMed  PubMed Central  Google Scholar  * Brass AL, Zhu AQ, Singh H . Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting

function _in vivo_ using fused dimers. _EMBO J_ 1999; 18: 977–991. Article  CAS  PubMed  PubMed Central  Google Scholar  * Brass AL, Kehrli E, Eisenbeis CF, Storb U, Singh H . Pip, a

lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1. _Genes Dev_ 1996; 10: 2335–2347. Article 

CAS  PubMed  Google Scholar  * Ochiai K, Maienschein-Cline M, Simonetti G, Chen J, Rosenthal R, Brink R _et al_. Transcriptional regulation of germinal center B and plasma cell fates by

dynamical control of IRF4. _Immunity_ 2013; 38: 918–929. Article  CAS  PubMed  PubMed Central  Google Scholar  * Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P _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. Article  CAS  PubMed 

PubMed Central  Google Scholar  * Scott EW, Simon MC, Anastasi J, Singh H . Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. _Science_ 1994;

265: 1573–1577. Article  CAS  PubMed  Google Scholar  * Scott EW, Fisher RC, Olson MC, Kehrli EW, Simon MC, Singh H . PU.1 functions in a cell-autonomous manner to control the

differentiation of multipotential lymphoid-myeloid progenitors. _Immunity_ 1997; 6: 437–447. Article  CAS  PubMed  Google Scholar  * Polli M, Dakic A, Light A, Wu L, Tarlinton D, Nutt S .

The development of functional B lymphocytes in conditional PU.1 knock-out mice. _Blood_ 2005; 106: 2083–2090. Article  CAS  PubMed  Google Scholar  * Ye M, Ermakova O, Graf T . PU.1 is not

strictly required for B cell development and its absence induces a B-2 to B-1 cell switch. _J Exp Med_ 2005; 202: 1411–1422. Article  CAS  PubMed  PubMed Central  Google Scholar  * Sokalski

KM, Li SKH, Welch I, Cadieux-Pitre H-AT, Gruca MR, Dekoter RP . Deletion of genes encoding PU.1 and Spi-B in B cells impairs differentiation and induces pre-B cell acute lymphoblastic

leukemia. _Blood_ 2011; 118: 1–33. Article  Google Scholar  * Johnson K, Hashimshony T, Sawai CM, Pongubala JM, Skok JA, Aifantis I _et al_. Regulation of immunoglobulin light-chain

recombination by the transcription factor IRF-4 and the attenuation of interleukin-7 signaling. _Immunity_ 2008; 28: 335–345. Article  CAS  PubMed  Google Scholar  * Lu R, Medina KL, Lancki

DW, Singh H . IRF-4,8 orchestrate the pre-B-to-B transition in lymphocyte development. _Genes Dev_ 2003; 17: 1703–1708. Article  CAS  PubMed  PubMed Central  Google Scholar  * Ma S, Pathak

S, Trinh L, Lu R . Interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to down-regulate pre-B-cell receptor and promote cell-cycle withdrawal in pre-B-cell

development. _Blood_ 2008; 111: 1396–1403. Article  CAS  PubMed  PubMed Central  Google Scholar  * Ma S, Turetsky A, Trinh L, Lu R . IFN regulatory factor 4 and 8 promote Ig light chain

kappa locus activation in pre-B cell development. _J Immunol_ 2006; 177: 7898–7904. Article  CAS  PubMed  Google Scholar  * Cook WD, McCaw BJ, Herring C, John DL, Foote SJ, Nutt SL _et al_.

PU.1 is a suppressor of myeloid leukemia, inactivated in mice by gene deletion and mutation of its DNA binding domain. _Blood_ 2004; 104: 3437–3444. Article  CAS  PubMed  Google Scholar  *

Holtschke T, Löhler J, Kanno Y, Fehr T, Giese N, Rosenbauer F _et al_. Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene.

_Cell_ 1996; 87: 307–317. Article  CAS  PubMed  Google Scholar  * Metcalf D, Dakic A, Mifsud S, Di Rago L, Wu L, Nutt S . Inactivation of PU.1 in adult mice leads to the development of

myeloid leukemia. _Proc Natl Acad Sci USA_ 2006; 103: 1486–1491. Article  CAS  PubMed  PubMed Central  Google Scholar  * Rosenbauer F, Koschmieder S, Steidl U, Tenen DG . Effect of

transcription-factor concentrations on leukemic stem cells. _Blood_ 2005; 106: 1519–1524. Article  CAS  PubMed  PubMed Central  Google Scholar  * Rosenbauer F, Wagner K, Kutok JL, Iwasaki H,

Le Beau MM, Okuno Y _et al_. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. _Nat Genet_ 2004; 36: 624–630. Article  CAS  PubMed  Google

Scholar  * Scheller M, Schonheit J, Zimmermann K, Leser U, Rosenbauer F, Leutz A . Cross talk between Wnt/beta-catenin and Irf8 in leukemia progression and drug resistance. _J Exp Med_

2013; 210: 2239–2256. Article  CAS  PubMed  PubMed Central  Google Scholar  * Steidl U, Steidl C, Ebralidze A, Chapuy B, Han HJ, Will B _et al_. A distal single nucleotide polymorphism

alters long-range regulation of the PU.1 gene in acute myeloid leukemia. _J Clin Invest_ 2007; 117: 2611–2620. Article  CAS  PubMed  PubMed Central  Google Scholar  * Mullighan CG, Zhang J,

Kasper LH, Lerach S, Payne-Turner D, Phillips LA _et al_. CREBBP mutations in relapsed acute lymphoblastic leukaemia. _Nature_ 2011; 471: 235–239. Article  CAS  PubMed  PubMed Central 

Google Scholar  * Zhang J, Mullighan CG, Harvey RC, Wu G, Chen X, Edmonson M _et al_. Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from

the Children's Oncology Group. _Blood_ 2011; 118: 3080–3087. Article  CAS  PubMed  PubMed Central  Google Scholar  * Bouamar H, Abbas S, Lin AP, Wang L, Jiang D, Holder KN _et al_. A

capture-sequencing strategy identifies IRF8, EBF1, and APRIL as novel IGH fusion partners in B-cell lymphoma. _Blood_ 2013; 122: 726–733. Article  CAS  PubMed  PubMed Central  Google Scholar

  * Niebuhr B, Kriebitzsch N, Fischer M, Behrens K, Gunther T, Alawi M _et al_. Runx1 is essential at two stages of early murine B-cell development. _Blood_ 2013; 122: 413–423. Article  CAS

  PubMed  Google Scholar  * Shukla V, Ma S, Hardy RR, Joshi SS, Lu R . A role for IRF4 in the development of CLL. _Blood_ 2013; 122: 2848–2855. Article  CAS  PubMed  PubMed Central  Google

Scholar  * Shaffer AL, Emre NC, Romesser PB, Staudt LM . IRF4: Immunity. Malignancy! Therapy? _Clin Cancer Res_ 2009; 15: 2954–2961. Article  CAS  PubMed  PubMed Central  Google Scholar  *

Adamaki M, Lambrou GI, Athanasiadou A, Tzanoudaki M, Vlahopoulos S, Moschovi M . Implication of IRF4 aberrant gene expression in the acute leukemias of childhood. _PLoS One_ 2013; 8: e72326.

Article  CAS  PubMed  PubMed Central  Google Scholar  * Acquaviva J, Chen X, Ren R . IRF-4 functions as a tumor suppressor in early B-cell development. _Blood_ 2008; 112: 3798–3806. Article

  CAS  PubMed  PubMed Central  Google Scholar  * Pathak S, Ma S, Trinh L, Eudy J, Wagner KU, Joshi SS _et al_. IRF4 is a suppressor of c-Myc induced B cell leukemia. _PLoS One_ 2011; 6:

e22628. Article  CAS  PubMed  PubMed Central  Google Scholar  * Jo SH, Schatz JH, Acquaviva J, Singh H, Ren R . Cooperation between deficiencies of IRF-4 and IRF-8 promotes both myeloid and

lymphoid tumorigenesis. _Blood_ 2010; 116: 2759–2767. Article  CAS  PubMed  PubMed Central  Google Scholar  * Xu LS, Sokalski KM, Hotke K, Christie DA, Zarnett O, Piskorz J _et al_.

Regulation of B cell linker protein transcription by PU.1 and Spi-B in murine B cell acute lymphoblastic leukemia. _J Immunol_ 2012; 189: 3347–3354. Article  CAS  PubMed  Google Scholar  *

Carotta S, Willis SN, Hasbold J, Inouye M, Pang SH, Emslie D _et al_. The transcription factors IRF8 and PU.1 negatively regulate plasma cell differentiation. _J Exp Med_ 2014; 211:

2169–2181. Article  PubMed  PubMed Central  Google Scholar  * Dakic A, Metcalf D, Di Rago L, Mifsud S, Wu L, Nutt SL . PU.1 regulates the commitment of adult hematopoietic progenitors and

restricts granulopoiesis. _J Exp Med_ 2005; 201: 1487–1502. Article  CAS  PubMed  PubMed Central  Google Scholar  * Mittrücker HW, Matsuyama T, Grossman A, Kündig TM, Potter J, Shahinian A

_et al_. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. _Science_ 1997; 275: 540–543. Article  PubMed  Google Scholar  * Hobeika E, Thiemann S,

Storch B, Jumaa H, Nielsen PJ, Pelanda R _et al_. Testing gene function early in the B cell lineage in mb1-cre mice. _Proc Natl Acad Sci USA_ 2006; 103: 13789–13794. Article  CAS  PubMed 

PubMed Central  Google Scholar  * DeKoter RP, Lee HJ, Singh H . PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. _Immunity_ 2002; 16: 297–309. Article  CAS 

PubMed  Google Scholar  * Anderson KL, Nelson SL, Perkin HB, Smith KA, Klemsz MJ, Torbett BE . PU.1 is a lineage-specific regulator of tyrosine phosphatase CD45. _J Biol Chem_ 2001; 276:

7637–7642. Article  CAS  PubMed  Google Scholar  * Mullighan CG, Goorha S, Radtke I, Miller CB, Coustan-Smith E, Dalton JD _et al_. Genome-wide analysis of genetic alterations in acute

lymphoblastic leukaemia. _Nature_ 2007; 446: 758–764. Article  CAS  PubMed  Google Scholar  * Decker T, Pasca di Magliano M, McManus S, Sun Q, Bonifer C, Tagoh H _et al_. Stepwise activation

of enhancer and promoter regions of the B cell commitment gene Pax5 in early lymphopoiesis. _Immunity_ 2009; 30: 508–520. Article  CAS  PubMed  Google Scholar  * Minegishi Y, Rohrer J,

Coustan-Smith E, Lederman HM, Pappu R, Campana D _et al_. An essential role for BLNK in human B cell development. _Science_ 1999; 286: 1954–1957. Article  CAS  PubMed  Google Scholar  *

Pappu R, Cheng AM, Li B, Gong Q, Chiu C, Griffin N _et al_. Requirement for B cell linker protein (BLNK) in B cell development. _Science_ 1999; 286: 1949–1954. Article  CAS  PubMed  Google

Scholar  * Jumaa H, Wollscheid B, Mitterer M, Wienands J, Reth M, Nielsen PJ . Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65.

_Immunity_ 1999; 11: 547–554. Article  CAS  PubMed  Google Scholar  * Jumaa H, Bossaller L, Portugal K, Storch B, Lotz M, Flemming A _et al_. Deficiency of the adaptor SLP-65 in pre-B-cell

acute lymphoblastic leukaemia. _Nature_ 2003; 423: 452–456. Article  CAS  PubMed  Google Scholar  * Wang JH, Nichogiannopoulou A, Wu L, Sun L, Sharpe AH, Bigby M _et al_. Selective defects

in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. _Immunity_ 1996; 5: 537–549. Article  CAS  PubMed  Google Scholar  * Ma S, Pathak S, Mandal M,

Trinh L, Clark MR, Lu R . Ikaros and Aiolos inhibit pre-B-cell proliferation by directly suppressing c-Myc expression. _Mol Cell Biol_ 2010; 30: 4149–4158. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Heizmann B, Kastner P, Chan S . Ikaros is absolutely required for pre-B cell differentiation by attenuating IL-7 signals. _J Exp Med_ 2013; 210: 2823–2832. Article

  CAS  PubMed  PubMed Central  Google Scholar  * Joshi I, Yoshida T, Jena N, Qi X, Zhang J, Van Etten RA _et al_. Loss of Ikaros DNA-binding function confers integrin-dependent survival on

pre-B cells and progression to acute lymphoblastic leukemia. _Nat Immunol_ 2014; 15: 294–304. Article  CAS  PubMed  PubMed Central  Google Scholar  * Schwickert TA, Tagoh H, Gultekin S,

Dakic A, Axelsson E, Minnich M _et al_. Stage-specific control of early B cell development by the transcription factor Ikaros. _Nat Immunol_ 2014; 15: 283–293. Article  CAS  PubMed  PubMed

Central  Google Scholar  * Mullighan C, Downing J . Ikaros and acute leukemia. _Leuk Lymphoma_ 2008; 49: 847–849. Article  PubMed  Google Scholar  * Mullighan CG, Su X, Zhang J, Radtke I,

Phillips LAA, Miller CB _et al_. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. _N Engl J Med_ 2009; 360: 470–480. Article  CAS  PubMed  PubMed Central  Google Scholar  *

DeKoter R, Singh H . Regulation of B lymphocyte and macrophage development by graded expression of PU.1. _Science_ 2000; 288: 1439–1441. Article  CAS  PubMed  Google Scholar  * Rosenbauer F,

Owens BM, Yu L, Tumang JR, Steidl U, Kutok JL _et al_. Lymphoid cell growth and transformation are suppressed by a key regulatory element of the gene encoding PU.1. _Nat Genet_ 2006; 38:

27–37. Article  CAS  PubMed  Google Scholar  * Schebesta A, McManus S, Salvagiotto G, Delogu A, Busslinger GA, Busslinger M . Transcription factor Pax5 activates the chromatin of key genes

involved in B cell signaling, adhesion, migration, and immune function. _Immunity_ 2007; 27: 49–63. Article  CAS  PubMed  Google Scholar  * Greig KT, de Graaf CA, Murphy JM, Carpinelli MR,

Pang SH, Frampton J _et al_. Critical roles for c-Myb in lymphoid priming and early B-cell development. _Blood_ 2010; 115: 2796–2805. Article  CAS  PubMed  PubMed Central  Google Scholar  *

Ferreiros-Vidal I, Carroll T, Taylor B, Terry A, Liang Z, Bruno L _et al_. Genome-wide identification of Ikaros targets elucidates its contribution to mouse B-cell lineage specification and

pre-B-cell differentiation. _Blood_ 2013; 121: 1769–1782. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank M Reth and T Mak for mice, J Leahy for animal

husbandry and the institute flow cytometry facility for excellent technical assistance. We thank Markus Jaritz for bioinformatic analysis. This work was supported by program and project

grants (APP1054925 to SLN and 637345 to SC) and fellowships (APP1058238 to SLN) from the National Health and Medical Research Council (NHRMC) of Australia. SHMP was supported by the

Leukaemia Foundation of Australia and SC by an NHMRC Career Development Fellowship. Research of the Busslinger group was supported by Boehringer Ingelheim and an ERC Advanced Grant

(291740-LymphoControl). This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIIS. AUTHOR INFORMATION Author

notes * R A Dickins Present address: 5Current address: Monash University, Australian Center for Blood Diseases, The Alfred, Commercial Road, Melbourne, Victoria 3004, Australia, * S Carotta

Present address: 6Current address: Boehringer Ingelheim RCV, New Therapeutic Drug Concepts, Dr Boehringer Gasse 5-11, Vienna A-1121, Austria, AUTHORS AND AFFILIATIONS * Molecular Immunology

Department, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia S H M Pang, P Gangatirkar, Z Zheng, R A Dickins, L M Corcoran, N D Huntington, S L Nutt 

& S Carotta * Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia S H M Pang, P Gangatirkar, R A Dickins, L M Corcoran, N D Huntington, S L Nutt & 

S Carotta * The Institute of Molecular Pathology, Vienna, Austria M Minnich, A Ebert & M Busslinger * Department of Pathology, St Jude Children’s Research Hospital, Memphis, TN, USA G

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THIS ARTICLE Pang, S., Minnich, M., Gangatirkar, P. _et al._ PU.1 cooperates with IRF4 and IRF8 to suppress pre-B-cell leukemia. _Leukemia_ 30, 1375–1387 (2016).

https://doi.org/10.1038/leu.2016.27 Download citation * Received: 12 February 2015 * Revised: 14 November 2015 * Accepted: 08 January 2016 * Published: 02 March 2016 * Issue Date: June 2016

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