Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Oncogenes, Fusion Genes and Tumor Suppressor Genes

The leukemogenic AF4–MLL fusion protein causes P-TEFb kinase activation and altered epigenetic signatures

Abstract

Expression of the AF4–MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4–MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4–MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4me3 and H3K79me3 histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Meyer C, Kowarz E, Hofmann J, Renneville A, Zuna J, Trka J et al. New Insights into the MLL recombinome of acute leukemias. Leukemia 2009; 23: 1490–1499.

    Article  CAS  PubMed  Google Scholar 

  2. Nilson I, Reichel M, Ennas MG, Greim R, Knörr C, Siegler G et al. Exon/intron structure of the human AF-4 gene, a member of the AF-4/LAF-4/FMR-2 gene family coding for a nuclear protein with structural alterations in acute leukaemia. Br J Haematol 1997; 98: 157–169.

    Article  CAS  PubMed  Google Scholar 

  3. Chen W, Li Q, Hudson WA, Kumar A, Kirchhof N, Kersey JH . A murine Mll-AF4 knock-in model results in lymphoid and myeloid deregulation and hematologic malignancy. Blood 2006; 108: 669–677.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Metzler M, Forster A, Pannell R, Arends MJ, Daser A, Lobato MN et al. A conditional model of MLL-AF4 B-cell tumourigenesis using invertor technology. Oncogene 2006; 25: 3093–3103.

    Article  CAS  PubMed  Google Scholar 

  5. Krivtsov AV, Feng Z, Lemieux ME, Faber J, Vempati S, Sinha AU et al. H3K79 methylation profiles define murine and human MLL-AF4 leukemias. Cancer Cell 2008; 14: 355–368.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bursen A, Moritz S, Gaussmann A, Moritz S, Dingermann T, Marschalek R . Interaction of AF4 wild-type and AF4•MLL fusion protein with SIAH proteins: indication for t(4;11) pathobiology? Oncogene 2004; 23: 6237–6249.

    Article  CAS  PubMed  Google Scholar 

  7. Hsieh JJ, Cheng EH, Korsmeyer SJ . Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression. Cell 2003a; 115: 293–303.

    Article  CAS  PubMed  Google Scholar 

  8. Bursen A, Schwabe K, Rüster B, Henschler R, Ruthardt M, Dingermann T et al. The AF4-MLL fusion protein is capable of inducing ALL in mice without requirement of MLL-AF4. Blood 2010; 115: 3570–3579.

    Article  CAS  PubMed  Google Scholar 

  9. Erfurth F, Hemenway CS, de Erkenez AC, Domer PH . MLL fusion partners AF4 and AF9 interact at subnuclear foci. Leukemia 2004; 18: 92–102.

    Article  CAS  PubMed  Google Scholar 

  10. Mueller D, García-Cuéllar MP, Bach C, Buhl S, Maethner E, Slany RK . Misguided transcriptional elongation causes mixed lineage leukemia. PLoS Biol 2009; 7: e1000249.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Mueller D, Bach C, Zeisig D, Garcia-Cuellar MP, Monroe S, Sreekumar A et al. A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. Blood 2007; 110: 4445–4454.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Feng Q, Wang H, Ng HH, Erdjument-Bromage H, Tempst P, Struhl K et al. Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain. Curr Biol 2002; 12: 1052–1058.

    Article  CAS  PubMed  Google Scholar 

  13. Steger DJ, Lefterova MI, Ying L, Stonestrom AJ, Schupp M, Zhuo D et al. DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Mol Cell Biol 2008; 28: 2825–2839.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Estable MC, Naghavi MH, Kato H, Xiao H, Qin J, Vahlne A et al. MCEF, the newest member of the AF4 family of transcription factors involved in leukemia, is a positive transcription elongation factor-b-associated protein. J Biomed Sci 2002; 9: 234–245.

    Article  CAS  PubMed  Google Scholar 

  15. Marshall NF, Price DH . Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J Biol Chem 1995; 270: 12335–12338.

    Article  CAS  PubMed  Google Scholar 

  16. Bitoun E, Oliver PL, Davies KE . The mixed-lineage leukemia fusion partner AF4 stimulates RNA polymerase II transcriptional elongation and mediates coordinated chromatin remodeling. Hum Mol Genet 2007; 16: 92–106.

    Article  CAS  PubMed  Google Scholar 

  17. Rietschel B, Arrey TN, Meyer B, Bornemann S, Schuerken M, Karas M et al. Elastase digests: New ammunition for shotgun membrane proteomics. Mol Cell Proteomics 2009; 8: 1029–1043.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS . Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999; 20: 3551–3567.

    Article  CAS  PubMed  Google Scholar 

  19. Milne TA, Briggs SD, Brock HW, Martin ME, Gibbs D, Allis CD et al. MLL targets SET domain methyltransferase activity to Hox gene promoters. Mol Cell 2002; 10: 1107–1117.

    CAS  PubMed  Google Scholar 

  20. Obier N, Müller AM . Chromatin flow cytometry identifies changes in epigenetic cell states. Cells Tissues Organs 2010; 191: 167–174.

    Article  CAS  PubMed  Google Scholar 

  21. Flory E, Hoffmeyer A, Smola U, Rapp UR, Bruder JT . Raf-1 kinase targets GA-binding protein in transcriptional regulation of the human immunodeficiency virus type 1 promoter. J Virol 1996; 70: 2260–2268.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Breitkreutz BJ, Stark C, Reguly T, Boucher L, Breitkreutz A, Livstone M et al. The BioGRID Interaction Database: 2008 update. Nucl Acids Res 2008; 36: D637–D640.

    Article  CAS  PubMed  Google Scholar 

  23. Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J et al. STRING 8—a global view on proteins and their functional interactions in 630 organisms. Nucl Acids Res 2009; 37: D412–D416.

    Article  CAS  PubMed  Google Scholar 

  24. Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K . The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell 2005; 19: 523–534.

    Article  CAS  PubMed  Google Scholar 

  25. Wysocka J, Swigut T, Milne TA, Dou Y, Zhang X, Burlingame AL et al. WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development. Cell 2005; 121: 859–872.

    Article  CAS  PubMed  Google Scholar 

  26. Dou Y, Milne TA, Tackett AJ, Smith ER, Fukuda A, Wysocka J et al. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF. Cell 2005; 121: 873–885.

    Article  CAS  PubMed  Google Scholar 

  27. Rozenblatt-Rosen O, Rozovskaia T, Burakov D, Sedkov Y, Tillib S, Blechman J et al. The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex. Proc Natl Acad Sci USA 1998; 95: 4152–4157.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Adler HT, Chinery R, Wu DY, Kussick SJ, Payne JM, Fornace Jr AJ et al. Leukemic HRX fusion proteins inhibit GADD34-induced apoptosis and associate with the GADD34 and hSNF5/INI1 proteins. Mol Cell Biol 1999; 19: 7050–7060.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Takeda S, Chen DY, Westergard TD, Fisher JK, Rubens JA, Sasagawa S et al. Proteolysis of MLL family proteins is essential for taspase1-orchestrated cell cycle progression. Genes Dev 2006; 20: 2397–2409.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tyagi S, Chabes AL, Wysocka J, Herr W . E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases. Mol Cell 2007; 27: 107–119.

    Article  CAS  PubMed  Google Scholar 

  31. Kushura M, Nagasaki K, Limura K, Maass N, Manabe T, Ishikawa S et al. Cloning of Hemamethylene-bis-acetamide-inducible Transcript, HEXIM1, in human vascular smooth muscle cells. Biomed Res 1999; 20: 273–279.

    Article  Google Scholar 

  32. Covic M, Hassa PO, Saccani S, Buerki C, Meier NI, Lombardi C et al. Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-kappaB-dependent gene expression. EMBO J 2005; 24: 85–96.

    Article  CAS  PubMed  Google Scholar 

  33. Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K et al. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol Cell 2005; 19: 535–545.

    Article  CAS  PubMed  Google Scholar 

  34. Huang B, Yang X, Zhou MM, Ozato K, Chen LF . Brd4 coactivates transcriptional activation of NFkB via specific binding to acetylated RelA. Mol Cell Biol 2009; 29: 1375–1387.

    Article  CAS  PubMed  Google Scholar 

  35. Lin CY, Liang YC, Yung BY . Nucleophosmin/B23 regulates transcriptional activation of E2F1 via modulating the promoter binding of NF-kB, E2F1 and pRB. Cell Signal 2006; 18: 2041–2048.

    Article  CAS  PubMed  Google Scholar 

  36. Gurumurthy M, Tan CH, Ng R, Zeiger L, Lau J, Lee J et al. Nucleophosmin interacts with HEXIM1 and regulates RNA polymerase II transcription. J Mol Biol 2008; 378: 302–317.

    Article  CAS  PubMed  Google Scholar 

  37. Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia T, Wassell R et al. ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol Cell 2002; 10: 1119–1128.

    Article  CAS  PubMed  Google Scholar 

  38. Hsieh JJ, Ernst P, Erdjument-Bromage H, Tempst P, Korsmeyer SJ . Proteolytic cleavage of MLL generates a complex of N- and C-terminal fragments that confers protein stability and subnuclear localization. Mol Cell Biol 2003b; 23: 186–194.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ, Kitabayashi I et al. Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol Cell Biol 2004; 24: 5639–5649.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Jayne S, Rothgiesser KM, Hottiger MO . CARM1 but not its enzymatic activity is required for transcriptional coactivation of NF-kappaB-dependent gene expression. J Mol Biol 2009; 394: 485–495.

    Article  CAS  PubMed  Google Scholar 

  41. Gaussmann A, Wenger T, Eberle I, Bursen A, Bracharz S, Herr I et al. Combined effects of the two reciprocal t(4;11) fusion proteins MLL-AF4 and AF4-MLL confer resistance to apoptosis, cell cycling capacity and growth transformation. Oncogene 2007; 26: 3352–3363.

    Article  CAS  PubMed  Google Scholar 

  42. Lin C, Smith ER, Takahashi H, Lai KC, Martin-Brown S, Florens L et al. AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Mol Cell 2010; 37: 429–437.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zeisig DT, Bittner CB, Zeisig BB, García-Cuéllar MP, Hess JL, Slany RK . The eleven-nineteen-leukemia protein ENL connects nuclear MLL fusion partners with chromatin. Oncogene 2005; 24: 5525–5532.

    Article  CAS  PubMed  Google Scholar 

  44. Yokoyama A, Lin M, Naresh A, Kitabayashi I, Cleary ML . A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell 2010; 17: 198–212.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Jennifer Merkens for technical assistance. This study was supported by research Grants Ma1876/5–2, Ma1876/7–1 from the DFG and Grant 108400 from the Deutsche Krebshilfe eV to RM RM is PI within the CEF on Macromolecular Complexes funded by DFG grant EXC 115.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to R Marschalek.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Leukemia website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Benedikt, A., Baltruschat, S., Scholz, B. et al. The leukemogenic AF4–MLL fusion protein causes P-TEFb kinase activation and altered epigenetic signatures. Leukemia 25, 135–144 (2011). https://doi.org/10.1038/leu.2010.249

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2010.249

Keywords

This article is cited by

Search

Quick links