Molecular Biology

, Volume 51, Issue 3, pp 432–444 | Cite as

Identification of proteins associated with transcription factors HOXA9 and E2A-PBX1 by tandem affinity purification

  • E. A. Shestakova
  • M. Boutin
  • S. Bourassa
  • E. Bonneil
  • J. J. Bijl
Molecular Cell Biology
  • 74 Downloads

Abstract

Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cell leukemia in children. Using a transgenic mouse model, we previously demonstrated that homeobox (HOX) gene HOXA9 genetically interact with E2A-PBX1 gene in the development of B-cell leukemia in mice. HOXA9 itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly accelerated by its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimeric complexes in different combinations. Cooperative interaction between PBX1 and HOX proteins enhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 is retained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead to aberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidence that E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes are actually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger protein complexes acting in independent complementing oncogenic pathways. In this study we aim to search for other HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2A-PBX1 or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemic and HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attached FLAG/HA peptides. The protein composition of each complex was determined using tandem mass-spectrometry. In the E2A-PBX1 containing complex we identified lymphoid transcription factor IKAROS, chromatin remodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3 ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is the first time the protein partners of either E2A-PBX1 or HOXA9 oncoproteins were identified using an unbiased biochemical approach. The identification of translation initiation factors associated with HOXA9 might indicate a novel function for HOX proteins independent of their transcriptional activity.

Keywords

transcription factors oncoproteins HOXA9 E2A-PBX1 tandem affinity purification tandem mass-spectrometry associated proteins oncogenic potential 

Abbreviations

AD

activation domain

ALL

acute lymphoid leukemia

AML

acute myeloid leukemia

ESI MS/MS

electrospray ionization tandem mass spectrometry

HOX

homeobox

HCD

HOX Cooperative Domain

MS

mass spectrometry

TALE

the Three Amino acid Loop Extension

TAP

tandem affinity purification

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Copyright information

© Pleiades Publishing, Inc. 2017

Authors and Affiliations

  • E. A. Shestakova
    • 1
    • 2
  • M. Boutin
    • 3
  • S. Bourassa
    • 3
  • E. Bonneil
    • 4
  • J. J. Bijl
    • 1
    • 5
  1. 1.HMR Research CenterUniversity of MontrealMontrealCanada
  2. 2.Blokhin Russian Cancer Research CenterMoscowRussia
  3. 3.Proteomic PlatformCHU de Quebec Research CenterQuebecCanada
  4. 4.Proteomic Platform, IRICUniversity of MontrealMontrealCanada
  5. 5.Department of MedicineUniversity of MontrealMontrealCanada

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