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

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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.

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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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Authors and Affiliations

  1. HMR Research Center, University of Montreal, Montreal, QC, Canada

    E. A. Shestakova & J. J. Bijl

  2. Blokhin Russian Cancer Research Center, Moscow, 115478, Russia

    E. A. Shestakova

  3. Proteomic Platform, CHU de Quebec Research Center, Quebec, QC, Canada

    M. Boutin & S. Bourassa

  4. Proteomic Platform, IRIC, University of Montreal, Montreal, QC, Canada

    E. Bonneil

  5. Department of Medicine, University of Montreal, Montreal, QC, Canada

    J. J. Bijl

Authors
  1. E. A. Shestakova
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  2. M. Boutin
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  3. S. Bourassa
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  4. E. Bonneil
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  5. J. J. Bijl
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Correspondence to E. A. Shestakova.

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The article was translated by the authors.

Published in Russian in Molekulyarnaya Biologiya, 2017, Vol. 51, No. 3, pp. 490–501.

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Shestakova, E.A., Boutin, M., Bourassa, S. et al. Identification of proteins associated with transcription factors HOXA9 and E2A-PBX1 by tandem affinity purification. Mol Biol 51, 432–444 (2017). https://doi.org/10.1134/S002689331703013X

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  • DOI: https://doi.org/10.1134/S002689331703013X

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