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

Shestakova, E.A.; Boutin, M.; Bourassa, S.; Bonneil, E.; Bijl, J.J.

Авторы: Shestakova E.A., Boutin M., Bourassa S., Bonneil E., Bijl J.J.
Журнал: Molecular Biology
Том: 51
Номер: 3
Год издания: 2017
Издательство: Maik Nauka/Interperiodica Publishing
Местоположение издательства: Russian Federation
Первая страница: 432
Последняя страница: 444
DOI: 10.1134/s002689331703013x
Аннотация: Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cellleukemia 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. HOXA9itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly acceleratedby its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimericcomplexes in different combinations. Cooperative interaction between PBX1 and HOX proteinsenhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 isretained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead toaberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidencethat E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes areactually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger proteincomplexes acting in independent complementing oncogenic pathways. In this study we aim to search forother HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2APBX1or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemicand HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attachedFLAG/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, chromatinremodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is thefirst time the protein partners of either E2A-PBX1 or HOXA9 oncoproteins were identified using an unbiasedbiochemical approach. The identification of translation initiation factors associated with HOXA9 might indicatea novel function for HOX proteins independent of their transcriptional activity.Keywords: transcription factors, oncoproteins, HOXA9, E2A-PBX1, tandem affinity purification, tandemmass-spectrometry, associated proteins, oncogenic potential
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