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PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in hematopoietic cells

ABSTRACT: The global protein interactions of the oncofusion protein PML::RARA were identified using proximity labeling followed by mass spectrometry. Briefly, Lineage-depleted bone marrow cells from C57Bl/6 mice were transduced with MSCV-IRES-GFP retroviruses containing an enhanced biotin ligase (TurboID) fused to either the N-terminus or C-terminus of PML::RARAWT (TurboID-PML::RARAWT and PML::RARAWT-TurboID respectively), TurboID-PML::RARAC88A (mutation in the DNA binding domain of RARA), or a TurboID cDNA alone. A flexible Glycine/Serine linker placed between TurboID and PML::RARA allows TurboID to freely rotate, and biotinylate proteins within 10 angstroms of the TurboID-PML::RARA fusion protein. Four days following the first round of transduction, cells were flow sorted on GFP+ cells. Two days following the sort, 1-5 million cells were treated with 50 uM Biotin for four hours at 37 degrees C. Cell lysates were prepared, sonicated, and incubated with streptavidin agarose beads overnight. Beads were washed, peptides were eluted and digested, and then run on a timsTOF Pro 2.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Timothy J. Ley

PROVIDER: MSV000092739 | MassIVE | Wed Aug 23 20:20:00 BST 2023

SECONDARY ACCESSION(S): PXD044816

REPOSITORIES: MassIVE

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Project description:Identifying specific protein interactors and spatially or temporally restricted local proteomes contributes significantly to our understanding of cellular processes in virtually all aspects of life. Obtaining such data is challenging, especially when the protein, cell type or event of interest is rare. In recent years, different proximity labeling techniques have been developed that have greatly improved our ability to tackle these questions. However, while effective in mammalian systems, use in plants has been extremely limited due to technical challenges. Recent technological improvements in the form of two highly active versions of the biotin ligase BirA* (TurboID and miniTurboID), prompted us to test this new system on two challenging but widely asked questions in plants: what are interaction partners of low-abundant proteins and what are organellar proteomes in rare and transient cell types. To address the second question, we used nuclei of developing stomatal guard cells, which express the transcription factor FAMA, as our test case. FAMA is a master regulator of guard cell development and promotes terminal differentiation of the guard cell precursor by both activating and repressing hundreds of genes. FAMA-expressing young guard cells are rare and restricted to the epidermis of developing aerial tissues, which makes them a good model system to test TurboID applicability under material-limiting conditions. For this experiment, young Arabidopsis seedlings expressing nuclear TurboID under the FAMA or UBIQUITIN10 (UBQ10) promoter were treated with biotin to label nuclear proteomes. Col-0 wild type was used as controls for unspecific binding of proteins to the beads. Biotinylated proteins were isolated by affinity purification with streptavidin-coupled beads and identified by LC-MS/MS. By targeting TurboID to the nucleus we were able to purify nuclear proteins with relatively high specificity. Enrichment of FAMA-stage specific proteins, when nuclear TurboID was driven by the FAMA compared to the UBQ10 promoter, supports general applicability of TurboID for identification of subcellular proteomes.

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PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in hematopoietic cells

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