IKZF1 and IKZF3 are transcription factors highly expressed in myeloma and contribute to myeloma cell survival. To better understand the function of IKZFs, we sought out to identify novel physiologic partners. To this purpose we generated a human MM cell line, ARP-1, stably expressing physiologic levels of FLAG-HA tagged human IKZF1 or IKZF3 via retroviral delivery. FLAG-peptide eluates from anti-FLAG affinity purifications, either from a nuclear extract (nucleoplasm) or benzonase-extracted detergent insoluble fraction (DNA-bound), were trypsinized and subjected to mass spectrometry analysis. Relative to FLAG-immunoprecipitates from ARP-1 cells infected with an empty virus, we identified peptides corresponding to the NuRD and SWI/SNF complexes, known members of IKZF1 and IKZF3 complex. Surprisingly, we identified the transcription factor RUNX1, RUNX3 and CBF-beta as novel interactors of the IKZFs.
In brief, TCA-precipitated protein eluates were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. The peptide mixtures were loaded onto microcapillary fused silica columns (100um i.d.), packed with C18 reverse phase (Aqua; Phenomenx), SCX (Luna; Phenomenex) and C18-RP, placed in-line with an Agilent 11000 quaternary pump, and analyzed by a 10-step MudPIT on linear ion traps. MS/MS datasets were searched using SEQUEST (v. 27.9) against a non-redudant human protein database (NCBI, 2015-03-25) containing 160 usual contaminants. To estimate false discover rates (FDRs), the amino acid sequence of each non-redundant protein was randomized. Peptide/spectrum matches were sorted and selected using DTASelect with the following criteria set: spectra/peptide matches were retained only if they had a DeltCn of at least 0.8, and minimum XCorr of 1.8 for singly, 2.0 for doubly, and 3.0 for triply charged spectra. Additionally, the peptides had to be minimum 7 amino acids in length and fully tryptic.
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: IKZFs ; RUNX1 ; transcription factors ; multiple myeloma
|Principal Investigators:||Laurence Florens, The Stowers Institute for Medical Research, USA|
Zhou N, Gutierrez-Uzquiza A, Zheng XY, Chang R, Vogl DT, Garfall AL, Bernabei L, Saraf A, Florens L, Washburn MP, Illendula A, Bushweller JH, Busino L.
RUNX proteins desensitize multiple myeloma to lenalidomide via protecting IKZFs from degradation.
Leukemia. Epub 2019 Feb 13.
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