FOXP1 is a transcriptional Coactivator of Wnt/β-catenin signaling in Diffuse Large B-cell Lymphoma

ABSTRACT: We used a mass spectrometry-coupled lentiviral ‘CD-tagging’ mutagenesis approach to identify genes that activate Wnt/β-catenin signaling. Human A375 melanoma cells containing a β-catenin-driven GFP (green fluorescent protein) transcriptional reporter were transduced with CDBF lentivirus. When integrated near an expressed and spliced gene, the cytomegalovirus (CMV) promoter of the CDBF vector drives constitutive BFP (blue fluorescence protein) expression and by virtue of the splice donor (SD) sequence, an overexpressed FLAG-tagged fusion of the targeted gene. Depending on where within the gene locus the CDBP vector integrates, the resulting overexpressed gene product may be full length or amino-terminal trunctated. Fluorescence activated cell sorting (FACS) was used to isolate BFP+/GFP+ (Wnt active) or BFP+/GFP- (Wnt inactive) A375 cells. We reasoned that if successful, FLAG epitope tag immunopurification and mass spectrometry-based identification of the overexpressed fusion proteins would be cheaper, faster and provide more information than traditional PCR-based detection. Five FLAG immunopurification followed by a series of high salt washes, on-bead tryptic digestion and shotgun mass spectrometry (MS) identified 20 high-confidence proteins specific to Wnt-active cells. The high-salt washes removed associated proteins from the FLAG-tagged bait proteins. The FOXP1 transcription factor ranked as the top screen hit, as determined by spectral count abundance and the CompPASS WD-score.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Melanoma

SUBMITTER: Dennis Goldfarb  

LAB HEAD: Michael Ben Major

PROVIDER: PXD001485 | Pride | 2016-07-05

REPOSITORIES: Pride