Profiling of potential interacting partners of the stress-response transcription coactivator LEDGF/p75 using transcription factor protein arrays Free

2095

Prostate cancer (PCa) is the second leading cause of male cancer deaths in the United States, and is considered a health disparity disease because it disproportionately affects African American men. The identification of transcriptions factors that regulate stress survival pathways in prostate cancer cells is essential for the development of novel therapeutic interventions for PCa. Our group has shown previously that the transcription co-activator LEDGF/p75 (lens-epithelium derived growth factor of 75kD) is overexpressed in prostate tumor cells. LEDGF/p75 is upregulated under conditions of increased oxidative stress and transactivates genes encoding stress response proteins that are likely to promote tumor survival and growth in such environment. The mechanisms by which LEDGF/p75 contributes to the transcriptional activation of stress genes are not well-understood, due in part to the scarce knowledge of endogenous interacting partners of this protein. We used transcription factor protein arrays for the profiling of potential binding partners of LEDGF/p75. Histidine-tagged LEDGF/p75 protein was produced in E. coli and purified by nickel-based immobilized metal affinity chromatography. The purified protein was then used to probe membranes containing transcription factor protein arrays (acquired from Active Motif and Panomics). By modifying the arrays to identify protein-protein interactions via immunoblotting, we were able to detect His-LEDGF/p75 bound to specific transcription factors using a human autoantibody highly specific for this protein. Detection of positive interactions was achieved using a secondary goat anti-human antibody labeled with horseradish peroxidase (HRP), followed by chemiluminescence. Potential interacting partners were identified by their strong reactivity with LEDGF/p75 in at least two independent experiments. These included RNA-polymerase II and the transcription factors STAT1, PC4, Hand1, Hand2, MECP2, KLF12, LHX2, CDX2, CRSP9, GTF2H2, PXR2, and RYBP. Co-immunoprecipitation and domain mapping analyses are currently being conducted to confirm these interactions and identify the binding domains within LEDGF/p75. These studies highlight potential interacting proteins of LEDGF/p75, many of which have been implicated in cancer, and provide the basis for elucidating the mechanisms by which this protein contributes at the transcriptional level to tumor cell survival under an environment of increased oxidative stress.