Investigation of PKNOX2 protein interactions with ATM and RNF8 in bone marrow derived mesenchymal stem cells

Recent research has shown that the transcription factor PKNOX2 is a tumor suppressor protein and decreases in bone marrow-derived mesenchymal stem cells (BM-MSCs) of Fanconi anemia patients. In the previous unpublished study of Günel-Özcan Group, it was suggested that PKNOX2 may play a role in the DNA repair pathway due to the low 'coverage' of ATM and RNF8 proteins in the proteome analysis list obtained after PKNOX2 co-immunoprecipitation. The aim of this study was to confirm the interaction of PKNOX2 protein with ATM and RNF8 proteins in order to investigate the DNA damage and status of this interaction in BM-MSCs. To confirm the interactions, the PKNOX2 overexpression plasmid was transferred to BM-MSCs by electroporation, protein isolation was performed from these cells and, co-immunoprecipitation and Western blotting were performed. While PKNOX2 and ATM interaction was confirmed by Western Blot analysis after co-IP, PKNOX2 and ATM or RNF8 interactions was also confirmed by in situ proximity ligation assay (PLA) due to IgG heavy chain contamination possibility. These interactions were determined by PLA method both in the cytoplasm and in the nucleus. In order to examine the changes in the interactions with DNA damage, DNA damage was detected by flow cytometry in ɣ-irradiatiated. In the flow cytometry analyses performed with PI staining, it was thought that the cells damaged DNA were arrested in the G2 phase, although it was not possible to conclude that there was no sharp dissociation in the cell cycle peaks. In situ proximity ligation assay did not detect PKNOX2 and ATM interaction in ɣ-irradiated cells but increase in PKNOX2-RNF8 interaction was observed. In conclusion, it was shown for the first time in the literature that PKNOX2 transcription factor interacts with ATM and RNF8. Changes in interactions with DNA damage should be confirmed by further studies.