Fjóla Dögg Aðalsteinsdóttir, Thejus Venkatesh, Drífa Hrund Guðmundsóttir, Linda Viðarsdóttir and Stefán Sigurðsson
Genome stability is essential for the normal function of all cells and the DNA damage response is critical for maintaining this stability. It has been shown that microphthalmia-associated transcription factor (MITF) interacts with proteins that take part in the DNA damage response. There are nine isoforms of MITF and some isoforms are tissue-specific like MITF-M which is specific for melanocytes and melanoma cells, however, MITF-A seems to be expressed ubiquitously. The A-isoform is the longest of the nine isoforms with a unique N-terminus. The objective of this study is to gain a better understanding of the role MITF has in DNA repair.
We used mass spectrometry after expressing FLAG tagged MITF-A and MITF-M in U2OS cells. To validate the results from the mass spectrometry we conducted co-immunoprecipitation assays to isolate the FLAG-tagged proteins along with their interacting proteins. These proteins were probed for using Western blot and immunostaining using antibodies specific to repair proteins of interest.
We validated the interaction of both the A and M isoforms of MTIF to known DNA repair proteins. Both isoforms interact with Ku70 and Ku80, proteins involved in non-homologous end joining of DNA double-strand breaks. Interestingly, isoform A seems to have more affinity for RPA1 and PARP1, compared with the M isoform.
Our results indicate that isoform A of MITF has stronger interaction with DNA repair proteins compared to the M isoform, implying a potential role for MITF in the repair process. Next step is to identify the specific binding site on the A-isoform.
