Identification of the transcriptional regulators of ATB7B gene by genomic locus proteomics and their effect on cisplatin resistance

ATP7B is a copper pump that plays a vital part in cellular homeostasis. It removes the excess metal from the cells to prevent the toxic results of copper accumulation. Insufficient ATP7B activity results in copper gathering in the body, culminating in Wilson's disease. Upregulation of ATP7B conversely causes the removal of platinum-based drugs along with copper, leading to drug resistance. Understanding the transcriptional regulation of ATP7B may shed light on the mechanisms of disease progression. Metal Regulatory Transcription Factor 1 (MTF1) is a well-studied regulator of ATP7B; however, its expression does not always correlate with ATP7B expression in cancers. The expression of a transcription factor does not always have to be correlated with the expression of the target gene. But the probability that ATP7B is regulated by a single transcription factor is also very low, thus indicating the necessity to identify novel regulators of ATP7B. An in-silico analysis using TRANSFAC/PROMO software was performed to find these additional factors. It was found that several transcription factors may bind to the ATP7B promoter, which was focused around -3000 to +1, indicating that the regulatory sequences are most likely located in this region. A novel proximity labeling methodology called "Genomic Locus Proteomics" was used in this thesis to identify the transcription factors that regulate ATP7B expression. In this technique, the deadCas9 (dCas9) protein is fused to the APEX2 enzyme, and the protein is guided to the ATP7B promoter via specific gRNAs. The efficient gRNAs spanning the -3000 to +1 region were determined by T7E assay and ChIP-qPCR experiments. The proteins near the dCas9-targeted regions of the ATP7B promoter were subsequently biotinylated with the APEX2 enzyme and pulled down with streptavidin-magnetic beads. The marked proteins were then identified with mass spectrometry, and several proteins were selected based on their enrichment scores and their associations with cancer progression and drug resistance in cancers. The target gene PINX1 (Pin2/TRF1-Interacting Protein) was analyzed to illuminate its relationship with ATP7B activity, and it was shown that overexpression of this gene leads to an increase in ATP7B expression. Furthermore, PINX1 overexpression in the HEK293T and Huh7 cells increased the cell viability against cisplatin treatment. This resistance to cisplatin is attributed to the heightened ATP7B activity in the cells. Our findings indicate a transcriptional connection between PINX1 and ATP7B. This relationship may play a role in the progression of the disease and could potentially be utilized to develop more effective therapies.