UNDERSTANDING THE PALB2 PROMOTER REGION AND ITS EXPRESSION REGULATION THROUGHOUT THE CELL CYCLE

INTRODUCTION AND OBJECTIVES: Germline mutations in the PALB2 tumor suppressor gene are associated with breast and pancreatic cancer predisposition. PALB2 encodes for a homonymous protein with a pivotal role in genomic integrity maintenance through the homologous recombination (HR) DNA repair pathway. The protein works as a scaffold protein, connecting BRCA1 and BRCA2 to promote the recruitment of the RAD51 recombinase to DNA-damaged sites. Despite the growing interest in PALB2, both the promoter region of gene, and its expression regulation are not fully understood. The present study seeks to characterize the PALB2 promoter locus, defining its minimal promoter region, and also analyze expression regulation. MATERIAL AND METHODS: The Genome Browser platform was used to track down in silico H3K27Ac signal (euchromatin marker) upstream PALB2 gene and, therefore, identify the putative PALB2 promoter region. The selected region was generated through PCR using BJ cells genomic DNA as a template; different deletion fragments were further generated to map the minimal region with promoter activity. The activity was assessed through a luciferase reporter expression assay in HEK293FT cells. RESULTS AND CONCLUSION: We identified a sequence enclosing a 1001 nucleotide upstream of PALB2 as the putative gene promoter region (named 1001). We demonstrated that 1001 has a promoter activity by its ability to activate the luciferase transcription (the reporter gene). To better map these putative PALB2 promoter region, we generated different fragments and identified a minimal regulatory region (1001366) that exhibits similar levels of transcription activation observed for 1001. We also demonstrated that the segment 1001 and smaller different fragments showed an increase in transcriptional activity upon DNA damage induced by treatment with ionizing radiation (IR) when compared to non-IR cells. Further, we also observed an increase in PALB2 mRNA and protein levels after IR (10Gy) damage and PARP inhibition (Olaparib 2,5uM – 10uM). Interestingly, we demonstrated that the levels of PALB2 mRNA and PALB2 protein varied throughout the cell cycle, showing an increase during the S and G2/M phases, in which HR is active. In summary, our data identified a minimal regulatory region of PALB2 and suggested that it is responsive to different DNA damage agents, particularly during the S and G2/M phases of the cell cycle.