Analysis of UV-induced DNA damage in human cells using CPD-seq 2.0
Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their formation mechanism and functional impacts are not fully understood. Here, we used UV damage sequencing data and analyzed cyclobutane pyrimidine dimer (CPD) formation, DNA repair, and CPD deamination in human cells at single-nucleotide resolution. Our data shows prominent CPD hotspots immediately after UV irradiation at ETS binding sites, particularly at sites with a conserved TTCCGG motif, which correlate with mutation hotspots identified in cutaneous melanoma. Additionally, CPDs are repaired slower at ETS binding sites than in flanking DNA. Cytosine deamination in CPDs to uracil is suggested as an important step for UV mutagenesis. However, we found that CPD deamination is significantly suppressed at ETS binding sites, particularly for the CPD hotspot on the 5' side of the ETS motif, arguing against a role for CPD deamination in promoting ETS-associated UV mutations.  Finally, we analyzed a subset of frequently mutated promoters, including the ribosomal protein genes RPL13A and RPS20, and found that mutations in the ETS motif can significantly reduce the promoter activity. Thus, our data identifies high UV damage and low repair, but not CPD deamination, as the main mechanism for ETS-associated mutations in melanoma and uncover new roles of often-overlooked mutation hotspots in perturbing gene transcription. Overall design: Human skin fibroblast cells were grown in DMEM/F-12 medium with 10% FBS. Media was removed and cells were exposed to UV-C at 6 and 8 J/m2 to induce UV damage. A portion of the cells were harvested immediately after UV treatment as time 0 (i.e., no repair). The rest of cells were incubated in fresh DMEM for repair. Repair time points were taken at 6 and 24 h post UV irradiation. Genomic DNA was isolated from cells and used for the preparation of CPD-seq 2.0 libraries. After Illumina sequencing, locations of UV-induced photolesions were identified using our published protocol. Formation and repair of UV damage was analyzed in gene promoters, particularly those containing ETS binding sites.