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Characterization of cyclobutane pyrimidine dimer-dependent modulation of gene expression in human keratinocytes following UVB exposure

ABSTRACT: To address CPD-dependent UVB activities, a model system was established in which transfection of keratinocytes with pseudouridine-modified mRNA (M-NM-(-mRNA) encoding CPD-photolyase resulted in 90% reduction of CPDs within 6 and 24 hours after UVB exposure. Microarray analysis of this model system demonstrated that more than 50 % of the gene expression altered by UVB were changed in a CPD-dependent manner. The expression of most of the CPD-dependent genes was changed at 6 h after UVB as compared to 24 h likely due to the higher CPD levels. Nine genes (ATF3, CCNE1, CDKN2B, EGR1, ID2, PTGS2, RUNX1, SNAI1, SNAI2) regulated by CPDs were selected for further investigation (qPCR, Western blot) based on the microarray data. Gene expression modulated by UVB irradiation in HaCaT keratinocytes was measured at 6 and 24 hours after the exposure to dose of 20 mJ/cm2 UVB. Three independent experiments were performed at each time (6 or 24 hours) using different passages for each experiment.

ORGANISM(S): Homo sapiens

SUBMITTER: Gabor Boros

PROVIDER: E-GEOD-65034 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Characterization of cyclobutane pyrimidine dimer-dependent modulation of gene expression in human keratinocytes following UVB exposure

Project description:To address CPD-dependent UVB activities, a model system was established in which transfection of keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase resulted in 90% reduction of CPDs within 6 and 24 hours after UVB exposure. Microarray analysis of this model system demonstrated that more than 50 % of the gene expression altered by UVB were changed in a CPD-dependent manner. The expression of most of the CPD-dependent genes was changed at 6 h after UVB as compared to 24 h likely due to the higher CPD levels. Nine genes (ATF3, CCNE1, CDKN2B, EGR1, ID2, PTGS2, RUNX1, SNAI1, SNAI2) regulated by CPDs were selected for further investigation (qPCR, Western blot) based on the microarray data.

2015-01-16 | GSE65034 | GEO

CPD-capture-seq map of CPD formation in UV-irradiated primary melanocytes, normal human skin fibroblasts and UV-irradiated genomic DNA

Project description:UV-induced CPDs were mapped in primary skin melanocytes or normal human skin fibroblasts following either UVC or UVB irradiation and in isolated human genomic DNA (naked DNA control) that was UVB or UVC irradiated. CPDs were mapped across the human genome using the CPD-capture-seq method and the resulting libraries were captured for ~4000 genomic regions of interest (~3 Mbp) of the human genome by the company Rapid Genomics prior to Illumina sequencing

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Melanoma mutations are caused by deamination of cytosine in pyrimidine dimers

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Project description:Considering that human skin cancer is predominantly attributed to UV radiation from sunlight, additional investigations are needed to elucidate the role of P2RY6 in UVB-induced skin carcinogenesis. Surprisingly, we find that P2ry6 deletion exhibits marked promotion to UVB-induced skin papilloma formation compared with wild-type mice, suggesting its tumor-suppressive role in UVB-induced skin cancer. Additionally, P2ry6 knockout promotes mouse skin hyperplasia induced by short-term UVB irradiation, while UDP, the ligand of P2RY6, can inhibit UVB-induced skin damage. Furthermore, UVB irradiation can significantly upregulate P2RY6 expression in mouse and human skin cells. These results indicate that P2RY6 plays a crucial protective role in resisting UVB-induced skin damage and carcinogenesis. At the molecular level, P2RY6 deletion inhibits ubiquitination and expression of XPC after UVB irradiation in keratinocytes, resulting in the accumulation of CPDs (cyclobutane pyrimidine dimers). P2RY6 deletion also activates PI3K/AKT signaling pathway in vitro and in vivo. The CPD accumulation and inflammatory responses enhanced by P2RY6 deletion are reversed by an AKT inhibitor. These findings suggest that P2RY6 acts as a tumor suppressor in UVB-induced skin cancer by regulating PI3K/AKT signaling pathway.

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Genomic Sites Ultrasensitive to Ultraviolet Radiation

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Loss of E3 ubiquitin protein ligase Cbl-b provides protection against UVB-induced cytotoxicity and inflammation.

Project description:The exposure of skin to ultraviolet (UV) radiation results in sunburn cell (SBC) and cyclobutane pyrimidine dimer (CPD) formation and local immune suppression. However, the pathways that regulate these events are poorly understood. Here, we exposed mice to UVB to study the early cytotoxic effects in absence of E3 ubiquitin protein ligase Cbl-b. We observed that Cbl-b-/- mice developed significantly less SBCs and CPDs compared to WT mice. In addition, microarray analysis revealed that loss of Cbl-b-induced cell tolerance genes rather than DNA repair genes. Taken together, our results suggest a novel role of Cbl-b in regulating UV cytotoxicity.

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CPD-seq mapping of UV damage repair in rad16 mutant yeast

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CPD-seq mapping of UV damage formation and repair in yeast

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2016-08-19 | E-GEOD-79977 | biostudies-arrayexpress

Analysis of UV-induced DNA damage in human cells using CPD-seq 2.0

Project description: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.

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