UV-induced Mutations Research Articles

Ultraviolet Mutagenesis Coupled with Next-Generation Sequencing as a Method for Functional Interrogation of Powdery Mildew Genomes.

Powdery mildews are obligate biotrophic fungal pathogens causing important diseases of plants worldwide. Very little is known about the requirements for their pathogenicity at the molecular level. This is largely due to the inability to culture these organisms in vitro or to modify them genetically. Here, we describe a mutagenesis procedure based on ultraviolet (UV) irradiation to accumulate mutations in the haploid genome of the barley powdery mildew pathogen Blumeria graminis f. sp. hordei. Exposure of B. graminis f. sp. hordei conidia to different durations of UV-C radiation (10 s to 12 min) resulted in a reduced number of macroscopically visible fungal colonies. B. graminis f. sp. hordei colony number was negatively correlated with exposure time and the total number of consecutive cycles of UV irradiation. Dark incubation following UV exposure further reduced fungal viability, implying that photoreactivation is an important component of DNA repair in B. graminis f. sp. hordei. After several rounds of UV mutagenesis, we selected two mutant isolates in addition to the parental B. graminis f. sp. hordei K1 isolate for whole-genome resequencing. By combining automated prediction of sequence variants and their manual validation, we identified unique UV-induced mutations in the genomes of the two isolates. Most of these mutations were in the up- or downstream regions of genes or in the intergenic space. Some of the variants detected in genes led to predicted missense mutations. As an additional insight, our bioinformatic analyses revealed a complex population structure within supposedly clonal B. graminis f. sp. hordei isolates.

113 Aspirin protects melanocytes and keratinocytes from UV-induced DNA damage in vivo

Ultraviolet (UV) radiation promotes skin cancer through multiple mechanisms, which can be secondary to inflammation, oxidative stress, and DNA damage. UV-induced DNA mutations may arise from disrepair of cyclobutane pyrimidine dimers (CPD) or 8-oxoguanine (8-OG). We investigated whether the anti-inflammatory activities of aspirin (ASA) could protect against UV-induced DNA damage and skin cancer in mouse models. Skin from adult C57BL/6 mice receiving 0.4 mg ASA daily by gavage had reduced inflammation (CD3+ T-lymphocytes, CD272+ lymphocytes/NK cells, Ly6G+ neutrophils, F4/80+ macrophages), fewer sunburn cells, and lower 8-OG levels than skin from control animals (gavaged with only water) 48 h after acute UVB exposure (600 J/m2).

Noninvasive Assessment of Epidermal Genomic Markers of UV Exposure in Skin

The measurement of UV-induced DNA damage as a dosimeter of exposure and predictor of skin cancer risk has been proposed by multiple groups. Although UV-induced mutations and adducts are present in normal-appearing UV-exposed epidermis, sampling normal nonlesional skin requires noninvasive methods to extract epidermal DNA for analysis. Here, we demonstrate the feasibility of such an approach, termed surfactant-based tissue acquisition for molecular profiling. Sampling in patients was performed using a felt-tip pen soaked in a mixture of surfactants (Brij-30/N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate). In mice, we show that the epidermis can be selectively removed without scarring, with complete healing within 2 weeks. We exposed hairless mice to low-dose UV radiation over a period of 3 months and serially sampled them through up to 2 months following the cessation of UV exposure, observing a progressive increase in a UV signature mutational burden. To test whether surfactant-based tissue acquisition for molecular profiling could be applied to human patients, samples were collected from sun-exposed and sun-protected areas, which were then subjected to high-depth targeted exome sequencing. Extensive UV-driven mosaicism and substantially increased mutational loads in sun-exposed versus sun-protected areas were observed, suggesting that genomic measures, as an integrated readout of DNA damage, repair, and clonal expansion, may be informative markers of UV exposure.

Regulation of UV damage repair in quiescent yeast cells

Non-growing quiescent cells face special challenges when repairing lesions produced by exogenous DNA damaging agents. These challenges include the global repression of transcription and translation and a compacted chromatin structure. We investigated how quiescent yeast cells regulated the repair of DNA lesions produced by UV irradiation. We found that UV lesions were excised and repaired in quiescent cells before their re-entry into S phase, and that lesion repair was correlated with high levels of Rad7, a recognition factor in the global genome repair sub-pathway of nucleotide excision repair (GGR-NER). UV exposure led to an increased frequency of mutations that included C->T transitions and T > A transversions. Mutagenesis was dependent on the error-prone translesion synthesis (TLS) DNA polymerase, Pol zeta, which was the only DNA polymerase present in detectable levels in quiescent cells. Across the genome of quiescent cells, UV-induced mutations showed an association with exons that contained H3K36 or H3K79 trimethylation but not with those bound by RNA polymerase II. Together, the data suggest that the distinct physiological state and chromatin structure of quiescent cells contribute to its regulation of UV damage repair.

Epidermal structure and differentiation.

In this Primer, Moreci and Lechler follow the lifetime of an epidermal cell from its birth to its ultimate death, and detail how this journey is necessary for epidermal function.

Molecular Biology of Basal and Squamous Cell Carcinomas.

The prevalent keratinocyte-derived neoplasms of the skin are basal cell carcinoma and squamous cell carcinoma. Both so-called non-melanoma skin cancers comprise the most common cancers in humans by far. Common risk factors for both tumor entities include sun exposure, DNA repair deficiencies leading to chromosomal instability, or immunosuppression. Yet, fundamental differences in the development of the two different entities have been and are currently unveiled. The constitutive activation of the sonic hedgehog signaling pathway by acquired mutations in the PTCH and SMO genes appears to represent the early basal cell carcinoma developmental determinant. Although other signaling pathways are also affected, small hedgehog inhibitory molecules evolve as the most promising basal cell carcinoma treatment options systemically as well as topically in current clinical trials. For squamous cell carcinoma development, mutations in the p53 gene, especially UV-induced mutations, have been identified as early events. Yet, other signaling pathways including epidermal growth factor receptor, RAS, Fyn, or p16INK4a signaling may play significant roles in squamous cell carcinoma development. The improved understanding of the molecular events leading to different tumor entities by de-differentiation of the same cell type has begun to pave the way for modulating new molecular targets therapeutically with small molecules.

Human genes differ by their UV sensitivity estimated through analysis of UV-induced silent mutations in melanoma

Human genes differ by their UV sensitivity estimated through analysis of UV-induced silent mutations in melanoma

Merkel-cell carcinoma

Merkel-cell carcinoma (MCC) is arare and aggressive neuroendocrine carcinoma named for its Merkel-cell-like ultrastructure. The neuroendocrine Merkel cell was previously believed to be the cell of origin. However, Merkel cells are postmitotic and thus probably not the cell of origin of MCC. It is derived from an epidermal stem cell, which also might represent the cell of origin of MCC. Further putative cells of origin are dermal stem cells and pre/pro‑B cells, the latter showing some similar markers (e.g. PAX5).About 80% of MCCs are induced by the integration of DNA of the Merkel cell polyoma virus (MCPyV) into the genome. On the other hand, about 20% of MCCs show UV-induced mutations in numerous genes (e.g. TP53, RB1). In routine histology, MCC appears monomorphic and the diagnosis is confirmed by immunohistochemistry showing CK20 arranged in typical paranuclear plaques, together with the presence of neurofilaments and chromograninA. Virus-positive and virus-negative MCC are not different histologically.UV-induced and viral neoantigens cause the strong immunogenicity of MCC. Moreover, over the last few years, the presence of PD-1 and PD-L1 has been demonstrated within tumor and immune cells. For the checkpoint inhibitors pembrolizumab and avelumab, responses of about 50% have been shown, independent of virus state. Circulating tumor cells (CTCs) seem to be helpful in tumor tracking. Further immunological and molecular studies are necessary for future individual therapies, also concerning immunocompromised patients.

554 Skin ageing continues long after ultraviolet radiation damage

Wrinkling has been attributed to the cumulative effects of acute Ultraviolet Radiation (UVR) exposure on the dermis. Previous studies have shown that the collagenase MMP-1 is secreted by dermal fibroblasts and degrades collagen immediately after UV exposure. However, skin ages gradually, and not intermittently after sun exposure. In this study we sought to examine the mechanism underpinning the gradual appearance of wrinkles, and to determine whether this is a result of intrinsic changes in the biology of the dermis. In vitro studies were performed using patient-derived human dermal fibroblasts from healthy skin, HFF and HDF cell lines. Cell lines were UV-protected or repeatedly exposed to UVB. Dermal fibroblasts from fairer skin types had higher levels of UV-induced mutations which, in multivariate analysis, was linked to higher expression of extracellular matrix degradation genes. This suggests that the background UVR exposure history influences the composition of the dermis as it ages. We investigated the mechanism driving collagen degradation in dermal fibroblasts with and without a history of UV damage. Fibroblasts with high background UV exposure have elevated levels of MMP-1 at rest. In addition, they show increased collagen degradation activity, which persists in the absence of acute UV exposure. Our study shows that dermal fibroblasts with a history of chronic UV exposure have higher levels of collagenase expression and activity, even in the absence of acute UV exposure. Importantly, these findings explain the mechanism driving gradual collagen degradation and wrinkling in the absence of UV light.

Molecular genetic investigations as the basis for targeted treatment of basal cell carcinoma of the eye

Basal cell carcinomas are generally the most common malignant human cancers. They grow destructively and invasively into surrounding tissue. The rising incidence of basal cell carcinomas in an aging society demands new, less destructive treatment approaches especially for advanced and difficult to resect basal cell carcinomas at surgically demanding locations, such as those growing or metastasizing on the eyelid. New key technologies, such as next generation sequencing (NGS) enable high-throughput genetic analyses of tumors. In this way new knowledge on the molecular genetic pathogenesis of basal cell carcinomas is gained, which enables the development of new targeted treatment of the affected signal pathway. In line with the multistep photocarcinogenesis theory, basal cell carcinomas possess ahigh load of UV-induced gene mutations (75%). Independent of the genesis 85% of basal cell carcinomas harbor activating mutations of the hedgehog signaling pathway. Accordingly, two hedgehog inhibitors for the treatment of difficult to resect or metastasized basal cell carcinomas have been licensed (vismodegib and sonidegib); however, only 60% of patients respond to this treatment. This is due to the high mutational load with 85% of the tumors harboring additional mutations in other signaling pathways. Molecular genetic analyses will enable the identification of further targeted therapies for advanced basal cell carcinomas. Due to the high mutational load checkpoint inhibitors (e. g. cemiplimab) are also effective in the treatment of basal cell carcinomas. Nicotinamide and UV protection can reduce the mutational load and hence decrease the risk for tumor development.

130 Novel method for non-invasive assessment of molecular markers of UV exposure in normal skin

The use of markers of UV-induced DNA damage as a dosimeter of exposure and predictor of skin cancer risk has been proposed by multiple groups. While UV-induced mutations and adducts are well described and present in normal appearing UV-exposed epidermis, sampling normal non-lesional skin requires non-invasive methods to extract epidermal DNA for analysis. Here we demonstrate the feasibility of such an approach. Sampling was performed using a felt-tip pen soaked in a mixture of surfactants (Brij-30/DPS). In mice, we show that the epidermis can be selectively removed without scarring, with complete healing within 2 weeks. We exposed Hairless mice to low-dose solar simulated light (Newport) over a period of 3 months for a total exposure of 175 kJ/m2, a dose which normally produces tumors within 3 months. Over 2 micrograms / cm2 of DNA could be purified and exome sequenced. We noted a progressive increase in a UV-signature mutational burden over the following 2 months following the cessation of UV exposure, consistent with accumulation of mutant clones. Next we sought to assess whether this was practical to perform in human patients. Skin samples were collected from neck or shoulder (sun-exposed areas with dermatoheliosis) and lateral chest or back (minimally sun-exposed areas) with no prior preparation. Approximately 300 ng of total DNA could be isolated from an approximately 1 cm2 area of sampled skin. These samples were subjected to targeted exome sequencing using a commercially available panel (QIAseq Cancer Panel) to a depth of approximately 3500X and referenced to saliva. We identified considerable heterogeneity in multiple samples obtained from the same patient, suggesting widespread mosaicism, but also an enrichment for UV-signature mutations in key tumor suppressors including TP53 and NOTCH1/2. Our next steps are to use hypermutated areas of the genome to further refine this dosimetry method and assess its dynamic range across a larger spectrum of patients.

748 Duplex sequencing reveals the effects of caffeine on reducing UV-induced mutations of cancer-relevant genes

Epidemiological studies have shown that daily caffeinated coffee intake is associated with decreased risks of developing skin cancer. Importantly, this cancer-preventive effect is attributable to caffeine (no benefit with decaffeinated coffee) and is dose-dependent. However, the mechanism by which caffeine prevents skin cancer remains elusive. To determine the effects of caffeine on chronic UV-induced genetic mutations and skin cancer development, we irradiated Xpc–/– (DNA repair-deficient) mice with 25–250 mJ/cm2 of UVB per day, twice a week for 19 weeks and treated with or without caffeine topically immediately after each UV irradiation. Chronic UV irradiation to mouse back skin caused cutaneous squamous cell carcinoma (cSCC). Topical application of caffeine significantly inhibited cSCC development compared to vehicle treatment (n = 70 mice per group). We collected UV-irradiated but nontumorous skin from these mice for Duplex Sequencing, a next-generation sequencing method capable of detecting ultralow-frequency mutations. Among seven genes analyzed (Trp53, Cdkn2a, Tert, Sftpc, Ambp, B2m, and Cyc1), the most prevalent mutation in UV-irradiated skin was the UV signature C>T mutation. C>T mutation on the noncoding strand was essentially absent in transcriptionally active genes, likely due to transcription-coupled repair that removes UV-induced DNA lesions only on the transcribed (noncoding) strand. Caffeine did not drastically change the overall mutation frequency in UV-irradiated skin. Instead, caffeine reduced the frequency of mutations in the p53 DNA-binding domain and the p16/Cdkn2a promoter. These findings indicate mechanisms by which caffeine may have selectively sensitized p53-mutant or p16-deficient keratinocytes to death in vivo, a general concept that has been suggested in prior studies performed in cell lines in vitro.

HPV and skin carcinogenesis

Epidemiological and biological studies provide several lines of evidence for the involvement of cutaneous beta human papillomaviruses (HPVs), together with ultraviolet (UV) radiation, in the development of cutaneous squamous cell carcinoma. These viruses appear to act with a hit-and-run mechanism, being necessary at an early stage of carcinogenesis and being dispensable for the maintenance of the malignant phenotype. Studies in experimental models show that beta HPVs, mainly via the E6 and E7 oncoproteins, are able to promote proliferation and to circumvent cellular stresses induced by UV radiation. These findings support a model of skin carcinogenesis in which beta HPV-infected keratinocytes remain alive despite the accumulation of UV-induced DNA mutations. In this manner, these cells become highly susceptible to progression towards malignancy. Thus, UV radiation is the main driver of skin cancer development, while beta HPVs act as facilitators of the accumulation of UV-induced DNA mutations.

The Protein Tyrosine Phosphatase H1 PTPH1 Supports Proliferation of Keratinocytes and is a Target of the Human Papillomavirus Type 8 E6 Oncogene.

Human papillomaviruses (HPV) replicate their DNA in the suprabasal layer of the infected mucosa or skin. In order to create a suitable environment for vegetative viral DNA replication HPV delay differentiation and sustain keratinocyte proliferation that can lead to hyperplasia. The mechanism underlying cell growth stimulation is not well characterized. Here, we show that the E6 oncoprotein of the βHPV type 8 (HPV8), which infects the cutaneous skin and is associated with skin cancer in Epidermodysplasia verruciformis patients and immunosuppressed organ transplant recipients, binds to the protein tyrosine phosphatase H1 (PTPH1), which resulted in increased protein expression and phosphatase activity of PTPH1. Suppression of PTPH1 in immortalized keratinocytes reduced cell proliferation as well as the level of epidermal growth factor receptor (EGFR). Furthermore, we report that HPV8E6 expressing keratinocytes have increased level of active, GTP-bound Ras. This effect was independent of PTPH1. Therefore, HPV8E6-mediated targeting of PTPH1 might result in higher level of EGFR and enhanced keratinocyte proliferation. The HPV8E6-mediated stimulation of Ras may be an additional step to induce cell growth. Our results provide novel insights into the mechanism how βHPVE6 proteins support proliferation of infected keratinocytes, thus creating an environment with increased risk of development of skin cancer particularly upon UV-induced DNA mutations.

Overproduction of thermoalkalophilic lipase secreted by Bacillus atrophaeus FSHM2 using UV-induced mutagenesis and statistical optimization of medium components

Microbial enzymes of extremophilic origin serve as a vital source of stable industrial enzymes. The present study focused on overproduction of a thermoalkalophilic lipase produced by Bacillus atrophaeus FSHM2 through UV-induced random mutagenesis (5–45 min exposure to UV light) and factorial experimental design augmented to response surface methodology. Firstly, a UV-induced mutant (designated as UV-45) was developed after the exposure of wild strain to UV irradiation for 45 min which was able to secrete 3484.8 U/L lipase. Afterward, Plackett–Burman experimental approach augmented to central composite design was employed to optimize medium components (olive oil, maltose, glucose, sucrose, yeast extract, tryptone, urea, (NH4)2SO4, NaCl, CaCl2, and ZnSO4) for lipase production by the UV-45 mutant strain. The maximum lipase production of 5505.3 U/L were predicted in medium containing 5% of olive oil, 0.69% of glucose, 0.69% of sucrose, 2.5% of maltose, yeast extract (0.7 g/L), urea (0.44 g/L), (NH4)2SO4 (2.44 g/L), tryptone (1.19 g/L), NaCl (1.61 g/L), CaCl2 (3.81 g/L), and ZnSO4 (1.42 g/L). A mean value of 5161.3 ± 83.3 U/L of lipolytic activity was acquired from real experiments. To sum up, the lipolytic activity of wild type strain (1720.4 U/L) increased by 3-fold after UV-induced mutagenesis and medium components optimization (5161.3 U/L).

Mutational Patterns in Metastatic Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma from the head and neck typically metastasize to the lymph nodes of the neck and parotid glands. When a primary is not identified, they are difficult to distinguish from metastases of mucosal origin and primary salivary gland squamous cell carcinoma. UV radiation causes a mutation pattern that predominantly features cytosine to thymine transitions at dipyrimidine sites and has been associated with cutaneous squamous cell carcinoma. In this study, we used whole genome sequencing data from 15 cutaneous squamous cell carcinoma metastases and show that a UV mutation signature is pervasive across the cohort and distinct from mucosal squamous cell carcinoma. The mutational burden was exceptionally high and concentrated in some regions of the genome, especially insulator elements (mean 162 mutations/megabase). We therefore evaluated the likely impact of UV-induced mutations on the dipyrimidine-rich binding site of the main human insulator protein, CCCTC-binding factor, and the possible implications on CCCTC-binding factor function and the spatial organization of the genome. Our findings suggest that mutation signature analysis may be useful in determining the origin of metastases in the neck and the parotid gland. Furthermore, UV-induced DNA damage to insulator binding sites may play a role in the carcinogenesis and progression of cutaneous squamous cell carcinoma.

A New Efficient Method for Measuring Oxygen Consumption Rate Directly ex vivo in Human Epidermal Biopsies.

Skin cells are constantly exposed to environmental influences such as air pollution, chemicals, pathogens and UV radiation. UV radiation can damage different biological structures, but most importantly cellular DNA. Mitochondria contain their own genome and accumulate UV-induced DNA mutations to a large extent. This can result, e.g., in accelerated skin aging. Understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on the development of age-related diseases. Previous studies have been carried out on cell cultures derived from primary cells, which does not fully represent the real situation in the skin, while the mitochondrial parameters were considered barely or not at all. Here we describe a method to measure mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using an Agilent Seahorse XF24 Flux Analyzer. Before the assay, epidermis and dermis are separated enzymatically, we then used the XF24 Islet capture microplates to position the epidermis samples to measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). In these plates, small nets can be fixed to the plate bottom. The epidermis was placed with the vital-basal-side on the net. Active ingredients in the three ports were injected consecutively to determine the effect of each compound. This allows determining the efficiency of the individual complexes within the respiratory chain. This protocol enables the testing of toxic substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.

Excision of the 59-kb fdxN DNA element is required for transcription of the nifD gene in Anabaena PCC 7120 Heterocysts

ABSTRACTIn the heterocyst-forming cyanobacterium Anabaena (also Nostoc) sp. strain PCC 7120, the nitrogen-fixation (nif) genes are expressed specifically in heterocysts during the late stages of development. We used gfp-reporter fusions to examine the regulation of the nifH and nifD genes. Plasmid-borne reporter fusions containing up to 500-bp of the nifH upstream region, which extended into the nifU gene, did not show developmentally regulated GFP fluorescence after removal of a nitrogen source from the growth medium, which indicated that sequences essential for transcriptional regulation are outside of the tested regions. Therefore, a gfp-reporter fusion was engineered into the chromosome at the 5′ end of nifD to produce strain AMC1774. This reporter construct at the native locus showed strong developmentally regulated GFP expression in differentiating heterocysts by 18 h after removal of combined nitrogen. We then screened for UV-induced mutants of AMC1774 that differentiated heterocysts but failed to show GFP-reporter fluorescence. Several dark mutants were obtained and then complemented with an expression library. One mutant was complemented with a clone that contained the xisF and alr1460 genes. The xisF gene is required for excision of a 59,428-bp DNA element present within the fdxN gene in the nifB-fdxN-nifS-nifU gene cluster, which is upstream of the nifHDK gene cluster. Analysis of the original UV-induced mutant showed that it was defective for excision of the fdxN DNA element. Together, our data shows that transcription of the nifHDK genes requires a distant promoter upstream of the fdxN DNA element. We used a gfp transcriptional reporter to demonstrate developmentally regulated promoter activity from the intergenic region upstream of nifB. We also show that the promoter for a second nifH gene, nifH2 (alr0874), lies proximal to nifH2 in its upstream intergenic region.

Nucleosome positions establish an extended mutation signature in melanoma

Ultraviolet (UV) light-induced mutations are unevenly distributed across skin cancer genomes, but the molecular mechanisms responsible for this heterogeneity are not fully understood. Here, we assessed how nucleosome structure impacts the positions of UV-induced mutations in human melanomas. Analysis of mutation positions from cutaneous melanomas within strongly positioned nucleosomes revealed a striking ~10 base pair (bp) oscillation in mutation density with peaks occurring at dinucleotides facing away from the histone octamer. Additionally, higher mutation density at the nucleosome dyad generated an overarching “translational curvature” across the 147 bp of DNA that constitutes the nucleosome core particle. This periodicity and curvature cannot be explained by sequence biases in nucleosomal DNA. Instead, our genome-wide map of UV-induced cyclobutane pyrimidine dimers (CPDs) indicates that CPD formation is elevated at outward facing dinucleotides, mirroring the oscillation of mutation density within nucleosome-bound DNA. Nucleotide excision repair (NER) activity, as measured by XR-seq, inversely correlated with the curvature of mutation density associated with the translational setting of the nucleosome. While the 10 bp periodicity of mutations is maintained across nucleosomes regardless of chromatin state, histone modifications, and transcription levels, overall mutation density and curvature across the core particle increased with lower transcription levels. Our observations suggest structural conformations of DNA promote CPD formation at specific sites within nucleosomes, and steric hindrance progressively limits lesion repair towards the nucleosome dyad. Both mechanisms create a unique extended mutation signature within strongly positioned nucleosomes across the human genome.

Epidermal Tissue Adapts to Restrain Progenitors Carrying Clonal p53 Mutations

SummaryAging human tissues, such as sun-exposed epidermis, accumulate a high burden of progenitor cells that carry oncogenic mutations. However, most progenitors carrying such mutations colonize and persist in normal tissue without forming tumors. Here, we investigated tissue-level constraints on clonal progenitor behavior by inducing a single-allele p53 mutation (Trp53R245W; p53∗/wt), prevalent in normal human epidermis and squamous cell carcinoma, in transgenic mouse epidermis. p53∗/wt progenitors initially outcompeted wild-type cells due to enhanced proliferation, but subsequently reverted toward normal dynamics and homeostasis. Physiological doses of UV light accelerated short-term expansion of p53∗/wt clones, but their frequency decreased with protracted irradiation, possibly due to displacement by UV-induced mutant clones with higher competitive fitness. These results suggest multiple mechanisms restrain the proliferation of p53∗/wt progenitors, thereby maintaining epidermal integrity.