Transcriptional profiling of UV-B response of Populus alba.
ABSTRACT: Microarray technology was used to assess transcriptome changes in poplar (Populus alba L.) under a realistic simulation of increased UV-B radiation. Plants were UV-Bbe (UV-B biologically effective radiation) supplemented with a dose of 6 kJ/m2/day for 12 hours per day and allowed to recover during the night. Poplar plants were UV-B treated using a refined controlled environment able to guarantee a realistic simulation of natural conditions, especially for light parameters such as presence of background UV-B radiation for control plants and balanced PAR/UV-A/UV-B ratio. A time course experiment was planned to look both at the rapid and delayed response of poplar to UVB; two time points after 3 h (T3h) and 30 h (6th hour of the third day of treatment, T30h) were considered. 4 independent biological replicates were analysed for each time point. Competitive hybridisations were carried out using the PICME 28K microarray. Keywords: Time course experiment, stress response Two condition experiment: UVB supplemented plants vs normal UV-B level plants. Biological replicates: 4 UVB suplemented plants, 4 control plants, two time points, one replicate per array. Dye swap between replicates.
Project description:Microarray technology was used to assess transcriptome changes in poplar (Populus alba L.) under a realistic simulation of increased UV-B radiation. Plants were UV-Bbe (UV-B biologically effective radiation) supplemented with a dose of 6 kJ/m2/day for 12 hours per day and allowed to recover during the night. Poplar plants were UV-B treated using a refined controlled environment able to guarantee a realistic simulation of natural conditions, especially for light parameters such as presence of background UV-B radiation for control plants and balanced PAR/UV-A/UV-B ratio. A time course experiment was planned to look both at the rapid and delayed response of poplar to UVB; two time points after 3 h (T3h) and 30 h (6th hour of the third day of treatment, T30h) were considered. 4 independent biological replicates were analysed for each time point. Competitive hybridisations were carried out using the PICME 28K microarray. Keywords: Time course experiment, stress response Overall design: Two condition experiment: UVB supplemented plants vs normal UV-B level plants. Biological replicates: 4 UVB suplemented plants, 4 control plants, two time points, one replicate per array. Dye swap between replicates.
Project description:The â??small perturbationâ?? approach is critical in studying the â??steady stateâ?? of a biological system. In our experiments, small perturbations were generated by applying a series of repeating intermittent small doses of ultraviolet radiation to a human keratinocyte cell line, HaCaT. The biological response was assessed by monitoring the gene expression profiles using a high reliability and high resolution cDNA microarray system. Following intermittent 10 J/m2 UVB small perturbations, two opposite classes of genes, down-regulated and up-regulated, exhibited an immediate response followed by relaxation between each small perturbation, but were prolonged down- or up-regulated without relaxation while larger doses (233 or 582.5 J/m2) of UVB were applied. A repeated cycle pattern of gene expression following small perturbations is an indication of the existence of steady states. This cycle pattern is suppressed when large perturbations are applied. We believe that this is a universal phenomenon. In our experiments, the functions of up-regulated genes were mainly associated with anti-proliferation, anti-mitogenesis, and apoptosis. On the other hand, down-regulated genes were mainly related to proliferation, mitogenesis, and anti-apoptosis. In conclusion, this study experimentally proves the concept of steady state at the transcription level and demonstrates the feasibility of using small perturbation approaches for investigating steady states. This study could also set a foundation of computational systems biology, which has implicitly used the concept of steady state. Keywords: time course Three UVB exposures are indicated by UV at time points of 0, 8 and 16 hours. T1, T2, T3, T4, T5, and T6 denote the sampling time points. T1, T3 and T5 are allocated 30 minutes after the corresponding UVB irradiation. T2, T4 and T6 are allocated 8 hours after each UVB irradiation. At each sampling time point, two samples (control and UV-irradiated) are collected. See supplementary file Loop_design.pdf for further explanation.
Project description:Ultraviolet (UV) radiation is a major melanoma risk factor, yet underlying mechanisms remain poorly understood. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon-response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-gamma (IFN-gamma), but not type-I interferons. IFN-gamma was produced by macrophages recruited to neonatal skin by UVB-induced chemokine receptor Ccr2 ligands. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-gamma blockade abolished macrophage-associated melanoma growth and survival. IFN-gamma-producing macrophages were identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-gamma in promoting melanocytic cell survival/immunoevasion, and suggest IFN-gamma-R signaling represents a novel therapeutic melanoma target. Biologic replicates of UVA- and UVB-treated mouse melanocytes, as well as untreated mouse melanocytes and mouse keratinocytes, were used to define melanocyte expression signatures associated with UV treatment.
Project description:Ultraviolet (UV) wavebands in sunlight are immunomodulatory. About half the amount of UVA within a minimum erythemal dose of sunlight is systemically immunosuppressive, while higher doses protect from UVB immunosuppression in mice. We have previously shown that these responses to UVA are genetically restricted as they occur in C57BL/6 but not Balb/c mice. We used gene set enrichment analysis of microarray data and real-time RT-PCR confirmation to determine the molecular mechanisms associated with UVA immunomodulation. We found up-regulation of mRNA for the alternative complement pathway. The core-enriched genes complement component 3, properdin and complement factor B were all activated by the immunosuppressive dose of UVA only in UVA-responsive C57BL/6 but not unresponsive BALB/c mice. This therefore matched the genetic restriction and dose responsiveness of UVA immunosuppression. The immune-protective higher UVA dose prevented UVB from down regulating chemokine receptor 7 and IL-12B, and decreased IL-10, supporting previous identification of IL-12 and IL-10 in high dose UVA protection from UVB immunosuppression. Our study has identified activation of the alternative complement pathway as a trigger of UVA-induced systemic immunosuppression and suggests that this pathway is likely to be an important sensor of UVA-induced damage to the skin. 24 hours after UVA, UVB and ssUV irradiation, a 1 cm2 uniform section of skin was excised from the dorsal surface of irradiated and control mice. Total RNA was then extracted from the whole skin using TRIzol reagent (Gibco Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions, purified, DNase treated and reverse transcribed into cDNA. For the microarray study a direct incorporation of Cyanine 3-dCTP and Cyanine 5-dCTP fluorescent dyes (Perkin Elmer Life Sciences, Inc. Boston, MA, USA) was used for cDNA synthesis. For each UV dose, a reference design was used to compare an unirradiated control against an irradiated sample. Microarray experiments used compugen 22k mouse oligonucleotide microarray slides (The Clive and Vera Ramaciotti Centre for Gene Function Analysis, Sydney Australia (http://www.ramaciotti.unsw.edu.au). Lower and higher UVA doses were used. C57BL/6 mice were irradiated with lower UVA, higher UVA, UVB, or ssUV; Balb/C mice were irradiated with lower or higher UVA. Experiments were replicated 6 times for each UV dose. A fluorescent dye swap was done for each alternate hybridisation to reduce systematic dye bias of incorporated fluorescent dyes.
Project description:To test the hypothesis that different mechanisms and/or factors might be involved in physiological pigmentary responses of the skin to different types of UV, we used whole human genome microarrays and immunohistochemical analyses to characterize human skin in situ to examine how melanocyte-specific proteins and paracrine melanogenic factors are regulated by repetitive exposure to suberythemal doses of different types of UV (UVA, UVB or SSR). Six volunteers with skin type II-III were irradiated with SSR, UVA or UVB radiation for 2 weeks (5 times per week, 10 times total) after preliminary determination of their MEDs. Biopsies were taken 3 days after the last irradiation.
Project description:Previous studies showed that SV40 transformed cells have unique DNA damage responses; further inspecting these responses by microarray provides an opportunity to discover transciprtional insights of DNA damage responses after UVB irradiation. This study is used to comapre to GSE7589, our previous study of human normal lung fibroblast after UVB irradiation. We used a loop design in this study, cDNA microarray experiment consisted of eight RNA samples, including UVB-irradiated samples and their corresponding controls of 4 time points after UV irradiation.
Project description:We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after UVB at different time points (6, 12 and 24h) to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of ultraviolet-B radiation (UVB). Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. We analysed six lines of melanocytes isolated from lightly pigmented neonatal foreskin (LM), and six lines from darkly pigmented neonatal foreskin (DM), at 0, 6, 12 and 24 hours post UV irradiation. We also assessed the effect of different keratinocyte-conditioned media (KCM+ or KCM-)
Project description:Betaine (trimethylglycine) is a non-toxic, highly water-soluble organic osmolyte widely used in skin care due to its assumed moisturizing and protective properties, but only few studies have addressed its specific effects in skin. Here, the cellular and molecular targets of betaine were analyzed by genome-wide expression analysis in organotypic cultures of rat epidermal keratinocytes (REK). In this model, we also examined whether betaine modifies the impacts of acute UVB exposure. The expression of several genes relevant to epidermal biology, proliferation/differentiation or malignancy as well as solute transport were verified by independent methods (qRT-PCR, western blotting). The data concerning changes in calcium metabolism after UVB exposure has been published separately (Bart et al., Br. J. Dermatol. 171:376-387, 2014). Organotypic cultures of rat epidermal keratinocytes (REK) were prepared by plating cells on a collagen-coated insert (submerged in medium), lifting the confluent cell layer to the air-liquid interface 3 days after plating. These 3D cultures were divided in four treatment groups with three replicates in each: control, betaine (10 mM), UVB (30 mJ/cm2) and betaine + UVB. Betaine was added to the cultures for 11 days, starting from day 4 until sample collection. UVB exposure was performed 24 h prior to sample collection for 2-week-old 3D cultures with a well-formed epidermal layer. For further details on the experimental set-up and characteristics of the UV source, please refer to Bart et al. (Br. J. Dermatol. 171:376-387, 2014) and Rauhala et al. (J. Biol. Chem. 288:17999-18012, 2013).
Project description:The goal of this thesis is to study the response of a human keratinocyte cell line (HaCaT cells) after UVB irradiation. In order to develop a system for UV irradiation, we firtstly studied the UV illumination system to find out a stable and reliable condition for UV irradiation experiments. Further, we found out the proper dose of UVB radiation and time points after UVB irradiation by performing MTT assay and trypan blue viability test. According to the results of MTT assay and trypan blue viability test, the cellular activity as well as survival rate of UVB-irradiated cells were in proportion to the radiation doses within a lower UVB dose range. We wanted to find out the possible reasons for this phenomenon by using cDNA microarray. We hope the thesis could be a guide for the following researches, and be useful in the clinical studies about the UV damage. Keywords: dose response and time course Loop-designed microarray experiments were performed in our study. Twenty-one samples are arranges as seven small loops and one large loop, with three and seven microarray slides for each small and large loop, respectively. This set of loop-designed microarray experiment contains one control untreated (non UVB-treated) samples and two UVB-treated samples (one low dose and one high dose UVB-treated) at each time point (total seven time points).
Project description:Abstract Background: Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair has been described to quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. However, the precise mechanism by which RNA polymerase II (Pol II) transcription is affected following UV irradiation during the repair processes genome-wide is not well understood. Results: We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We find that about 90% of the promoters of expressed genes show reduced Pol II occupancy 2-4 hours following UVB irradiation, and that the presence of Pol II is restored to “normal”, or higher, levels 5-6 hours after irradiation. We also identified a smaller set of genes, where the presence of Pol II at the promoter regions does not decrease after UVB irradiation, but often increases throughout the entire transcription units. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH but not TBP follows the behavior of Pol II suggesting that at these genes TFIIH may be sequestered for DNA repair upon UVB treatment. Conclusions: Thus, our study reveals a global negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed genes following sublethal UVB irradiation, and a small subset of genes (including regulators of repair, cell growth and survival), where Pol II escapes this negative regulation. Following genome-wide RNA Polymerase II redistribution over time upon UVB irradiation