Project description:LAP-35 and SK-N_MC cells were treated with 10 J/m2 UV light versus untreated Alternative pre-mRNA processing plays a key role in the response to DNA damage as well as in neoplastic transformation. We found that two Ewing Sarcoma (ES) cell lines exhibit different sensitivity to UV light irradiation, with SK-N-MC cells being more sensitive than LAP-35 cells. RNA profiling during the response to low doses of UV light irradiation revealed genes differentially regulated between the two cell lines. In particular, UV light irradiation induced a novel isoform of the RNA helicase DHX9 which is targeted to nonsense-mediated decay (NMD) and therefore causes down-regulation of DHX9 in SK-N-MC cells, but not in LAP-35 cells. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription, and we found that down-regulation of DHX9 by UV light irradiation in SK-N-MC cells impairs the recruitment of EWS-FLI1 to target genes and increases sensitivity to DNA damage. Notably, sensitivity of SK-N-MC cells to irradiation correlated with enhanced phosphorylation and decreased processivity of RNAPII upon irradiation, which in turn causes inclusion of the novel DHX9 exon in SK-N-MC cells exposed to UV light, an observation that could be recapitulated in LAP35 cells by pharmacological reduction of RNAPII processivity. Our data suggest that EWS-FLI1 oncogene activity could be targeted by modulation of DHX9 gene expression.
Project description:Plants use sunlight as a source of energy for photosynthesis but also as an important environmental cue to regulate growth, development and light acclimation. Wavelengths in the UV-B (280-315 nm) and UV-A/blue (315-500 nm) regions of the spectrum are perceived by UV RESISTANCE LOCUS 8 (UVR8) and cryptochromes (CRY1 and CRY2), respectively. Despite recent advances in our understanding of how these photoreceptors promote photomorphogenesis, very little is known about the molecular mechanisms regulated by UVR8 and CRYs in sunlight exposed plants. Here, a factorial experiment was designed to assess the roles of UVR8 and CRYs in regulating transcriptome wide changes, hormone accumulation, and growth competence of Arabidopsis thaliana plants exposed to solar UV-B, UV-A, and blue radiation.
Project description:human hepatoma Hep3B cells were treated with 10 J/m2 UV light versus untreated Alternative pre-mRNA processing plays a key role in the response to DNA damage as well as in neoplastic transformation. We found that two Ewing Sarcoma (ES) cell lines exhibit different sensitivity to UV light irradiation, with Hep3B_UV_40J-N-MC cells being more sensitive than LAP-35 cells. RNA profiling during the response to low doses of UV light irradiation revealed genes differentially regulated between the two cell lines. In particular, UV light irradiation induced a novel isoform of the RNA helicase DHX9 which is targeted to nonsense-mediated decay (NMD) and therefore causes down-regulation of DHX9 in Hep3B_UV_40J-N-MC cells, but not in LAP-35 cells. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription, and we found that down-regulation of DHX9 by UV light irradiation in Hep3B_UV_40J-N-MC cells impairs the recruitment of EWS-FLI1 to target genes and increases sensitivity to DNA damage. Notably, sensitivity of Hep3B_UV_40J-N-MC cells to irradiation correlated with enhanced phosphorylation and decreased processivity of RNAPII upon irradiation, which in turn causes inclusion of the novel DHX9 exon in Hep3B_UV_40J-N-MC cells exposed to UV light, an observation that could be recapitulated in LAP35 cells by pharmacological reduction of RNAPII processivity. Our data suggest that EWS-FLI1 oncogene activity could be targeted by modulation of DHX9 gene expression.
Project description:human hepatoma Hep3B cells were treated with 40 J/m2 UV light versus untreated Alternative pre-mRNA processing plays a key role in the response to DNA damage as well as in neoplastic transformation. We found that two Ewing Sarcoma (ES) cell lines exhibit different sensitivity to UV light irradiation, with Hep3B_UV_40J-N-MC cells being more sensitive than LAP-35 cells. RNA profiling during the response to low doses of UV light irradiation revealed genes differentially regulated between the two cell lines. In particular, UV light irradiation induced a novel isoform of the RNA helicase DHX9 which is targeted to nonsense-mediated decay (NMD) and therefore causes down-regulation of DHX9 in Hep3B_UV_40J-N-MC cells, but not in LAP-35 cells. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription, and we found that down-regulation of DHX9 by UV light irradiation in Hep3B_UV_40J-N-MC cells impairs the recruitment of EWS-FLI1 to target genes and increases sensitivity to DNA damage. Notably, sensitivity of Hep3B_UV_40J-N-MC cells to irradiation correlated with enhanced phosphorylation and decreased processivity of RNAPII upon irradiation, which in turn causes inclusion of the novel DHX9 exon in Hep3B_UV_40J-N-MC cells exposed to UV light, an observation that could be recapitulated in LAP35 cells by pharmacological reduction of RNAPII processivity. Our data suggest that EWS-FLI1 oncogene activity could be targeted by modulation of DHX9 gene expression.
Project description:Ultraviolet (UV) light induces the formation of bulky UV photoproducts in the genome that interfere with DNA replication and transcription. It is well-established how human cells repair UV light-induced DNA lesions, however the signaling pathways and mechanisms that regulate transcription after exposure to UV light are poorly understood. Here, we provide a systematic view on dynamic protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on canonical DNA damage kinases and the p38 MAP kinase pathway. Notably, we demonstrate that p38 and its downstream effector kinase MK2 are responsible for one quarter of protein phosphorylation induced by UV light. We identify RNA binding proteins as primary targets and 14-3-3 family proteins as direct readers of UV light-induced, p38-MK2-dependent phosphorylation. Importantly, we demonstrate that UV light triggers rapid and dynamic phosphorylation of the negative elongation factor (NELF) complex subunit NELFE on serine 115 that mediates its binding to 14-3-3. NELFE interaction with 14-3-3 stabilizes NELFE and RNA pol II interaction on the chromatin and inhibits transcriptional elongation, thereby promoting cell survival after UV light.
Project description:Ultraviolet (UV) light induces the formation of bulky UV photoproducts in the genome that interfere with DNA replication and transcription. It is well-established how human cells repair UV light-induced DNA lesions, however the signaling pathways and mechanisms that regulate transcription after exposure to UV light are poorly understood. Here, we provide a systematic view on dynamic protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on canonical DNA damage kinases and the p38 MAP kinase pathway. Notably, we demonstrate that p38 and its downstream effector kinase MK2 are responsible for one quarter of protein phosphorylation induced by UV light. We identify RNA binding proteins as primary targets and 14-3-3 family proteins as direct readers of UV light-induced, p38-MK2-dependent phosphorylation. Importantly, we demonstrate that UV light triggers rapid and dynamic phosphorylation of the negative elongation factor (NELF) complex subunit NELFE on serine 115 that mediates its binding to 14-3-3. NELFE interaction with 14-3-3 stabilizes NELFE and RNA pol II interaction on the chromatin and inhibits transcriptional elongation, thereby promoting cell survival after UV light.
Project description:This experiment was conducted to test multiple hypotheses: 1) long-wave 365 nm UV light exposure at low fluences does not alter gene expression of hMSC, 2) presence of radical species during polymerization causes DNA damage in hMSC, 3) 3D encapsulation of hMSC causes changes in gene expression of hMSC compared with traditional 2D culture, 4) Differencesin 3D hydrogel networks induce gene expression changes in hMSC The first publication derived from this data set concerns UV exposure and reactive radical species. Light is a non-invasive tool that is widely used in a range of biomedical applications. Techniques such as photopolymerization, photodegradation and photouncaging can be used to alter the chemical and physical properties of biomaterials in the presence of live cells. Long-wave UV light is an easily accessible and commonly used wavelength. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies only investigate cell viability, ignoring other possible non-toxic effects. Since light exposure could potentially induce phenotypic changes (i.e. if damage repair mechanisms are activated), we examined changes in gene expression of human mesenchymal stem cells exposed to light under various 2D and 3D culture conditions. While exposure to long-wave UV light did not induce any significant changes in gene expression regardless of culture conditions, significant changes were observed due to scaffold fabrication chemistry and between cells plated in 2D versus 3D scaffolds.