Cryptochrome-Timeless structure reveals circadian clock timing mechanisms.

Circadian rhythms influence many behaviours and diseases1,2. They arise from oscillations in gene expression caused by repressor proteins that directly inhibit transcription of their own genes. The fly circadian clock offers a valuable model for studying these processes, wherein Timeless (Tim) plays a critical role in mediating nuclear entry of the transcriptional repressor Period (Per) and the photoreceptor Cryptochrome (Cry) entrains the clock by triggering Tim degradation in light2,3. Here, through cryogenic electron microscopy of the Cry-Tim complex, we show how a light-sensing cryptochrome recognizes its target. Cry engages a continuous core of amino-terminal Tim armadillo repeats, resembling how photolyases recognize damaged DNA, and binds a C-terminal Tim helix, reminiscent of the interactions between light-insensitive cryptochromes and their partners in mammals. The structure highlights how the Cry flavin cofactor undergoes conformational changes that couple to large-scale rearrangements at the molecular interface, and how a phosphorylated segment in Tim may impact clock period by regulating the binding of Importin-α and the nuclear import of Tim-Per4,5. Moreover, the structure reveals that the N terminus of Tim inserts into the restructured Cry pocket to replace the autoinhibitory C-terminal tail released by light, thereby providing a possible explanation for how the long-short Tim polymorphism adapts flies to different climates6,7.

Blue light-emitting diode irradiation promotes transcription factor EB-mediated lysosome biogenesis and lysosomal cell death in murine photoreceptor-derived cells.

Exposure to blue light from light-emitting diodes (LEDs) is a source of damage for human eyes in today's modern life. Although it is well known that blue light can cause cellular damage and death, the molecular mechanism underlying this is still not fully understood. Here, we demonstrated that exposure to blue LED light increased lysosome levels and perinuclear cluster formation in 661W murine photoreceptor-derived cells. Irradiation with blue LED light promoted the nuclear transport of transcription factor EB (TFEB) and a subsequent increase in lysosomal-related gene expression. Moreover, blue LED light induced morphological changes in lysosomal structure and lysosomal membrane permeabilization (LMP). These effects were suppressed by an antioxidant, N-acetylcysteine (NAC). Finally, a calcium ion chelator, BAPTA-AM, attenuated blue LED light-induced lysosomal biogenesis and cell death. Taken together, these findings suggest that oxidative stress under blue LED light increases lysosome levels via the TFEB pathway in a calcium-dependent manner, resulting in the accumulation of damaged lysosomes and subsequently lysosomal cell death. Our results imply that lysosomal homeostasis plays a key role in the maintenance of eye function and the progression of retinal diseases.

Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants.

Phytochromes are bilin-binding photosensory receptors which control development over a broad range of environmental conditions and throughout the whole plant life cycle. Light-induced conformational changes enable phytochromes to interact with signaling partners, in particular transcription factors or proteins that regulate them, resulting in large-scale transcriptional reprograming. Phytochromes also regulate promoter usage, mRNA splicing and translation through less defined routes. In this review we summarize our current understanding of plant phytochrome signaling, emphasizing recent work performed in Arabidopsis. We compare and contrast phytochrome responses and signaling mechanisms among land plants and highlight open questions in phytochrome research.

Post-transcriptional regulation of Ghd7 protein stability by phytochrome and OsGI in photoperiodic control of flowering in rice.

Rice (Oryza sativa) is a facultative short-day (SD) plant, flowering early under SD and late under long-day (LD) conditions. Ghd7 is a major regulator of flowering time in rice, which strongly delays flowering under LD. Induction of Ghd7 expression by phytochromes has been shown to contribute to photoperiodic regulation of flowering in rice. Here, we show that Ghd7 also is regulated by phytochromes at a post-transcriptional level. We found that constitutive expression of Ghd7 delays flowering in the wild-type (WT) background, but not in the se5 mutant background (deficient in functional phytochromes) under LD and that Ghd7 protein fails to accumulate in the se5 mutant. We also found that co-expressing OsGIGANTEA (OsGI) with Ghd7 causes reduced accumulation of Ghd7 protein and partially suppresses the delayed flowering phenotype in the WT background, suggesting that phytochromes and OsGI play antagonist roles in regulating Ghd7 protein stability and flowering time. We show that OsPHYA, OsPHYB and OsGI could directly interact with Ghd7. Interestingly, OsPHYA and OsPHYB could inhibit the interaction between OsGI and Ghd7, thus helping to stabilize Ghd7 protein. Our results revealed a new level of Ghd7 regulation by phytochromes and OsGI in photoperiodic control of flowering in rice.

Ultraviolet-C (UVC) ray acts as a synchronizing cue for circadian rhythm control in murine fibroblast.

Ultraviolet-C (UVC) electromagnetic radiation is the most damaging type of the UV radiation and causes many cellular and physiological responses. UVC has been using for sterilization and disinfection, and the risk of exposure to the UVC is increasing. Here, we determined the effect of the UVC on the cellular circadian clock system. UVC irradiation synchronized the biological clock system and induced time-dependent expression of clock genes including Clock, Cry1, and Per1. The rhythmic expression of clock genes is also followed by time-dependent mRNA degradation or non-canonical translation initiation of clock genes. Furthermore, we show a translocation of PERIOD1 (PER1) protein after UVC irradiation, which mediates the rhythmic feedback loop of clock genes. Our results suggest that UVC can synchronize the circadian clock system, and induces rhythmic expression of clock genes via time-dependent transcription, post-transcription, and post-translational modification.

Stress-Induced Phosphorylation of Nuclear YB-1 Depends on Nuclear Trafficking of p90 Ribosomal S6 Kinase.

Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology.

Light-controllable Transcription System by Nucleocytoplasmic Shuttling of a Truncated Phytochrome B.

Transcriptional regulation is a useful strategy for gene therapy and for biomedical research. Unlike chemically regulated transcriptional approaches, spatiotemporal control of transcription using optogenetic tools is a powerful technology for the analysis of single cells. For light to penetrate into tissues, it is desired to use photoreceptors absorbing red/far-red light with a low-molecular mass applicable for the use of virus vectors, and a photoswitch using the photoreceptor needs to be constructed as a single expression vector. Herein, we describe an optogenetic tool based on Arabidopsis thaliana phytochrome (Phy) B and its binding partner, phytochrome-interacting factor (PIF) 6. We generated a truncated PhyB, which allowed for reversible association with PIF6 by red/far-red light illumination. The red light illumination only for 5 min induced PhyB translocation from the cytoplasm into the nucleus by the association with PIF6, resulting in transcriptional activation based on Gal4 DNA-binding domain and the upstream activating sequence of Gal system. The nucleocytoplasmic shuttling vector using PhyB and PIF6 might be applicable for transcriptional regulation in tissue experiments.

Insight into nuclear body formation of phytochromes through stochastic modelling and experiment.

Spatial relocalization of proteins is crucial for the correct functioning of living cells. An interesting example of spatial ordering is the light-induced clustering of plant photoreceptor proteins. Upon irradiation by white or red light, the red light-active phytochrome, phytochrome B, enters the nucleus and accumulates in large nuclear bodies (NBs). The underlying physical process of nuclear body formation remains unclear, but phytochrome B is thought to coagulate via a simple protein-protein binding process. We measure, for the first time, the distribution of the number of phytochrome B-containing NBs as well as their volume distribution. We show that the experimental data cannot be explained by a stochastic model of nuclear body formation via simple protein-protein binding processes using physically meaningful parameter values. Rather modelling suggests that the data is consistent with a two step process: a fast nucleation step leading to macroparticles followed by a subsequent slow step in which the macroparticles bind to form the nuclear body. An alternative explanation for the observed nuclear body distribution is that the phytochromes bind to a so far unknown molecular structure. We believe it is likely this result holds more generally for other nuclear body-forming plant photoreceptors and proteins.

Multilayered Reprogramming in Response to Persistent DNA Damage in C. elegans.

DNA damage causally contributes to aging and age-related diseases. Mutations in nucleotide excision repair (NER) genes cause highly complex congenital syndromes characterized by growth retardation, cancer susceptibility, and accelerated aging in humans. Orthologous mutations in Caenorhabditis elegans lead to growth delay, genome instability, and accelerated functional decline, thus allowing investigation of the consequences of persistent DNA damage during development and aging in a simple metazoan model. Here, we conducted proteome, lipidome, and phosphoproteome analysis of NER-deficient animals in response to UV treatment to gain comprehensive insights into the full range of physiological adaptations to unrepaired DNA damage. We derive metabolic changes indicative of a tissue maintenance program and implicate an autophagy-mediated proteostatic response. We assign central roles for the insulin-, EGF-, and AMPK-like signaling pathways in orchestrating the adaptive response to DNA damage. Our results provide insights into the DNA damage responses in the organismal context.

Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation.

EGF receptor (EGFR) endocytosis is induced by stress in a manner dependent on the p38 MAPK family. Ligand and stresses such as X-rays, reportedly promote nuclear trafficking of endocytosed EGFR for regulation of gene transcription and DNA repair. We fail to detect EGFR endocytosis or nuclear transport following X-ray treatment of HeLa or head and neck cancer cells, despite extensive DNA damage induction. Apparent nuclear staining with EGFR extracellular domain antibody remained present despite reduced/absent EGFR expression, and so did not represent nuclear EGFR. UVB and UVC, but not X-ray or UVA, treatment induced p38 activation and EGFR endocytosis, although all of these stresses induced DNA damage, indicating that DNA damage alone is not sufficient to induce EGFR endocytosis. Increased reactive oxygen species (ROS) levels following UVB treatment, compared to that seen with X-rays, do not alone explain differences in p38 activation. UVB, like UVC, induced EGFR accumulation predominantly in perinuclear endosomes, rather than in the nucleus. Our morphological techniques identifying major changes in receptor distribution do not exclude the possibility that small but biologically relevant amounts of EGFR enter the nucleus. This study highlights the importance and limitations of morphological analyses of receptor distribution in understanding signaling outcome.

TIGAR knockdown radiosensitizes TrxR1-overexpressing glioma in vitro and in vivo via inhibiting Trx1 nuclear transport.

The up-regulation of thioredoxin reductase-1 (TrxR1) is detected in more than half of gliomas, which is significantly associated with increased malignancy grade and recurrence rate. The biological functions of NADPH-dependent TrxR1 are mainly associated with reduced thioredoxin-1 (Trx1) which plays critical roles in cellular redox signaling and tumour radio-resistance. Our previous work has proved that TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown could notably radiosensitize glioma cells. However, whether TrxR1-overexpressing glioma cells could be re-radiosensitized by TIGAR silence is still far from clear. In the present study, TrxR1 was stably over-expressed in U-87MG and T98G glioma cells. Both in vitro and in vivo data demonstrated that the radiosensitivity of glioma cells was considerably diminished by TrxR1 overexpression. TIGAR abrogation was able to radiosensitize TrxR1-overexpressing gliomas by inhibiting IR-induced Trx1 nuclear transport. Post-radiotherapy, TIGAR low-expression predicted significant longer survival time for animals suffering from TrxR1-overexpessing xenografts, which suggested that TIGAR abrogation might be a promising strategy for radiosensitizing TrxR1-overexpressing glial tumours.

Autophagy Promotes the Repair of Radiation-Induced DNA Damage in Bone Marrow Hematopoietic Cells via Enhanced STAT3 Signaling.

Autophagy protects hematopoietic cells from radiation damage in part by promoting DNA damage repair. However, the molecular mechanisms by which autophagy regulates DNA damage repair remain largely elusive. Here, we report that this radioprotective effect of autophagy depends on STAT3 signaling in murine bone marrow mononuclear cells (BM-MNCs). Specifically, we found that STAT3 activation and nuclear translocation in BM-MNCs were increased by activation of autophagy with an mTOR inhibitor and decreased by knockout of the autophagy gene Atg7. The autophagic regulation of STAT3 activation is likely mediated by induction of KAP1 degradation, because we showed that KAP1 directly interacted with STAT3 in the cytoplasm and knockdown of KAP1 increased the phosphorylation and nuclear translocation of STAT3. Subsequently, activated STAT3 transcriptionally upregulated the expression of BRCA1, which increased the ability of BM-MNCs to repair radiation-induced DNA damage. This novel finding that activation of autophagy can promote DNA damage repair in BM-MNCs via the ATG-KAP1-STAT3-BRCA1 pathway suggests that autophagy plays an important role in maintaining genomic integrity of BM-MNCs and its activation may confer protection of BM-MNCs against radiation-induced genotoxic stress.

Apoptosis-inducing factor (AIF) nuclear translocation mediated caspase-independent mechanism involves in X-ray-induced MCF-7 cell death.

PURPOSE: Breast cancer is the most common cancer among women and radiotherapy is a conventional therapy following surgery. Previous studies have demonstrated that except the caspase-dependent pathway, caspase-independent pathway is also involved in the cell death responding to irradiation, despite the unclear mechanism. The purpose of the present study was to observe the role of apoptosis-inducing factor (AIF), the first identified caspase-independent molecule, in X-ray-induced breast cancer cell (MCF-7) cell death. MATERIALS AND METHODS: In this study, WST-1 assay, DAPI nuclear staining and clonogenic survival assay were used to test the cell response to different treatments; Western blot was used to detect the protein expression; RT-PCR and plasmid transfection were used to observe the role of AIF. RESULTS: X-ray-induced AIF transferred from the mitochondrion to the nucleus. Inhibition of AIF expression reduced X-ray-induced MCF-7 cell death. Further, AIF nuclear translocation is in a caspase-independent manner in this process, but not caspase-dependent manner. CONCLUSIONS: The present study revealed that AIF nuclear translocation proceeded in X-ray-induced MCF-7 cell death in a caspase-independent manner.

COP1 is required for UV-B-induced nuclear accumulation of the UVR8 photoreceptor.

The UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) promotes UV-B acclimation and tolerance in Arabidopsis thaliana UVR8 localizes to both cytosol and nucleus, but its main activity is assumed to be nuclear. UV-B photoreception stimulates nuclear accumulation of UVR8 in a presently unknown manner. Here, we show that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is required for UV-B-induced nuclear accumulation of UVR8, but bypassing the COP1 requirement for UVR8 nuclear accumulation did not rescue the cop1 mutant UV-B phenotype. Using a glucocorticoid receptor (GR)-based fusion protein system to conditionally localize GR-UVR8 to the nucleus, we have demonstrated that both photoactivation and nuclear localization of UVR8 are required for UV-B-induced photomorphogenic responses. In contrast, there was no UV-B response when UV-B-activated UVR8 was artificially retained in the cytosol. In agreement with a predominantly nuclear activity, constitutively active UVR8(W285A) accumulated in the nucleus also in the absence of UV-B. Furthermore, GR-COP1 expression lines suggested that UV-B-activated UVR8 can be coimported into the nucleus by COP1. Our data strongly support localization of UVR8 signaling in the nucleus and a dual role for COP1 in the regulation of UV-B-induced UVR8 nuclear accumulation and in UVR8-mediated UV-B signaling.

Optogenetic Control of Nuclear Protein Import in Living Cells Using Light-Inducible Nuclear Localization Signals (LINuS).

Many biological processes are regulated by the timely import of specific proteins into the nucleus. The ability to spatiotemporally control the nuclear import of proteins of interest therefore allows study of their role in a given biological process as well as controlling this process in space and time. The light-inducible nuclear localization signal (LINuS) was developed based on a natural plant photoreceptor that reversibly triggers the import of proteins of interest into the nucleus with blue light. Each LINuS is a small, genetically encoded domain that is fused to the protein of interest at the N or C terminus. These protocols describe how to carry out initial microscopy-based screening to assess which LINuS variant works best with a protein of interest. © 2016 by John Wiley & Sons, Inc.

Melatonin enhances mitochondrial ATP synthesis, reduces reactive oxygen species formation, and mediates translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (γ-GCS, HO-1, NQO1) in ultraviolet radiation-treated normal human epidermal keratinocytes (NHEK).

Melatonin is an ubiquitous molecule with a variety of functions including potent antioxidative properties. Due to its lipophilic character, it easily crosses cellular and intracellular membranes and reaches all subcellular organelles. Because of its ability to scavenge free radicals, melatonin protects against oxidative stress, for example, induced by ultraviolet radiation (UVR). Here, we investigated, in a dose-dependent (0, 10, 25, and 50 mJ/cm(2) ) and time-dependent (0, 4, 24, 48 hr post-UVR) manner, whether melatonin prevents the UVR-mediated alterations in ATP synthesis and the generation of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEK). Additionally, we evaluated the molecular mechanism of action of melatonin with regard to activation of phase-2 antioxidative enzymes via nuclear erythroid 2-related factor (Nrf2). We found that (i) melatonin counteracted UVR-induced alterations in the ATP synthesis and reduced free radical formation; (ii) melatonin induced the translocation of Nrf2 transcription factor from the cytosol into the nucleus resulting in, (iii) melatonin enhanced gene expression of phase-2 antioxidative enzymes including γ-glutamylcysteine synthetase (γ-GCS), heme oxygenase-1 (HO-1), and NADPH: quinone dehydrogenase-1 (NQO1) representing an elevated antioxidative response of keratinocytes. These results suggest that melatonin not only directly scavenges ROS, but also significantly induces the activation of phase-2 antioxidative enzymes via the Nrf2 pathway uncovering a new action mechanism that supports the ability of keratinocytes to protect themselves from UVR-mediated oxidative stress.

Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expression in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy.

ATDC (Ataxia Telangiectasia Group D Complementing) Promotes Radioresistance through an Interaction with the RNF8 Ubiquitin Ligase.

Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The ataxia telangiectasia group D complementing gene (ATDC, also called TRIM29) is highly expressed in many malignancies. It participates in the DNA damage response downstream of ataxia telangiectasia-mutated (ATM) and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (amino acids 348-588) of ATDC and the RING domain of RNF8 and was disrupted by mutation of ATDC Ser-550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.

Red Light-Regulated Reversible Nuclear Localization of Proteins in Mammalian Cells and Zebrafish.

Protein trafficking in and out of the nucleus represents a key step in controlling cell fate and function. Here we report the development of a red light-inducible and far-red light-reversible synthetic system for controlling nuclear localization of proteins in mammalian cells and zebrafish. First, we synthetically reconstructed and validated the red light-dependent Arabidopsis phytochrome B nuclear import mediated by phytochrome-interacting factor 3 in a nonplant environment and support current hypotheses on the import mechanism in planta. On the basis of this principle we next regulated nuclear import and activity of target proteins by the spatiotemporal projection of light patterns. A synthetic transcription factor was translocated into the nucleus of mammalian cells and zebrafish to drive transgene expression. These data demonstrate the first in vivo application of a plant phytochrome-based optogenetic tool in vertebrates and expand the repertoire of available light-regulated molecular devices.