Interaction of p53 with the CCT complex promotes protein folding and wild-type p53 activity.

p53 is a transcription factor that mediates tumor suppressor responses. Correct folding of the p53 protein is essential for these activities, and point mutations that induce conformational instability of p53 are frequently found in cancers. These mutant p53s not only lose wild-type activity but can also acquire the ability to promote invasion and metastasis. We show that folding of wild-type p53 is promoted by an interaction with the chaperonin CCT. Depletion of this chaperone in cells results in the accumulation of misfolded p53, leading to a reduction in p53-dependent gene expression. Intriguingly, p53 proteins mutated to prevent the interaction with CCT show conformational instability and acquire an ability to promote invasion and random motility that is similar to the activity of tumor-derived p53 mutants. Our data therefore suggest that both growth suppression and cell invasion may be differentially regulated functions of wild-type p53.

STING recognition of cytoplasmic DNA instigates cellular defense.

How the cell recognizes cytosolic DNA including DNA-based microbes to trigger host-defense-related gene activation remains to be fully resolved. Here, we demonstrate that STING (stimulator of interferon genes), an endoplasmic reticulum translocon-associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self- (apoptotic, necrotic) or pathogen-related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and proinflammatory genes in addition to type I IFN. Our data indicate that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.

Circadian rhythms identified in Caenorhabditis elegans by in vivo long-term monitoring of a bioluminescent reporter.

Circadian rhythms are based on endogenous clocks that allow organisms to adjust their physiology and behavior by entrainment to the solar day and, in turn, to select the optimal times for most biological variables. Diverse model systems-including mice, flies, fungi, plants, and bacteria-have provided important insights into the mechanisms of circadian rhythmicity. However, the general principles that govern the circadian clock of Caenorhabditis elegans have remained largely elusive. Here we report robust molecular circadian rhythms in C elegans recorded with a bioluminescence assay in vivo and demonstrate the main features of the circadian system of the nematode. By constructing a luciferase-based reporter coupled to the promoter of the suppressor of activated let-60 Ras (sur-5) gene, we show in both population and single-nematode assays that C elegans expresses ∼24-h rhythms that can be entrained by light/dark and temperature cycles. We provide evidence that these rhythms are temperature-compensated and can be re-entrained after phase changes of the synchronizing agents. In addition, we demonstrate that light and temperature sensing requires the photoreceptors LITE and GUR-3, and the cyclic nucleotide-gated channel subunit TAX-2. Our results shed light on C elegans circadian biology and demonstrate evolutionarily conserved features in the circadian system of the nematode.

Nucleic acid clamp-mediated recognition and stabilization of the physiologically relevant MYC promoter G-quadruplex.

The MYC proto-oncogene is upregulated, often at the transcriptional level, in ∼80% of all cancers. MYC's promoter is governed by a higher order G-quadruplex (G4) structure in the NHE III1 region. Under a variety of conditions, multiple isoforms have been described to form from the first four continuous guanine runs (G41-4) predominating under the physiologically relevant supercoiled conditions. In the current study, short oligonucleotides complementing the 5'- and 3'-regions flanking the G4 have been connected by an abasic linker to form G4 clamps, varying both linker length and G4 isoform being targeted. Clamp A with an 18 Šlinker was found to have marked affinity for its target isomer (G41-4) over the other major structures (G42-5 and G41-5, recognized by clamps B and C, respectively), and to be able to shift equilibrating DNA to foster greater G4 formation. In addition, clamp A, but not B or C, is able to modulate MYC promoter activity with a significant and dose-dependent effect on transcription driven by the Del4 plasmid. This linked clamp-mediated approach to G4 recognition represents a novel therapeutic mechanism with specificity for an individual promoter structure, amenable to a large array of promoters.

Rational design of novel N-alkyl-N capped biostable RNA nanostructures for efficient long-term inhibition of gene expression.

Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24-29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3'-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs.

Role of pulmonary macrophages in initiation of lung metastasis in anaplastic thyroid cancer.

Several clinical studies have demonstrated that increased macrophage infiltration into tumors confers metastatic potential and poor prognosis in cancer. Preclinical studies are needed to develop new strategies for countering metastasis. Our study was designed to investigate the impact of pulmonary macrophages on lung metastasis of anaplastic thyroid cancer (ATC). ATC (CAL-62) and macrophage (Raw264.7) were transfected with the effluc (CAL-62/effluc, Raw264.7/effluc). Coculture and migration assays were used to assess the effect of Raw264.7 or THP1 (human macrophage) (or conditioned medium) on the proliferation and/or migration of CAL-62/effluc cells in vitro. The effect of clodro-lipo or PBS-lipo on macrophage depletion was confirmed in vitro and in vivo. CAL-62/effluc cells (1 × 10(6) ) were intravenously injected into nude mice 24 h after clodro-lipo or PBS-lipo administration. Effect of clodro-lipo on the lung metastasis of CAL-62/effluc was assessed by bioluminescence imaging (BLI). Micro computed tomography (micro-CT) and histology. BLI signals of CAL-62/effluc and Raw264.7/effluc increased to cell number. Raw264.7 cells and THP1 cells promoted CAL-62/effluc proliferation, and conditioned medium of Raw264.7 cells promoted CAL-62/effluc migration. Clodro-lipo significantly depleted pulmonary macrophages in vitro and in vivo. Intensity of BLI signals in ATC lung metastasis was weaker in the clodro-lipo group than PBS-lipo control. Micro-CT imaging and hematoxylin/eosin staining revealed smaller tumor masses in the clodro-lipo group than PBS-lipo control. Our findings indicate that pulmonary macrophages have an important role in initiation of lung metastasis of ATC. New therapeutic strategies that preclude initiation of pulmonary metastasis could potentially be developed by targeting pulmonary macrophages.

Bioluminescent imaging of drug efflux at the blood-brain barrier mediated by the transporter ABCG2.

ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter.

Photochemical Regulation of Gene Expression Using Caged siRNAs with Single Terminal Vitamin†E Modification.

Caged siRNAs with a single photolabile linker and/or vitamin E (vitE) modification at the 5' terminal were rationally designed and synthesized. These virtually inactive caged siRNAs were successfully used to photoregulate both firefly luciferase and GFP gene expression in cells with up to an 18.6-fold enhancement of gene silencing activity, which represents one of the best reported photomodulation of gene silencing efficiencies to date. siRNA tracking and vitE competition experiments indicated that the inactivity of vitE-modified siRNAs was not due to the bulky moiety of vitE; rather, the involvement of vitE-binding proteins has a large contribution to caged siRNA inactivation by preventing the dissociation of siRNA/lipo complexes and/or siRNA release. Further patterning experiments revealed the ability to spatially regulate gene expression through simple light irradiation.

Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses.

Chronic infection with hepatitis B virus (HBV) puts individuals at high risk for complicating cirrhosis and liver cancer, but available treatment to counter the virus rarely eliminates infection. Although harnessing RNA interference (RNAi) to silence HBV genes has shown the potential, achieving efficient and durable silencing of viral genes remains an important goal. Here we report on the propagation of lentiviral vectors (LVs) that successfully deliver HBV-targeting RNAi activators to liver cells. Mono- and tricistronic artificial primary microRNAs (pri-miRs) derived from pri-miR-31, placed under transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, caused efficient inhibition of HBV replication markers. The tricistronic cassette was capable of silencing a mutant viral target and the effects were observed without disrupting the function of an endogenous miR (miR-16). The mTTR promoter stably expressed a reporter transgene in mouse livers over a study period of 1 year. Good silencing of HBV genes, without evidence of toxicity, was demonstrated following intravenous injection of LVs into neonatal HBV transgenic mice. Collectively, these data indicate that LVs may achieve sustained inhibition of HBV replication that is appealing for their therapeutic use.

Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects.

The ciliopathies are a group of heterogeneous diseases with considerable variations in phenotype for allelic conditions such as Meckel-Gruber syndrome (MKS) and Joubert syndrome (JBTS) even at the inter-individual level within families. In humans, mutations in TMEM67 (also known as MKS3) cause both MKS and JBTS, with TMEM67 encoding the orphan receptor meckelin (TMEM67) that localizes to the ciliary transition zone. We now describe the Tmem67(tm1(Dgen/H)) knockout mouse model that recapitulates the brain phenotypic variability of these human ciliopathies, with categorization of Tmem67 mutant animals into two phenotypic groups. An MKS-like incipient congenic group (F6 to F10) manifested very variable neurological features (including exencephaly, and frontal/occipital encephalocele) that were associated with the loss of primary cilia, diminished Shh signalling and dorsalization of the caudal neural tube. The 'MKS-like' group also had high de-regulated canonical Wnt/ß-catenin signalling associated with hyper-activated Dishevelled-1 (Dvl-1) localized to the basal body. Conversely, a second fully congenic group (F > 10) had less variable features pathognomonic for JBTS (including cerebellar hypoplasia), and retention of abnormal bulbous cilia associated with mild neural tube ventralization. The 'JBTS-like' group had de-regulated low levels of canonical Wnt signalling associated with the loss of Dvl-1 localization to the basal body. Our results suggest that modifier alleles partially determine the variation between MKS and JBTS, implicating the interaction between Dvl-1 and meckelin, or other components of the ciliary transition zone. The Tmem67(tm1(Dgen/H)) line is unique in modelling the variable expressivity of phenotypes in these two ciliopathies.

Functional significance of SPINK1 promoter variants in chronic pancreatitis.

Chronic pancreatitis is a progressive inflammatory disorder of the pancreas, which often develops as a result of genetic predisposition. Some of the most frequently identified risk factors affect the serine protease inhibitor Kazal type 1 (SPINK1) gene, which encodes a trypsin inhibitor responsible for protecting the pancreas from premature trypsinogen activation. Recent genetic and functional studies indicated that promoter variants in the SPINK1 gene might contribute to disease risk in carriers. Here, we investigated the functional effects of 17 SPINK1 promoter variants using luciferase reporter gene expression assay in four different cell lines, including three pancreatic acinar cell lines (rat AR42J with or without dexamethasone-induced differentiation and mouse 266-6) and human embryonic kidney 293T cells. We found that most variants caused relatively small changes in promoter activity. Surprisingly, however, we observed significant variations in the effects of the promoter variants in the different cell lines. Only four variants exhibited consistently reduced promoter activity in all acinar cell lines, confirming previous reports that variants c.-108G>T, c.-142T>C, and c.-147A>G are risk factors for chronic pancreatitis and identifying c.-52G>T as a novel risk variant. In contrast, variant c.-215G>A, which is linked with the disease-associated splice-site mutation c.194 + 2T>C, caused increased promoter activity, which may mitigate the overall effect of the pathogenic haplotype. Our study lends further support to the notion that sequence evaluation of the SPINK1 promoter region in patients with chronic pancreatitis is justified as part of the etiological investigation.

In Vivo Imaging of Human MDR1 Transcription in the Brain and Spine of MDR1-Luciferase Reporter Mice.

P-glycoprotein (Pgp) [the product of the MDR1 (ABCB1) gene] at the blood-brain barrier (BBB) limits central nervous system (CNS) entry of many prescribed drugs, contributing to the poor success rate of CNS drug candidates. Modulating Pgp expression could improve drug delivery into the brain; however, assays to predict regulation of human BBB Pgp are lacking. We developed a transgenic mouse model to monitor human MDR1 transcription in the brain and spinal cord in vivo. A reporter construct consisting of ∼10 kb of the human MDR1 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line (MDR1-luc). Fluorescence in situ hybridization localized the MDR1-luciferase transgene on chromosome 3. Reporter gene expression was monitored with an in vivo imaging system following D-luciferin injection. Basal expression was detectable in the brain, and treatment with activators of the constitutive androstane, pregnane X, and glucocorticoid receptors induced brain and spinal MDR1-luc transcription. Since D-luciferin is a substrate of ABCG2, the feasibility of improving D-luciferin brain accumulation (and luciferase signal) was tested by coadministering the dual ABCB1/ABCG2 inhibitor elacridar. The brain and spine MDR1-luc signal intensity was increased by elacridar treatment, suggesting enhanced D-luciferin brain bioavailability. There was regional heterogeneity in MDR1 transcription (cortex > cerebellum) that coincided with higher mouse Pgp protein expression. We confirmed luciferase expression in brain vessel endothelial cells by ex vivo analysis of tissue luciferase protein expression. We conclude that the MDR1-luc mouse provides a unique in vivo system to visualize MDR1 CNS expression and regulation.

Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways.

PURPOSE: Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing. METHODS: We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways. RESULTS: PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms. CONCLUSIONS: The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network.

Cation-dependent folding of 3' cap-independent translation elements facilitates interaction of a 17-nucleotide conserved sequence with eIF4G.

The 3'-untranslated regions of many plant viral RNAs contain cap-independent translation elements (CITEs) that drive translation initiation at the 5'-end of the mRNA. The barley yellow dwarf virus-like CITE (BTE) stimulates translation by binding the eIF4G subunit of translation initiation factor eIF4F with high affinity. To understand this interaction, we characterized the dynamic structural properties of the BTE, mapped the eIF4G-binding sites on the BTE and identified a region of eIF4G that is crucial for BTE binding. BTE folding involves cooperative uptake of magnesium ions and is driven primarily by charge neutralization. Footprinting experiments revealed that functional eIF4G fragments protect the highly conserved stem-loop I and a downstream bulge. The BTE forms a functional structure in the absence of protein, and the loop that base pairs the 5'-untranslated region (5'-UTR) remains solvent-accessible at high eIF4G concentrations. The region in eIF4G between the eIF4E-binding site and the MIF4G region is required for BTE binding and translation. The data support the model in which the eIF4F complex binds directly to the BTE which base pairs simultaneously to the 5'-UTR, allowing eIF4F to recruit the 40S ribosomal subunit to the 5'-end.

Area-specific cell stimulation via surface-mediated gene transfer using apatite-based composite layers.

Surface-mediated gene transfer systems using biocompatible calcium phosphate (CaP)-based composite layers have attracted attention as a tool for controlling cell behaviors. In the present study we aimed to demonstrate the potential of CaP-based composite layers to mediate area-specific dual gene transfer and to stimulate cells on an area-by-area basis in the same well. For this purpose we prepared two pairs of DNA-fibronectin-apatite composite (DF-Ap) layers using a pair of reporter genes and pair of differentiation factor genes. The results of the area-specific dual gene transfer successfully demonstrated that the cells cultured on a pair of DF-Ap layers that were adjacently placed in the same well showed specific gene expression patterns depending on the gene that was immobilized in the underlying layer. Moreover, preliminary real-time PCR results indicated that multipotential C3H10T1/2 cells may have a potential to change into different types of cells depending on the differentiation factor gene that was immobilized in the underlying layer, even in the same well. Because DF-Ap layers have a potential to mediate area-specific cell stimulation on their surfaces, they could be useful in tissue engineering applications.

A Photinus pyralis and Luciola italica chimeric firefly luciferase produces enhanced bioluminescence.

We report the enhanced bioluminescence properties of a chimeric enzyme (PpyLit) that contains the N-domain of recombinant Photinus pyralis luciferase joined to the C-domain of recombinant Luciola italica luciferase. Compared to the P. pyralis enzyme, the novel PpyLit chimera exhibited 1.8-fold enhanced flash-height specific activity, 2.0-fold enhanced integration-based specific activity, 2.9-fold enhanced catalytic efficiency (kcat/Km), and a 1.4-fold greater bioluminescence quantum yield. The results of this study provide an underlying basis of this unusual example of a chimeric enzyme with enhanced catalytic properties that are not simply the sum of the contributions of the two luciferases.

Identification of biologically relevant enhancers in human erythroid cells.

Identification of cell type-specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses, and candidate enhancers were identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the transcriptional start site (TSS) of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or phenotype. Candidate enhancers exhibited moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a set of erythroid-associated, biologically relevant, SNPs from the genome-wide association studies (GWAS) catalogue of NHGRI, National Institutes of Health. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function and provide insights into inherited and acquired hematologic disease.

Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake.

Pancreatic acinar cells (PAC) obtain thiamin from the circulation via a carrier-mediated process that involves thiamin transporters 1 and 2 (THTR-1 and THTR-2; products of SLC19A2 and SLC19A3, respectively). Chronic alcohol exposure of PAC inhibits thiamin uptake, and, on the basis of in vitro studies, this inhibition appears to be transcriptionally mediated. The aim of this study was to confirm the involvement of a transcriptional mechanism in mediating the chronic alcohol effect in in vivo settings and to delineate the molecular mechanisms involved. Using transgenic mice carrying full-length SLC19A2 and SLC19A3 promoters, we found that chronic alcohol feeding led to a significant reduction in the activity of SLC19A2 and SLC19A3 promoters (as well as in thiamin uptake and expression of THTR-1 and -2). Similar findings were seen in 266-6 cells chronically exposed to alcohol in vitro. In the latter studies, the alcohol inhibitory effect was found to be mediated via the minimal SLC19A2 and SLC19A3 promoters and involved the cis-regulatory elements stimulating protein 1 (SP1)/gut-enriched Kruppel-like factor and SP1-GG-box and SP1/GC, respectively. Chronic alcohol exposure of PAC also led to a significant reduction in the expression of the SP1 transcription factor, which upon correction (via expression) led to the prevention of alcohol inhibitory effects on not only the activity of SLC19A2 and SLC19A3 promoters but also on the expression of THTR-1 and -2 mRNA and thiamin uptake. These results demonstrate that the inhibitory effect of chronic alcohol exposure on physiological/molecular parameters of thiamin uptake by PAC is mediated via specific cis-regulatory elements in SLC19A2 and SLC19A3 minimal promoters.

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

Oxygen sensing by the prolyl-4-hydroxylase PHD2 within the nuclear compartment and the influence of compartmentalisation on HIF-1 signalling.

Hypoxia-inducible factors (HIFs) regulate more than 200 genes involved in cellular adaptation to reduced oxygen availability. HIFs are heterodimeric transcription factors that consist of one of three HIF-α subunits and a HIF-ß subunit. Under normoxic conditions the HIF-α subunit is hydroxylated by members of a family of prolyl-4-hydroxylase domain (PHD) proteins, PHD1, PHD2 and PHD3, resulting in recognition by von-Hippel-Lindau protein, ubiquitylation and proteasomal degradation. It has been suggested that PHD2 is the key regulator of HIF-1α stability in vivo. Previous studies on the intracellular distribution of PHD2 have provided evidence for a predominant cytoplasmic localisation but also nuclear activity of PHD2. Here, we investigated functional nuclear transport signals in PHD2 and identified amino acids 196-205 as having a crucial role in nuclear import, whereas amino acids 6-20 are important for nuclear export. Fluorescence resonance energy transfer (FRET) showed that an interaction between PHD2 and HIF-1α occurs in both the nuclear and cytoplasmic compartments. However, a PHD2 mutant that is restricted to the cytoplasm does not interact with HIF-1α and shows less prolyl hydroxylase activity for its target HIF-1α than wild-type PHD2 located in the nucleus. Here, we present a new model by which PHD2-mediated hydroxylation of HIF-1α predominantly occurs in the cell nucleus and is dependent on very dynamic subcellular trafficking of PHD2.