Optogenetic induction of caspase-8 mediated apoptosis by employing Arabidopsis cryptochrome 2.

Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.

Transcriptome analysis of drought-responsive and drought-tolerant mechanisms in maize leaves under drought stress.

Maize is a major crop essential for food and feed, but its production is threatened by various biotic and abiotic stresses. Drought is one of the most common abiotic stresses, causing severe crop yield reduction. Although several studies have been devoted to selecting drought-tolerant maize lines and detecting the drought-responsive mechanism of maize, the transcriptomic differences between drought-tolerant and drought-susceptible maize lines are still largely unknown. In our study, RNA-seq was performed on leaves of the drought-tolerant line W9706 and the drought-susceptible line B73 after drought treatment. We identified 3147 differentially expressed genes (DEGs) between these two lines. The upregulated DEGs in W9706 were enriched in specific processes, including ABA signaling, wax biosynthesis, CHO metabolism, signal transduction and brassinosteroid biosynthesis-related processes, while the downregulated DEGs were enriched in specific processes, such as stomatal movement. Altogether, transcriptomic analysis suggests that the different drought resistances were correlated with the differential expression of genes, while the drought tolerance of W9706 is due to the more rapid response to stimulus, higher water retention capacity and stable cellular environment under water deficit conditions.

Mutation in Mg-Protoporphyrin IX Monomethyl Ester (Oxidative) Cyclase Gene ZmCRD1 Causes Chlorophyll-Deficiency in Maize.

Chlorophyll molecules are non-covalently associated with chlorophyll-binding proteins to harvest light and perform charge separation vital for energy conservation during photosynthetic electron transfer in photosynthesis for photosynthetic organisms. The present study characterized a pale-green leaf (pgl) maize mutant controlled by a single recessive gene causing chlorophyll reduction throughout the whole life cycle. Through positional mapping and complementation allelic test, Zm00001d008230 (ZmCRD1) with two missense mutations (p.A44T and p.T326M) was identified as the causal gene encoding magnesium-protoporphyrin IX monomethyl ester cyclase (MgPEC). Phylogenetic analysis of ZmCRD1 within and among species revealed that the p.T326M mutation was more likely to be causal. Subcellular localization showed that ZmCRD1 was targeted to chloroplasts. The pgl mutant showed a malformed chloroplast morphology and reduced number of starch grains in bundle sheath cells. The ZmCRD1 gene was mainly expressed in WT and mutant leaves, but the expression was reduced in the mutant. Most of the genes involved in chlorophyll biosynthesis, chlorophyll degradation, chloroplast development and photosynthesis were down-regulated in pgl. The photosynthetic capacity was limited along with developmental retardation and production reduction in pgl. These results confirmed the crucial role of ZmCRD1 in chlorophyll biosynthesis, chloroplast development and photosynthesis in maize.

The role of mouse and human peroxisome proliferator-activated receptor-α in modulating the hepatic effects of perfluorooctane sulfonate in mice.

Perfluorooctane sulfonate (PFOS) is a stable environmental contaminant that can activate peroxisome proliferator-activated receptor alpha (PPARα). In the present work, the specific role of mouse and human PPARα in mediating the hepatic effects of PFOS was examined in short-term studies using wild type, Ppara-null and PPARA-humanized mice. Mice fed 0.006 % PFOS for seven days (∼10 mg/kg/day), or 0.003 % PFOS for twenty-eight days (∼5 mg/kg/day), exhibited higher liver and serum PFOS concentrations compared to controls. Relative liver weights were also higher following exposure to dietary PFOS in all three genotypes as compared vehicle fed control groups. Histopathological examination of liver sections from mice treated for twenty-eight days with 0.003 % PFOS revealed a phenotype consistent with peroxisome proliferation, in wild-type and PPARA-humanized mice that was not observed in Ppara-null mice. With both exposures, expression of the PPARα target genes, Acox1, Cyp4a10, was significantly increased in wild type mice but not in Ppara-null or PPARA-humanized mice. By contrast, expression of the constitutive androstane receptor (CAR) target gene, Cyp2b10, and the pregnane X receptor (PXR) target gene, Cyp3a11, were higher in response to PFOS administration in all three genotypes compared to controls for both exposure periods. These results indicate that mouse PPARα can be activated in the liver by PFOS causing increased expression of Acox1, Cyp4a10 and histopathological changes in the liver. While histopathological analyses indicated the presence of mouse PPARα-dependent hepatic peroxisome proliferation in wild-type (a response associated with activation of PPARα) and a similar phenotype in PPARA-humanized mice, the lack of increased Acox1 and Cyp4a10 mRNA by PFOS in PPARA-humanized mice indicates that the human PPARα was not as responsive to PFOS as mouse PPARα with this dose regimen. Moreover, results indicate that hepatomegaly caused by PFOS does not require mouse or human PPARα and could be due to effects induced by activation of CAR and/or PXR.

Roles of a Cryptochrome in Carbon Fixation and Sucrose Metabolism in the Liverwort Marchantia polymorpha.

In vascular plants, cryptochromes acting as blue-light photoreceptors have various functions to adapt plants to the fluctuating light conditions on land, while the roles of cryptochromes in bryophytes have been rarely reported. In this study, we investigated functions of a single-copy ortholog of cryptochrome (MpCRY) in the liverwort Marchantia polymorpha. Knock-out of MpCRY showed that a large number of the mutant plants exhibited asymmetric growth of thalli under blue light. Transcriptome analyses indicated that MpCRY is mainly involved in photosynthesis and sugar metabolism. Further physiological analysis showed that Mpcry mutant exhibited a reduction in CO2 uptake and sucrose metabolism. In addition, exogenous application of sucrose or glucose partially restored the symmetrical growth of the Mpcry mutant thalli. Together, these results suggest that MpCRY is involved in the symmetrical growth of thallus and the regulation of carbon fixation and sucrose metabolism in M. polymorpha.

E183K Mutation in Chalcone Synthase C2 Causes Protein Aggregation and Maize Colorless.

Flavonoids give plants their rich colors and play roles in a number of physiological processes. In this study, we identified a novel colorless maize mutant showing reduced pigmentation throughout the whole life cycle by EMS mutagenesis. E183K mutation in maize chalcone synthase C2 (ZmC2) was mapped using MutMap strategy as the causal for colorless, which was further validated by transformation in Arabidopsis. We evaluated transcriptomic and metabolic changes in maize first sheaths caused by the mutation. The downstream biosynthesis was blocked while very few genes changed their expression pattern. ZmC2-E183 site is highly conserved in chalcone synthase among Plantae kingdom and within species' different varieties. Through prokaryotic expression, transient expression in maize leaf protoplasts and stable expression in Arabidopsis, we observed that E183K and other mutations on E183 could cause almost complete protein aggregation of chalcone synthase. Our findings will benefit the characterization of flavonoid biosynthesis and contribute to the body of knowledge on protein aggregation in plants.

Regulation of the activity of maize glutamate dehydrogenase by ammonium and potassium.

Glutamate dehydrogenase (GDH) is an important enzyme in ammonium metabolism, the activity of which is regulated by multiple factors. In this study, we investigate the effects of ammonium and potassium on the activity of maize GDH. Our results show that both ammonium and potassium play multiple roles in regulating the activity of maize GDH, with the specific roles depending on the concentration of potassium. Together with the structural information of GDH, we propose models for the substrate inhibition of ammonium, and the elimination of substrate inhibition by potassium. These models are supported by the analysis of statistic thermodynamics. We also analyze the binding sites of ammonium and potassium on maize GDH, and the conformational changes of maize GDH. The findings provide insight into the regulation of maize GDH activity by ammonium and potassium and reveal the importance of the dose and ratio of nitrogen and potassium in crop cultivation.

Functions of COP1/SPA E3 Ubiquitin Ligase Mediated by MpCRY in the Liverwort Marchantia polymorpha under Blue Light.

COP1/SPA1 complex in Arabidopsis inhibits photomorphogenesis through the ubiquitination of multiple photo-responsive transcription factors in darkness, but such inhibiting function of COP1/SPA1 complex would be suppressed by cryptochromes in blue light. Extensive studies have been conducted on these mechanisms in Arabidopsis whereas little attention has been focused on whether another branch of land plants bryophyte utilizes this blue-light regulatory pathway. To study this problem, we conducted a study in the liverwort Marchantia polymorpha and obtained a MpSPA knock-out mutant, in which Mpspa exhibits the phenotype of an increased percentage of individuals with asymmetrical thallus growth, similar to MpCRY knock-out mutant. We also verified interactions of MpSPA with MpCRY (in a blue light-independent way) and with MpCOP1. Concomitantly, both MpSPA and MpCOP1 could interact with MpHY5, and MpSPA can promote MpCOP1 to ubiquitinate MpHY5 but MpCRY does not regulate the ubiquitination of MpHY5 by MpCOP1/MpSPA complex. These data suggest that COP1/SPA ubiquitinating HY5 is conserved in Marchantia polymorpha, but dissimilar to CRY in Arabidopsis, MpCRY is not an inhibitor of this process under blue light.

Transcriptomic analysis reveals somatic embryogenesis-associated signaling pathways and gene expression regulation in maize (Zea mays L.).

KEY MESSAGE: Transcriptome analysis of maize embryogenic callus and somatic embryos reveals associated genes reprogramming, hormone signaling pathways and transcriptional regulation involved in somatic embryogenesis in maize. Somatic embryos are widely utilized in propagation and genetic engineering of crop plants. In our laboratory, an elite maize inbred line Y423 that could generate intact somatic embryos was obtained and applied to genetic transformation. To enhance our understanding of regulatory mechanisms during maize somatic embryogenesis, we used RNA-based sequencing (RNA-seq) to characterize the transcriptome of immature embryo (IE), embryogenic callus (EC) and somatic embryo (SE) from maize inbred line Y423. The number of differentially expressed genes (DEGs) in three pairwise comparisons (IE-vs-EC, IE-vs-SE and EC-vs-SE) was 5767, 7084 and 1065, respectively. The expression patterns of DEGs were separated into eight major clusters. Somatic embryogenesis associated genes were mainly grouped into cluster A or B with an expression trend toward up-regulation during dedifferentiation. GO annotation and KEGG pathway analysis revealed that DEGs were implicated in plant hormone signal transduction, stress response and metabolic process. Among the differentially expressed transcription factors, the most frequently represented families were associated with the common stress response or related to cell differentiation, embryogenic patterning and embryonic maturation processes. Genes include hormone response/transduction and stress response, as well as several transcription factors were discussed in this study, which may be potential candidates for further analyses regarding their roles in somatic embryogenesis. Furthermore, the temporal expression patterns of candidate genes were analyzed to reveal their roles in somatic embryogenesis. This transcriptomic data provide insights into future functional studies, which will facilitate further dissections of the molecular mechanisms that control maize somatic embryogenesis.

iTRAQ-Based Quantitative Proteomic Analysis of Embryogenic and Non-embryogenic Calli Derived from a Maize (Zea mays L.) Inbred Line Y423.

Somatic embryos (SE) have potential to rapidly form a whole plant. Generally, SE is thought to be derived from embryogenic calli (EC). However, in maize, not only embryogenic calli (EC, can generate SE) but also nonembryogenic calli (NEC, can't generate SE) can be induced from immature embryos. In order to understand the differences between EC and NEC and the mechanism of EC, which can easily form SE in maize, differential abundance protein species (DAPS) of EC and NEC from the maize inbred line Y423 were identified by using the isobaric tags for relative and absolute quantification (iTRAQ) proteomic technology. We identified 632 DAPS in EC compared with NEC. The results of bioinformatics analysis showed that EC development might be related to accumulation of pyruvate caused by the DAPS detected in some pathways, such as starch and sucrose metabolism, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, fatty acid metabolism and phenylpropanoid biosynthesis. Based on the differentially accumulated proteins in EC and NEC, a series of DAPS related with pyruvate biosynthesis and suppression of acetyl-CoA might be responsible for the differences between EC and NEC cells. Furthermore, we speculate that the decreased abundance of enzymes/proteins involved in phenylpropanoid biosynthesis pathway in the EC cells results in reducing of lignin substances, which might affect the maize callus morphology.

iTRAQ-based quantitative proteomic analysis reveals new metabolic pathways responding to chilling stress in maize seedlings.

UNLABELLED: To date, transcriptome profile analysis of maize seedlings in response to cold stress have been well documented; however, changes in protein species abundance of maize seedlings in response to cold stress are still unknown. Herein, leaves from the maize inbred line W9816 (a cold-resistance genotype) were harvested at three-leaf stage, and were used to identify the differential abundance protein species (DAPS) between chilling stress (4°C) and control conditions (25°C). iTRAQ-based quantitative proteomic were used in this study. As a result, 173 DAPS were identified after chilling stress. Bioinformatic analysis showed that 159 DAPS were annotated in 38 Gene Ontology functional groups, 108 DAPS were classified into 20 clusters of orthologous groups of protein categories, 99 DAPS were enrichment in KEGG pathways. Antioxidants assays validated that the iTRAQ results were reliable. Based on functional analysis, we concluded that the adaptive response of maize seedlings to chilling stress might be related to alleviation of photodamage caused by the over-energized state of thylakoid membrane, more energy produced through glycolysis, increased abundance of stress-responsive protein species, and improvement in the overall ability to scavenge ROS. Posttranscriptional regulation and posttranslational modifications also play important roles for maize to adapt to chilling stress. BIOLOGICAL SIGNIFICANCE: The major challenge for maize breeders is the complexity of the response to chilling stress. Although extensive researches have been focus on maize chilling stress using segregating populations, epigenetics, transcriptomics, molecular biology, however, the molecular mechanism of chilling stress in maize remains to be further elucidated. In the present paper, a differential proteomic analysis was performed and the results revealed the adaptive response of maize seedlings to chilling stress might be related to alleviation of photodamage caused by the over-energized state of thylakoid membrane, more energy produced through glycolysis, increased abundance of stress-responsive protein species, improvement in the overall ability to scavenge ROS, including detoxifying enzymes and antioxidants. Posttranscriptional regulation and posttranslational modifications also play important roles for maize to adapt to chilling stress. This approach identified new protein species involved in posttranslational modifications, signal transduction, lipid metabolism, inorganic ion transport and metabolism and other biological processes that were not previously known to be associated with chilling stress response.

Isolation and functional characterization of a cold responsive phosphatidylinositol transfer-associated protein, ZmSEC14p, from maize (Zea may L.).

KEY MESSAGE: A Sec14-like protein, ZmSEC14p , from maize was structurally analyzed and functionally tested. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.

Bioinformatic analysis of microRNA networks following the activation of the constitutive androstane receptor (CAR) in mouse liver.

The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily that functions as a xenosensor, serving to regulate xenobiotic detoxification, lipid homeostasis and energy metabolism. CAR activation is also a key contributor to the development of chemical hepatocarcinogenesis in mice. The underlying pathways affected by CAR in these processes are complex and not fully elucidated. MicroRNAs (miRNAs) have emerged as critical modulators of gene expression and appear to impact many cellular pathways, including those involved in chemical detoxification and liver tumor development. In this study, we used deep sequencing approaches with an Illumina HiSeq platform to differentially profile microRNA expression patterns in livers from wild type C57BL/6J mice following CAR activation with the mouse CAR-specific ligand activator, 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzene (TCPOBOP). Bioinformatic analyses and pathway evaluations were performed leading to the identification of 51 miRNAs whose expression levels were significantly altered by TCPOBOP treatment, including mmu-miR-802-5p and miR-485-3p. Ingenuity Pathway Analysis of the differentially expressed microRNAs revealed altered effector pathways, including those involved in liver cell growth and proliferation. A functional network among CAR targeted genes and the affected microRNAs was constructed to illustrate how CAR modulation of microRNA expression may potentially mediate its biological role in mouse hepatocyte proliferation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423.

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize.

Δ(9)-THC modulation of fatty acid 2-hydroxylase (FA2H) gene expression: possible involvement of induced levels of PPARα in MDA-MB-231 breast cancer cells.

We recently reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2-hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ(9)-THC-treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (ß and γ). Δ(9)-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA-MB-231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ(9)-THC-mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ(9)-THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ(9)-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ(9)-THC-induced up-regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ(9)-THC up-regulation of FA2H in MDA-MB-231 cells.

Intronic DNA elements regulate Nrf2 chemical responsiveness of the human microsomal epoxide hydrolase gene (EPHX1) through a far upstream alternative promoter.

In humans, microsomal epoxide hydrolase (mEH) contributes important biological functions that underlie both detoxification and bioactivation fates arising from exposures to foreign chemicals. Previously, we discovered that human mEH gene transcription is initiated from alternative promoters. The respective transcripts are programmed with tissue specificity and the upstream E1b promoter contributes predominantly to mEH expression. The results presented demonstrate that exposures to the Nrf2 activators, sulforaphane (SFN) and tert-butylhydroquinone (tBHQ), markedly activate E1b transcription in human lung and liver cells. Genomic analyses identified two major DNase I hypersensitive regions (HS-1 and HS-2) within the ~15 kb intervening sequence separating E1b from the downstream E1 promoter. In BEAS-2B cells, the Nrf2 effectors, SFN and tBHQ, selectively activated the more distal HS-2 through an antioxidant response element (ARE). An activator protein 1/12-O-tetradecanoylphorbol-13-acetate interaction was further identified within the HS-2 enhancer that functioned to additionally contribute to ARE-mediated induction responsiveness of the E1b promoter. The results demonstrate that ARE modulation, integrated with additional transcriptional complexes, regulates the tissue-specific expression of mEH and that these processes likely coordinate both the protective and bioactivation functions contributed by mEH activities in human tissues.

Sp1 and Sp3 transcription factors regulate the basal expression of human microsomal epoxide hydrolase (EPHX1) through interaction with the E1b far upstream promoter.

Microsomal epoxide hydrolase (mEH, EPHX1) is a critical biotransformation enzyme, catalyzing the metabolism of many xenobiotics. Human mEH is transcribed using alternative promoters. The upstream E1 promoter is active in liver while the far upstream E1b promoter drives the expression of mEH in all tissues, including liver. Although several liver-specific transcription factors have been identified in the regulation of E1 transcription, little is known regarding the mechanisms of E1b transcriptional regulation. Genome-wide mapping of DNase I hypersensitive sites revealed an open chromatin region between nucleotide -300 upstream and +400 downstream of E1b. This area coincides with a previously described promoter region responsible for maintaining high basal promoter activity. In silico analysis of this location revealed several Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs suppressed the transactivation activity of the E1b proximal promoter, indicating their importance as contributors to E1b promoter regulation. Further, E1b promoter activities were increased significantly following Sp1 and Sp3 overexpression, while Mithramycin A, a selective Sp1 inhibitor, reduced the promoter activities. EMSA studies demonstrated that Sp1 bound to two putative Sp1/Sp3 binding sites. ChIP analysis confirmed that both endogenous Sp1 and Sp3 were bound to the proximal promoter region of E1b. Knockdown of Sp1 expression using siRNA did not alter the endogenous E1b transcriptional level, while knockdown of Sp3 greatly decreased E1b expression in different human cell lines. Taken together, these results support the concept that Sp1 and Sp3 are functionally involved as transcriptional integrators regulating the basal expression of the derived mEH E1b variant transcript.

Induction of the fatty acid 2-hydroxylase (FA2H) gene by Δ(9)-tetrahydrocannabinol in human breast cancer cells.

To investigate gene(s) being regulated by ∆(9)-tetrahydrocannabinol (∆(9)-THC), we performed DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are poorly differentiated breast cancer cells, treated with ∆(9)-THC for 48 hr at an IC50 concentration of approximately 25 µM. Among the highly up-regulated genes (> 10-fold) observed, fatty acid 2-hydroxylase (FA2H) was significantly induced (17.8-fold). Although the physiological role of FA2H has not yet been fully understood, FA2H has been shown to modulate cell differentiation. The results of Oil Red O staining after ∆(9)-THC exposure showed the distribution of lipid droplets (a sign of the differentiated phenotype) in cells. Taken together, the results obtained here indicate that FA2H is a novel ∆(9)-THC-regulated gene, and that ∆(9)-THC induces differentiation signal(s) in poorly differentiated MDA-MB-231 cells.

Cucumber mosaic virus movement protein severs actin filaments to increase the plasmodesmal size exclusion limit in tobacco.

Plant viral movement proteins (MPs) enable viruses to pass through cell walls by increasing the size exclusion limit (SEL) of plasmodesmata (PD). Here, we report that the ability of Cucumber mosaic virus (CMV) MP to increase the SEL of the PD could be inhibited by treatment with the actin filament (F-actin)-stabilizing agent phalloidin but not by treatment with the F-actin-destabilizing agent latrunculin A. In vitro studies showed that CMV MP bound globular and F-actin, inhibited actin polymerization, severed F-actin, and participated in plus end capping of F-actin. Analyses of two CMV MP mutants, one with and one without F-actin severing activities, demonstrated that the F-actin severing ability was required to increase the PD SEL. Furthermore, the Tobacco mosaic virus MP also exhibited F-actin severing activity, and its ability to increase the PD SEL was inhibited by treatment with phalloidin. Our data provide evidence to support the hypothesis that F-actin severing is required for MP-induced increase in the SEL of PD. This may have broad implications in the study of the mechanisms of actin dynamics that regulate cell-to-cell transport of viral and endogenous proteins.

Retinoid X receptor-alpha-dependent transactivation by a naturally occurring structural variant of human constitutive androstane receptor (NR1I3).

The constitutive androstane receptor (CAR) mediates the hepatic induction of various xenobiotic metabolizing enzymes and transporters after specific chemical exposures. Recent reports have established the existence of several human CAR mRNA splice variants, including a prominently expressed form termed CAR3, a receptor that possesses a 5 amino acid insertion within its ligand binding domain. In this study, we demonstrate that, in contrast to the constitutively active reference form of the receptor, CAR3 is ligand-activated, transactivating an optimized DR-4 x 3 reporter in response to the human CAR ligand 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3, 4-dichlorobenzyl)oxime (CITCO). The transactivation response requires the DNA binding domain and AF-2 motif of CAR3 and is markedly enhanced by retinoid X receptor-alpha (RXR) cotransfection. The stimulatory effects of RXR involve a unique mechanism, because they were completely dependent on the RXR AF-2 function but independent of both the RXR A/B domain and its C domain/heterodimerization region. Mammalian two-hybrid results demonstrated that RXR enhanced CITCO-dependent interaction of CAR3 with the receptor interaction domain of SRC-1, indicating that RXR augments CAR3 activity by facilitating coactivator recruitment. It is noteworthy that clotrimazole also functions as a ligand activator of CAR3, in contrast to the inverse agonist activity exhibited by this agent on the reference form of the receptor. Furthermore, results of transfection assays reveal that CAR3 is capable of transactivating the natural CYP2B6 and CYP3A4 gene enhancers, exhibiting both ligand- and RXR-dependence. These results demonstrate that CAR3, unlike CAR1, is a ligand-activated receptor and that CAR3 may regulate gene expression in vivo in a manner distinct from the reference form of the receptor.