EMSA Analysis Research Articles

Homology-based characterization of the cis-regulatory elements modulate flavone induction of CYP321A1 in Helicoverpa armigera

Xenobiotic response element (XRE) to flavone was the cis- regulatory elements that mediates the induction of the allelochemical-metabolizing CYP321A1 gene from Helicoverpa zea. However, it was unknown whether the XRE-Fla element existed in other species. Recently we have identified and cloned the CYP321A1 gene with promoter region in a related species, Helicoverpa armigera. Sequence similarity of two orthologous CYP321A1 genes was 97.27%, but the promoter sequence similarity was only 56.32%. Sequence alignment showed the XRE-Fla like element owns three mutations in H. armigera compared with H. zea. Progressive 5′ deletions and internal mutation indicated that H. armigera XRE-Fla was the essential element of CYP321A1 gene in response to flavone. XRE-Fla mutations and EMSA analysis confirmed that the H. armigera XRE-Fla element binding factor was stronger than H. zea. The findings indicate the XRE element mutations mainly contribute to the differences between the flavone-induced expressions of two CYP321A1 genes, which improve the flexibility and adaptability for allelochemical response of H. armigera.

Two Zn2Cys6-type transcription factors respond to aromatic compounds and regulate the expression of laccases in the white-rot fungus Trametes hirsuta.

White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o-toluidine exposure, in Trametes hirsuta AH28-2. Two Zn2Cys6-type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o-toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o-toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs.IMPORTANCEWhite-rot fungi differentially express laccase isoenzymes when exposed to aromatic compounds. Clarification of the molecular mechanisms underlying differential laccase expression is essential to elucidate how white-rot fungi respond to the environment. Our study shows that two Zn2Cys6-type transcription factors form heterodimers, interact with the promoters of laccase genes, and positively regulate laccase transcription in Trametes hirsuta AH28-2. Aromatic monomer addition induces faster heterodimer formation and rate of activity. These findings not only identify two new transcription factors involved in fungal laccase transcription but also deepen our understanding of the mechanisms underlying the response to aromatics exposure in white-rot fungi.

MnoSR removal in Mycobacterium smegmatis triggers broad transcriptional response to 1,3-propanediol and glucose as sole carbon sources.

The two-component signal transduction systems play an essential role in the adaptation of bacteria to changing environmental conditions. One of them is the MnoSR system involved in the regulation of methylotrophic metabolism in M. smegmatis. Mycobacterium smegmatis mutant strains ΔmnoS, ΔmnoR and ΔmnoS/R lacking functional mnoS, mnoR and both genes were generated using a homologous recombination approach. MnoR recombinant protein was purified by affinity column chromatography. The present study employs molecular biology techniques: cloning strategies, global RNA sequencing, qRT-PCR, EMSA, Microscale thermophoresis, and bioinformatics analysis. The ∆mnoS, ∆mnoR, and ∆mnoS/R mutant strains were generated and cultured in the presence of defined carbon sources. Growth curve analysis confirmed that inactivation of the MnoSR impairs the ability of M. smegmatis cells to use alcohols such as 1,3-propanediol and ethanol but improves the bacterial growth on ethylene glycol, xylitol, and glycerol. The total RNA sequencing method was employed to understand the importance of MnoSR in the global responses of mycobacteria to limited carbon access and in carbon-rich conditions. The loss of MnoSR significantly affected carbon utilization in the case of mycobacteria cultured on glucose or 1,3-propanediol as sole carbon sources as it influenced the expression of multiple metabolic pathways. The numerous transcriptional changes could not be linked to the presence of evident MnoR DNA-binding sites within the promotor regions for the genes outside of the mno operon. This was confirmed by EMSA and microscale thermophoresis with mutated MnoR binding consensus region. Our comprehensive analysis highlights the system's vital role in metabolic adaptability, providing insights into its potential impact on the environmental survival of mycobacteria.

The chromatin architectural regulator SND1 mediates metastasis in triple-negative breast cancer by promoting CDH1 gene methylation

BackgroundSND1 participates in tumorigenesis, tumour invasion and metastasis in different cancers. Previous studies have shown that SND1 can promote the invasion and migration of breast cancer cells. Triple-negative breast cancer (TNBC) is a specific breast cancer subtype with high metastatic potential and poor prognosis. However, the specific roles and mechanisms of SND1 in TNBC metastasis remain unaddressed.MethodsImmunostaining was used to detect the SND1 expression in tissue samples of 58 TNBC and 10 glioblastomas (GBM) as positive control. The correlation between SND1 expression and patient prognosis was assessed using the Kaplan–Meier estimator. The gene expression was evaluated by qRT-PCR, Western blot and immunofluorescence analyses. Gene Ontology analysis, ChIP, a dual-luciferase reporter assay, EMSA, and 3C analysis were applied to identify SND1-activated target genes. Bisulfite sequencing PCR and MeDIP were used to detect DNA methylation. We also used wound healing, Transwell and orthotopic implantation assays to investigate the function of SND1 in TNBC cell migration and invasion.ResultsThe data of immunohistochemistry manifested that SND1 is the overexpression in metastasized TNBC and an independent factor for TNBC prognosis. SND1 knockdown inhibited the migration and invasion of TNBC cells. We found that SND1 promotes the metastatic phenotype of TNBC cells by epigenetically altering chromatin conformational interactions, which in turn activates DNMT3A transcription. Then, DNMT3A attenuates CCND1 expression by inducing CCND1 gene methylation, leading to TNBC metastasis.ConclusionSND1 can promote the invasion and migration of TNBC cells by promoting DNMT3A expression and suppressing CDH1 activity. SND1 is a potential biomarker and a promising therapeutic target for TNBC.

Mechanisms of Regulation of the CHRDL1 Gene by the TWIST2 and ADD1/SREBP1c Transcription Factors.

Putative TWIST binding sites were found in the upstream region of the CHRDL1 gene and examined by electrophoretic mobility shift (EMSA) and reporter gene assays. EMSAs showed specific binding of TWIST1 and TWIST2 homodimers, as well as heterodimers with E12, to the more distal E-boxes. An adjoining E-box was bound by ADD1/SREBP1c. EMSA analysis suggested that TWIST2 and ADD1/SREBP1c could compete for binding. Luciferase (luc) reporter assays revealed that the CHRDL1 gene upstream region drives its expression and ADD1/SREBP1c increased it 2.6 times over basal levels. TWIST2, but not the TWIST2-Q119X mutant, blocked activation by ADD1/SREBP1c, but overexpression of TWIST2-Q119X increased luc gene expression. In addition, EMSA competition assays showed that TWIST2, but not TWIST1, competes with ADD1/SREBP1c for DNA binding to the same site. Formation of an inactive complex between the TWIST2 Q119X and Q65X mutant proteins and ADD1/SREBP1c may prevent repressor binding and allow the binding of other regulators to activate CHRDL1 gene expression.

Functional Mutations in the microRNA-155 Promoter Modulate its Transcription Efficiency and Expression.

Limited research has been conducted on porcine miR-155 promoters, and previous study from our group have identified two haplotypes (TT and CC) in different pig breeds, each associated with five fully linked mutation sites within or near the miR-155 gene (Li et al. Dev Comp Immunol 39(1):110-116, 2013). In this study, the promoter region of porcine miR-155 was screened, and two important transcription factors, Foxp3 and RelA, were identified. The binding ability of Foxp3 protein was found to be affected by the first mutation site (A/C) using EMSA analysis. In vitro experiments revealed that the expression level of miR-155 was significantly higher in the C haplotype compared to the T haplotype. Additionally, northern blotting assays indicated that both the first mutation site (A/C) and the fourth mutation site (G/T) had a significant impact on miR-155 expression levels. These findings provide further insights into the transcriptional regulation of porcine miR-155 and identify crucial mutation sites that influence miR-155 expression. This knowledge can serve as a basis for identifying potential molecular markers associated with disease resistance in swine.

Transcriptional regulatory mechanism of a variant transcription factor CsWRKY23 in citrus fruit resistance to Penicillium digitatum

As the most productive fruit in the world, there is a lack of research on disease resistance mechanisms in citrus. WRKY transcription factors are mediators of disease resistance in plants. In this research, CsWRKY23, which could respond to exogenous salicylic acid (SA) was identified. As a variant WRKY, the transient overexpression of CsWRKY23 in citrus peel enhanced the resistance to Penicillium digitatum. The potential targets of CsWRKY23 were identified by using DNA affinity purification sequencing (DAP-seq). In combination with RNA sequencing (RNA-seq) data, 48 direct target genes of CsWRKY23 were identified. In addition, five genes for EMSA and dual-luciferase report system analysis were selected and the results showed that CsWRKY23 activate CsAAE12, CsRbohD, CsSARD1, CsWRKY22 and CsIQM6 expressions. Further analysis revealed that CsWRKY23 contributed to SA synthesis pathway, reactive oxygen species (ROS) accumulation, and cell wall strengthening. This study presented a possible regulatory network for disease resistance involving the CsWRKY23.

A self-regulated transcription factor CsWRKY33 enhances resistance of citrus fruit to Penicillium digitatum

Green mold caused by Penicillium digitatum is a major postharvest disease in citrus fruit, but the molecular mechanism related to the induced disease resistance in citrus is still unclear. This study investigated the regulatory function of CsWRKY33 on citrus disease resistance. The results showed that CsWRKY33 was located in the nucleus, and the transient overexpression of CsWRKY33 significantly enhanced the host resistance against green mold. The DNA affinity purification sequencing and transcriptome sequencing analysis identified 485 direct target genes of CsWRKY33. KEGG pathway analysis revealed that the 250 up-regulated genes were mainly involved in the disease resistance response. And the 235 down regulated genes were mostly associated with plant growth and development. 9 up-regulated genes were selected for EMSA and DLR analysis, including CsRbohD, CsPODN1, CsPOD24, CsPOD, CsPAL, CsWRKY22, CsGSTU7, and CsHSP26A, and found CsWRKY33 activated the expression of these genes. Besides, PAL, POD, GST activities, H2O2 content and lignin content were increased when CsWRKY33 was transiently overexpressed. The results also found that CsWRKY33 could bind to its own promoter's W-box directly for positive regulation. In conclusion, CsWRKY33 plays an important regulatory role in citrus disease resistance, and the key mechanisms is the activation of disease resistance pathways such as phenylalanine metabolism and secondary metabolism.

S6K1 acts through FOXO to regulate juvenile hormone biosynthesis in the red flour beetle, Tribolium castaneum

As the downstream effector of the target of rapamycin complex 1 (TORC1) signaling pathway, the ribosomal protein S6 kinase (S6K) is an important regulator of insect reproduction, however, the underlying mechanism remains obscure. In this study, a S6K gene, named TcS6K1, was isolated from the red flour beetle, Tribolium castaneum. Analysis of temporal and spatial expression patterns revealed that TcS6K1 is expressed at the highest level in the one-day-old first instar larvae and head of 7-day-old females, respectively. RNAi-mediated knockdown of TcS6K1 in either female or male adults decreased the number of eggs laid, with a concomitant reduction of mRNA levelsof vitellogenin genes, TcVg1 and TcVg2, two male accessory gland secretory proteins, as well as the juvenile hormone (JH) biosynthesis-related gene, farnesol dehydrogenase (TcFDH). While the mRNA and protein levels of the transcription factor forkhead box O (TcFOXO) were not affected, suppression of TcS6K1 expression promoted TcFOXO nuclear translocation to exert its transcriptional action. Further RNAi and EMSA analysis revealed that TcFOXO negatively regulated the expression of TcFDH. These results indicate that S6K might regulate beetles’ reproduction through FOXO/JH signaling pathway.

Characterization and functional analysis of the MADS-box EgAGL9 transcription factor from the mesocarp of oil palm (Elaeis guineensis Jacq.)

Oil palm (Elaeis guineensis Jacq.) is one of the most important oil crops in the world, and compared to all oil crops, it has the highest productive efficiency. In the present study, a MADS-box transcription factor of the AGAMOUS class, named EgAGL9, was identified by expression profile analysis in the different developmental stages of oil palm mesocarp. Real-time quantitative PCR results confirmed that the expression of EgAGL9 increased rapidly during the last stages of oil palm mesocarp development. Then, three downstream genes, including EgSAD (Stearoyl-ACP desaturase), EgTSA (Tryptophan synthase) and EgSDH (Succinate dehydrogenase), were screened by ChIP-Seq and data analysis. EMSA analysis verified that EgAGL9 interacted with the promoter regions of EgSAD, EgTSA and EgSDH. Moreover, the expression levels of EgSAD, EgTSA and EgSDH were downregulated in EgAGL9-overexpressing protoplasts and calli of oil palm. Compared to WT, the total lipid content and ratio of unsaturated fatty acids in transgenic calli (including oleic acid, linoleic acid and linolenic acid) were significantly decreased. Together, these results revealed that these three EgAGL9-regulated genes are involved in regulatory pathways in the oil palm mesocarp. Compared with previous studies, the present study provides a new research strategy for understanding of the molecular regulatory pathways of lipid metabolism in mesocarp of oil palm. The obtained results will bring a new perspective for a comprehensive understanding of the regulation of the metabolic accumulation in the oil palm mesocarp.

ELONGATED HYPOCOTYL 5-mediated suppression of melatonin biosynthesis is alleviated by darkness and promotes cotyledon opening.

Melatonin (N-acetyl-5-methoxytryptamine) biosynthesis in plants is induced by darkness and high-intensity light; however, the underlying transcriptional mechanisms and upstream signalling pathways are unknown. We identified a dark-induced and highly expressed melatonin synthetase in Arabidopsis thaliana, AtSNAT6, encoding serotonin N-acetyltransferase. We assessed melatonin content and AtSNAT6 expression in mutants lacking key regulators of light/dark signalling. AtCOP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) and AtHY5 (ELONGATED HYPOCOTYL 5), which control light/dark transition and photomorphogenesis, promoted and suppressed melatonin biosynthesis, respectively. Using EMSA and ChIP-qPCR analysis, we showed that AtHY5 inhibits AtSNAT6 expression directly. An analysis of melatonin content in snat6 hy5 double mutant and AtHY5+AtSNAT6-overexpressing plants confirmed the regulatory function of AtHY5 and AtSNAT6 in melatonin biosynthesis. Exogenous melatonin further inhibited cotyledon opening in hy5 mutant and AtSNAT6-overexpressing seedlings, but partially reversed the promotion of cotyledon opening in AtHY5-overexpressing seedlings and snat6. Additionally, CRISPR/Cas9-mediated mutation of AtSNAT6 increased cotyledon opening in hy5 mutant, and overexpression of AtSNAT6 decreased cotyledon opening in AtHY5-overexpressing seedlings via changing melatonin biosynthesis, confirming that AtHY5 decreased melatonin-mediated inhibition of cotyledon opening. Our data provide new insights into the regulation of melatonin biosynthesis and its function in cotyledon opening.

Gram-Level Production of Balanol through Regulatory Pathway and Medium Optimization in Herb Fungus Tolypocladium ophioglossoides.

As a potential protein kinase C inhibitor, the fungus metabolite balanol has become more attractive in recent decades. In our previous work, we revealed its biosynthetic pathway through overexpression of the cluster-situated regulator gene blnR in Chinese herb fungus Tolypocladium ophioglossoides. However, information on the regulation of blnR is still largely unknown. In this study, we further investigated the regulation of balanol biosynthesis by BlnR through the analysis of affinity binding using EMSA and RNA-seq analysis. The results showed that BlnR positively regulates balanol biosynthesis through binding to all promoters of bln gene members, including its own promoter. Microscopic observation revealed blnR overexpression also affected spore development and hypha growth. Furthermore, RNA-seq analysis suggested that BlnR can regulate other genes outside of the balanol biosynthetic gene cluster, including those involved in conidiospore development. Finally, balanol production was further improved to 2187.39 mg/L using the optimized medium through statistical optimization based on response surface methodology.

MO581: Hyperphosphatemia Induces Lung Fibrosis in OLD Mice

Abstract BACKGROUND AND AIM Hyperphosphatemia is a common condition associated with chronic kidney disease (CKD) and also with aging. Furthermore, lung dysfunction is described in both cases; however, the relationship between high phosphate levels and lung function is still unknown. The aim of this work is to analyze the effect of hyperphosphatemia in the development of lung fibrosis related to aging. METHODS For in vivo studies, C57BL6 male mice of 5 months old (Young) and 24 months old (Old) fed with a standard diet containing 0.6% phosphate were used. Additionally, some old mice were fed with a hypophosphatemic diet containing 0.2% phosphate (Old-Low P) for the last 3 months. Serum phosphate levels were measured with DIPI-500 assay. Lung fibrosis was evaluated by Sirius Red staining and by expression of extracellular matrix proteins such as fibronectin or collagen V assessed by western blot. In vitro studies were performed on a human cell line from lung fibroblasts (Wi-38), which were treated with a phosphate donor called β-glycerophosphate (BGP, 10 mM) at different times. Fibrosis was detected by expression of fibronectin by western blot as in lung tissues. Oxidative stress was measured by confocal microscopy using the fluorogenic probe CellROX. EMSA assays was used to check the activation of fibronectin promoter by NFκB transcription factor. RESULTS Old mice that showed higher serum phosphate levels had more lung fibrosis than young mice. Old mice fed with the low phosphate diet, which had less serum phosphate levels, reduced significantly the lung fibrosis respect to old mice fed with the standard diet. In order to elucidate a possible mechanism, in vitro studies were performed in lung fibroblasts treated with BGP. BGP induced a significant increased on fibronectin expression. BGP also rose oxidative stress at short times. As NF-kB transcription factor can be activated by oxidative stress, we checked whether BGP induced the binding of this factor to fibronectin promoter to upregulate this gene. The EMSA analysis showed that BGP promoted the binding of NF-κB to fibronectin promoter. Besides, in the presence of an antioxidant such as catalase, BGP did not induce the binding of NF-kB to fibronectin promoter, suggesting a role for oxidative stress. CONCLUSION Old mice showed hyperphosphatemia and lung fibrosis, which both were improved using a low phosphate diet. In vitro studies in lung fibroblasts confirmed that fibrosis induced by BGP seems to be mediated by activation on NF-kB via oxidative stress. These results show a relationship between hyperphosphatemia and lung fibrosis in aging; however, more studies are necessary in order to elucidate the relationship with other chronic diseases related to aging.

MYB2 Is Important for Tapetal PCD and Pollen Development by Directly Activating Protease Expression in Arabidopsis.

Tapetal programmed cell death (PCD) is a complex biological process that plays an important role in pollen formation and reproduction. Here, we identified the MYB2 transcription factor expressed in the tapetum from stage 5 to stage 11 that was essential for tapetal PCD and pollen development in Arabidopsis thaliana. Downregulation of MYB2 retarded tapetal degeneration, produced defective pollen, and decreased pollen vitality. EMSA and transcriptional activation analysis revealed that MYB2 acted as an upstream activator and directly regulated expression of the proteases CEP1 and βVPE. The expression of these proteases was lower in the buds of the myb2 mutant. Overexpression of either/both CEP1 or/and βVPE proteases partially recover pollen vitality in the myb2 background. Taken together, our results revealed that MYB2 regulates tapetal PCD and pollen development by directly activating expression of the proteases CEP1 and βVPE. Thus, a transcription factor/proteases regulatory and activated cascade was established for tapetal PCD during another development in Arabidopsis thaliana. Highlight: MYB2 is involved in tapetal PCD and pollen development by directly regulating expression of the protease CEP1 and βVPE and establishes a transcription factor/proteases regulatory and activated cascade.

The Interaction Between CitMYB52 and CitbHLH2 Negatively Regulates Citrate Accumulation by Activating CitALMT in Citrus Fruit.

Citric acid plays significant roles in numerous physiological processes in plants, including carbon metabolism, signal transduction, and tolerance to environmental stress. For fruits, it has a major effect on fruit organoleptic quality by directly influencing consumer taste. Citric acid in citrus is mainly regulated by the balance between synthesis, degradation, and vacuolar storage. The genetic and molecular regulations of citric acid synthesis and degradation have been comprehensively elucidated. However, the transporters for citric acid in fruits are less well understood. Here, an aluminum-activated malate transporter, CitALMT, was characterized. Transient overexpression and stable transformation of CitALMT significantly reduced citrate concentration in citrus fruits and transgenic callus. Correspondingly, transient RNA interference-induced silencing of CitALMT and increased citrate significantly, indicating that CitALMT plays an important role in regulating citrate concentration in citrus fruits. In addition, dual-luciferase assays indicated that CitMYB52 and CitbHLH2 could trans-activate the promoter of CitALMT. EMSA analysis showed that CitbHLH2 could physically interact with the E-box motif in the CitALMT promoter. Bimolecular fluorescence complementation assays, yeast two-hybrid, coimmunoprecipitation and transient overexpression, and RNAi assay indicated that the interaction between CitMYB52 and CitbHLH2 could synergistically trans-activate CitALMT to negatively regulate citrate accumulation.

Promoter activity analysis and transcriptional profile of Ginkgo biloba 1-Deoxy-D- Xylulose 5-Phosphate reductoisomerase gene (GbDXR) under abiotic stresses

Terpene trilactones (TTL) is a pharmacological ingredient in Ginkgo biloba and its content has become one of the key indices for medicinal value evaluation of ginkgo. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first step specific for isopentenyl diphosphate production in methylerythritol phosphate pathway, which provide the basic structure required for TTLs biosynthesis. To understand the mechanism controlling the GbDXR gene expression, the GbDXR promoter sequence was isolated and subjected to transient expression with the green fluorescent protein (GFP) in tobacco plants. Characteristic analysis revealed various cis-acting elements that related to light-regulated transcription, hormone signaling (auxin, ethylene), adversity stress and defense signaling (heat/dehydration stress) in the GbDXR promoter region. In transient expression assay, deletion of different portions of the upstream GbDXR promoter identified that the promoter region -3230bp to -865bp conserve the positive regulation function, which could promote the expression of GFP in the cytoplasm of tobacco leaf epidermal cells. The regulation function of the promoter region -865bp to -262bp remained to be elucidated. EMSA analysis suggested possible interactions of GbERF10 and GbERF17 with the ERF-binding elements in the upstream of GbDXR promoter. For abiotic stresses treatment, the expression of GbDXR gene could be significantly induced by UV-B and drought stress. In general, the GbDXR gene expressed differently in different ginkgo tissues but exhibited the highest transcriptional level in the root, with the maximum TTLs content simultaneously. The positive relationship between gene expression level and TTLs content indicated that the GbDXR is responsible for TTLs biosynthesis in G. biloba.

Synthesis of flavour-related linalool is regulated by PpbHLH1 and associated with changes in DNA methylation during peach fruit ripening.

SummaryLinalool is one of the common flavour‐related volatiles across the plant kingdom and plays an essential role in determining consumer liking of plant foods. Although great process has been made in identifying terpene synthase (TPS) genes associated with linalool synthesis, much less is known about regulation of this pathway. We initiated study by identifying PpTPS3 encoding protein catalysing enantiomer (S)‐(+)‐linalool synthesis, which is a major linalool component (˜70%) observed in ripe peach fruit. Overexpression of PpTPS3 led to linalool accumulation, while virus‐induced gene silencing of PpTPS3 led to a 66.5% reduction in linalool content in peach fruit. We next identified transcription factor (TF) PpbHLH1 directly binds to E‐box (CACATG) in the PpTPS3 promoter and activates its expression based on yeast one‐hybrid assay and EMSA analysis. Significantly positive correlation was also observed between PpbHLH1 expression and linalool production across peach cultivars. Peach fruit accumulated more linalool after overexpressing PpbHLH1 in peach fruit and reduced approximately 54.4% linalool production after silencing this TF. DNA methylation analysis showed increased PpTPS3 expression was associated with decreased 5 mC level in its promoter during peach fruit ripening, but no reverse pattern was observed for PpbHLH1. Arabidopsis and tomato fruits transgenic for peach PpbHLH1 synthesize and accumulate higher levels of linalool compared with wild‐type controls. Taken together, these results would greatly facilitate efforts to enhance linalool production and thus improve flavour of fruits.

Abstract P-9: Excess of Nhp6 over Spt16/Pob3 is Necessary for Efficient Nucleosome Unfolding by yFACT

Background: DNA accessibility in chromatin is important for proper gene expression and is regulated by multiple factors. One of them is histone chaperone FACT, which conducts large-scale ATP-independent nucleosome unfolding that increases the accessibility of nucleosomal DNA. FACT binding results in dramatic DNA uncoiling from nucleosome, occurs without apparent loss of histones, and proceeds via an ‘all-or-none’ mechanism, but the detailed mechanism of this process is still unknown. FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and it is an important function of FACT in the processes of transcription, DNA replication, and repair in vivo. Methods: Nucleosome-positioning DNA sequences containing fluorescent labels (Cy3 and Cy5) at positions 13/91 or 35/112 from the nucleosome boundary were used. Nucleosomes were assembled by transfer of histone octamers from chicken chromatin to nucleosomal DNA during dialysis from 2M NaCl. After dialysis nucleosomes were gel purified and used at a final concentration of 0.5nM for spFRET measurements or at 10nM for EMSA analysis. For complex formation nucleosomes were incubated in the presence of Spt16/Pob3(0.13μM) and Nhp6(1.3μM) for 10 min at 30°C in buffer containing 17mM HEPES, 2mM Tris-HCl, 0.8mM Na3EDTA, 0.11mM 2-merсaptoethanol, 150mM KCl, 11mM NaCl, 1.1%glycerin, 12% sucrose. In spFRET microscopy analysis, the proximity ratio EPR from each nucleosome was calculated based on the intensity of the signals, corrected to the background and plotted as relative frequency distribution. Each plot was fitted as a sum of two Gaussians to describe two conformational states of nucleosomes. The fractions of nucleosomes in different states were estimated by calculating the surface of areas under the corresponding Gaussian peaks as a fraction of the total area of the plot. Results: Here we report the results of our analysis of nucleosome unfolding by yeast FACT (yFACT) at different ratios of Spt16/Pob3 and Nhp6 using single-particle Förster resonance energy transfer (spFRET) microscopy. Our analysis suggests that the optimal ratio of Spt16/Pob3 to Nhp6 for the most efficient FACT-dependent nucleosome unfolding is 1:10. Importantly, a mere increase in the concentration of FACT results in a decrease of the functional activity, suggesting that the formation of a functional complex having a certain stoichiometry of Spt16/Pob3 to Nhp6 is essential for efficient FACT-dependent nucleosome unfolding. Conclusion: We determined that a certain ratio of Spt16/Pob3 to Nhp6 is essential for efficient FACT-dependent nucleosome unfolding, suggesting the formation of a functional FACT: Nhp6 complex.

Molecular Characterization and Functional Analysis of Two Steroidogenic Genes TSPO and SMAD4 in Yellow Catfish.

The steroid hormones are required for gonadal development in fish. The present study was undertaken to characterize the cDNA and promoter sequences of TSPO and SMAD4 genes in yellow catfish Pelteobagrus fulvidraco, explored the mRNA tissue expression and deciphered their promoter regions. Yellow catfish TSPO and SMAD4 shared the similar domains to the corresponding genes from other vertebrates. The TSPO and SMAD4 mRNAs were widely expressed in the detected tissues, but at different levels. Several transcription factors were predicted, such as Sp, GATA, AP1, SOX1, SRY, STAT, HNF4α, PPARγ, Pu.1 and FOXL2. PPARγ overexpression increased but STAT3 overexpression reduced TSPO promoter activity, and FOXL2 overexpression inhibited the promoter activity of TSPO and SMAD4. The site mutation and EMSA analysis indicated that TSPO promoter possessed STAT3 and FOXL2 sites. Overall, our provided the novel understanding into the transcriptionally regulatory mechanisms of TSPO and SMAD4 in fish.

Isolation and Characterization of Three Sodium-Phosphate Cotransporter Genes and Their Transcriptional Regulation in the Grass Carp Ctenopharyngodon idella.

It is important to explore the regulatory mechanism of phosphorus homeostasis in fish, which help avoid the risk of P toxicity and prevent P pollution in aquatic environment. The present study obtained the full-length cDNA sequences and the promoters of three SLC20 members (slc20a1a, slc20a1b and slc20a2) from grass carp Ctenopharyngodon idella, and explored their responses to inorganic phosphorus (Pi). Grass carp SLC20s proteins possessed conservative domains and amino acid sites relevant with phosphorus transport. The mRNAs of three slc20s appeared in the nine tissues, but their expression levels were tissue-dependent. The binding sites of three transcription factors (SREBP1, NRF2 and VDR) were predicted on the slc20s promoters. The mutation and EMSA analysis indicated that: (1) SREBP1 binding site (−783/−771 bp) negatively but VDR (−260/−253 bp) binding site positively regulated the activities of slc20a1a promoter; (2) SREBP1 (−1187/−1178 bp), NRF2 (−572/−561 bp) and VDR(615/−609 bp) binding sites positively regulated the activities of slc20a1b promoter; (3) SREBP1 (−987/−977 bp), NRF2 (−1469/−1459 bp) and VDR (−1124/−1117 bp) binding sites positively regulated the activities of the slc20a2 promoter. Moreover, Pi incubation significantly reduced the activities of three slc20s promoters, and Pi-induced transcriptional inactivation of slc20s promoters abolished after the mutation of the VDR element but not SREBP1 and NRF2 elements. Pi incubation down-regulated the mRNA levels of three slc20s. For the first time, our study elucidated the transcriptional regulatory mechanisms of SLC20s and their responses to Pi, which offered new insights into the Pi homeostatic regulation and provided the basis for reducing phosphorus discharge into the waters.