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Abstract Background Iron (Fe) deficiency affects 30–50% of the world’s population. Genetic biofortification of staple crops is a promising strategy for improving human nutrition, but the number of effective precision breeding targets for Fe biofortification is small. Upstream open reading frames (uORFs) are cis-regulatory elements within the 5’ leader sequence (LS) of genes that generally repress translation of the main open reading frame (mORF). Results We aligned publicly available rice (Oryza sativa L.) ribo-seq datasets and transcriptomes to identify putative uORFs within important Fe homeostasis genes. A dual luciferase assay (DLA) was used to determine whether these uORFs cause repression of mORF translation and pinpoint LS regions that can be mutated for mORF derepression. A translationally repressive uORF region was identified in two positive regulators of the Fe-deficiency response: IDEF1 and IDEF2. The IDEF2-uORF peptide was highly conserved among monocots and a mutation series in the 5’ LS of the wheat (Triticum aestivum L.) TaIDEF2-A1 gene demonstrated variable mORF derepression. Conclusions Together these results reveal a possible regulatory mechanism by which IDEF2 transcription factors modulate the Fe deficiency response in monocots, and highlight novel precision breeding targets to improve crop nutrition and abiotic stress tolerance.

Background: Iron (Fe) deficiency affects 30–50% of the world's population. Genetic biofortification of staple crops is a promising strategy for improving human nutrition, but the number of effective precision breeding targets for Fe biofortification is small. Upstream open reading frames (uORFs) are cis-regulatory elements within the 5' leader sequence (LS) of genes that generally repress translation of the main open reading frame (mORF). Results: We aligned publicly available rice (Oryza sativa L.) ribo-seq datasets and transcriptomes to identify putative uORFs within important Fe homeostasis genes. A dual luciferase assay (DLA) was used to determine whether these uORFs cause repression of mORF translation and pinpoint LS regions that can be mutated for mORF derepression. A translationally repressive uORF region was identified in two positive regulators of the Fe-deficiency response: IDEF1 and IDEF2. The IDEF2-uORF peptide was highly conserved among monocots and a mutation series in the 5' LS of the wheat (Triticum aestivum L.) TaIDEF2-A1 gene demonstrated variable mORF derepression. Conclusions: Together these results reveal a possible regulatory mechanism by which IDEF2 transcription factors modulate the Fe deficiency response in monocots, and highlight novel precision breeding targets to improve crop nutrition and abiotic stress tolerance. Highlight: We searched for translationally repressive uORFs within Fe homeostasis genes to find precision breeding targets for improved crop nutrition and discovered uORFs in IDEF1 and IDEF2. [ABSTRACT FROM AUTHOR]

The Bardet–Biedl Syndrome 7 (Bbs7) gene was identified as the most likely candidate gene causing juvenile obesity in the Berlin Fat Mouse Inbred (BFMI) line. Bbs7 expression is significantly lower in the brain, adipose tissue, and liver of BFMI mice compared to lean C57BL/6NCrl (B6N) mice. A DNA sequence comparison between BFMI and B6N revealed 16 sequence variants in the Bbs7 promoter region. Here, we tested if these mutations contribute to the observed differential expression of Bbs7. In a cell-based dual-luciferase assay, we compared the effects of the BFMI and the B6N haplotypes of different regions of the Bbs7 promotor on the reporter gene expression. A single-nucleotide polymorphism (SNP) was identified causing a significant reduction in the reporter gene expression. This SNP (rs29947545) is located in the 5′ UTR of Bbs7 at Chr3:36.613.350. The SNP is not unique to BFMI mice but also occurs in several other mouse strains, where the BFMI allele is not associated with lower Bbs7 transcript amounts. Thus, we suggest a compensatory mutation in the other mouse strains that keeps Bbs7 expression at the normal level. This compensatory mechanism is missing in BFMI mice and the cell lines tested. [ABSTRACT FROM AUTHOR]

Santalum album L., a semi-parasitic evergreen tree, contains economically important essential oil, rich in sesquiterpenoids, such as (Z) α- and (Z) β-santalol. However, their transcriptional regulations are not clear. Several studies of other plants have shown that basic-helix-loop-helix (bHLH) transcription factors (TFs) were involved in participating in the biosynthesis of sesquiterpene synthase genes. Herein, bHLH TF genes with similar expression patterns and high expression levels were screened by co-expression analysis, and their full-length ORFs were obtained. These bHLH TFs were named SaMYC1, SaMYC3, SaMYC4, SaMYC5, SabHLH1, SabHLH2, SabHLH3, and SabHLH4. All eight TFs had highly conserved bHLH domains and SaMYC1, SaMYC3, SaMYC4, and SaMYC5, also had highly conserved MYC domains. It was indicated that the eight genes belonged to six subfamilies of the bHLH TF family. Among them, SaMYC1 was found in both the nucleus and the cytoplasm, while SaMYC4 was only localized in the cytoplasm and the remaining six TFs were localized in nucleus. In a yeast one-hybrid experiment, we constructed decoy vectors pAbAi-SSy1G-box, pAbAi-CYP2G-box, pAbAi-CYP3G-box, and pAbAi-CYP4G-box, which had been transformed into yeast. We also constructed pGADT7-SaMYC1 and pGADT7-SabHLH1 capture vectors and transformed them into bait strains. Our results showed that SaMYC1 could bind to the G-box of SaSSy, and the SaCYP736A167 promoter, which SaSSy proved has acted as a key enzyme in the synthesis of santalol sesquiterpenes and SaCYP450 catalyzed the ligation of santalol sesquiterpenes into terpene. We have also constructed pGreenII 62-SK-SaMYC1, pGreenII 0800-LUC-SaSSy and pGreenII 0800-LUC-SaCYP736A167 via dual-luciferase fusion expression vectors and transformed them into Nicotiana benthamiana using an Agrobacterium-mediated method. The results showed that SaMYC1 was successfully combined with SaSSy or SaCYP736A167 promoter and the LUC/REN value was 1.85- or 1.55-fold higher, respectively, than that of the control group. Therefore, we inferred that SaMYC1 could activate both SaSSy and SaCYP736A167 promoters. [ABSTRACT FROM AUTHOR]

To investigate the effect of IRX5 on the invasion and migration of hepatocellular carcinoma (HCC) cells and verify the targeted relationship between miR-136-5p and IRX5. Methods The SMMC7721 cells were divided into empty plasmid (pcDNA3.1) group and overexpress IRX5 (pcDNA3.1-IRX5) group in the overexpression IRX5 experiment. For knockdown IRX5 experiment a negative control (sh-NC) group and knockdown IRX5 (sh-IRX5) group were set up. The overexpression and knockdown efficiency rates of IRX5 were detected by Western blot assay. The effects of IRX5 on the invasion and migration of HCC cells were detected by wound healing assay and Transwell assay. miRNA and binding sites of IRX5 were predicted by miRanda and Targetscan. The 3' untranslated regions (UTR) of IRX5 were amplified by PCR and connected to pGL3-Control vector. To verified recombinant plasmid by enzyme digestion and gene sequencing methods. The 293T cells were divided into four groups: IRX5-3'UTR-Wt+NC group, IRX5-3'UTR-Wt+miR-136-5p group, IRX5- 3'UTR-Mut+NC group and IRX5-3'UTR-Mut+miR-136-5p group in dual luciferase assay. The luciferase activity was detected by dual luciferase reporter system. Results The expression level of IRX5 was significantly higher in the overexpression IRX5 group than that of the empty plasmid group (P＜0.05). Compared with the negative control group, the expression level of IRX5 was significantly reduced in the knockdown IRX5 group (P＜0.01). IRX5 promoted the invasion and migration of HCC cells (P＜0.05). The IRX5-3'UTR-Wt and IRX5-3'UTR-Mut were successfully constructed. The results of dual luciferase assay showed that miR-136-5p can reduce IRX5-3'UTR-Wt luciferase activity (P＜0.01), which showed no effect on IRX5-3'UTR-Mut luciferase activity (P＞0.05). Conclusion IRX5 promotes the invasion and migration of HCC cells. IRX5 acts as a target gene of miR-136-5p. miR-136-5p may inhibit the invasion and migration of HCC cells through the 3'UTR of IRX5. [ABSTRACT FROM AUTHOR]

Overaccumulation of lignin (a physiological disorder known as granulation) often occurs during fruit ripening and postharvest storage in pomelo (Citrus grandis). It causes an unpleasant fruit texture and taste. Previous studies have shown that lignin metabolism is closely associated with the process of juice sacs granulation. At present, the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate NAC transcription factor, CgNAC043, that is involved in the regulation of lignin biosynthesis in Citrus grandis, which has homologs in Arabidopsis and other plants. We used the fruit juice sacs of ‘San hong’ as the material, the staining for lignin with HCl−phloroglucinol of fruit juice sacs became dark red from the various developmental stages at 172 to 212 days post anthesis (DPA). The RT-qPCR was used to analyze the gene expression of CgNAC043 and its target gene CgMYB46 in fruit sacs, it was found that the expression trend of CgNAC043 was basically same as CgMYB46, which increased gradually and peaked at 212 DPA. The expression level of CgNAC043 in juice sacs obtained away from the core was the lowest, while those near the core and granulated area were highly expressed. The transcriptional activation activity of CgNAC043 and CgMYB46 was analyzed by a yeast two-hybrid system, with only CgNAC043 showing transcriptional activation activity in Y2H Gold yeast. A transformation vector, p1301- CgNAC043, was transformed into the mesocarp of ‘San hong’ by Agrobacterium-mediated transformation. Results showed that the expression of transcription factors CgMYB58 and CgMYB46 are all upregulated. Further experiments proved that CgNAC043 not only can directly trans-activate the promoter of CgMYB46 but also trans-activate the promoters for the lignin biosynthesis-related genes CgCCoAOMT and CgC3H by dual luciferase assay. We isolated the CgNAC043 gene in pomelo and found CgNAC043 regulates target genes conferring the regulation of juice sacs granulation.

BACKGROUND: Preliminary study has found that miR-146-5p is up-regulated in osteobIasts induced by sodium fIuoride. OBJECTIVE: To verify the targeting relationship between miR-146-5p and its potential target gene TNF receptor associated factor 6 (TRAF6) using a dual luciferase reporter gene through constructing a luciferase reporter plasmid for the 3' non-coding region (3'UTR) of TRAF6 gene. METHODS: Bioinformatics methods were used to predict the binding sites of miR-146-5p and TRAF6 genes. The 3'UTR fragment of TRAF6 gene was amplified by PCR and cloned into pYr-MirTarget vector to construct a wild type recombinant dual-luciferase reporter plasmid. There were six groups: (1) 6a-5p-nucleoside analogs + TRAF6-W 3'UTR co-transfection group; (2) non-nucleoside analogs mimics + TRAF6-W 3'UTR co-transfection group (control group); (3) miR-146a-5p inhibitor + TRAF6-W group; (4) TRAF6-W 3'UTR co-transfection group; (5) empty pYr-MirTarget transfection group; (6) normal cell group. The co-transfection of recombinant luciferase reporter plasmid and miR-146-5p or nucleoside analogs (mimics) with Saos-2 was made, respectively. At the same time, nucleoside analogs mimics (control group), miR-146-5p inhibitor (negative control group) and empty pYr-MirTarget-W 3'UTR and TRAF6-W 3'UTR were added simultaneously to detect luciferase activity in six groups of cells. Mimics, miR-146-5p inhibitor and nucleoside analogs mimics were transfected to the human osteoblast Saos-2 respectively. The protein expression level was detected by western blot assay after lysing cells to extract protein. RESULTS AND CONCLUSION: (1) Luciferase activity in Saos-2 cells co-transfected with miR-146-5p mimics was 10.103 0±0.558 5; the control group’s (NC mimics-TRAF6 3'UTR) luciferase activity was 13.140 0±0.720 4; the inhibitor group's luciferase activity was 13.707 1±0.434 8; and luciferase activity in Saos-2 cells of the wild-type TRAF6 3'UTR plasmid was 13.202 1±0.456 5. The luciferase activity of the cells transfected with the empty plasmid alone was 14.706 2±0.441 6. The original luciferase activity in Saos-2 cells was 1.126 4±0.126 2. The number of miR-146-5p mimics co-transfected significantly decreased (F=715.789, P < 0.000 1). The other three groups were compared with the control group, showing no significant difference (P > 0.05). (2) Western blot assay showed that compared with the control group, the expression level of TRAF6 protein was significantly down-regulated after transfection of miR-146-5p mimics. (3) To conclude, there is a targeted relationship between TRAF6 and miR-146-5p. [ABSTRACT FROM AUTHOR]

Highlights • TCF4 is associated with schizophrenia. • 3'UTR functional variants are risk factors for schizophrenia. • rs1272363 and rs373174212 have the ability to enhance the miRNA- mRNA binding. • rs1273263 significantly reduced the expression of TCF4. Abstract Transcription factor 4 (TCF4) gene plays an important role in nervous system development and it always associated with the risk of schizophrenia. Since miRNAs regulate targetgenes by binding to 3'UTRs of target mRNAs, the functional variants located in 3′UTR of TCF4 are highly suggested to affect the gene expressions in schizophrenia. To test the hypothesis regarding the effects of the variants located in 3'UTR of TCF4 , we conducted an in silico analysis to identify the functional variants and their predicted functions. In this study, we sequenced the 3'UTR of TCF4 in 13 multiplex schizophrenia families and 14 control families. We found two functional variants carried by three unrelated patients. We determined that the C allele of rs1272363 and the TC insert of rs373174214 might suppress post- transcriptional expression. Secondly, we cloned the region that flanked these two variants into a dual luciferase reporter system and compared the luciferase activities between the pmirGLO- TCF4 (control), pmirGLO- TCF4 -rs373174214 and pmirGLO- TCF4 -rs1273263. Both pmirGLO- TCF4 -rs373174214 and pmirGLO- TCF4 -rs1273263 caused lower reporter gene activities, as compared to the control. However, only the C allele of rs1272363 reduced the luciferase activity significantly (p = 0.0231). Our results suggested that rs1273263 is a potential regulator of TCF4 expression, and might be associated with schizophrenia. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) encodes a transcription trans-activator (Tat) with an essential role in the transcriptional elongation of viral RNA based on the viral promoter long terminal repeat (LTR). Tat-mediated transcription is conserved and can be distinguished from host transcription, so it is a therapeutic target for combating HIV replication. Traditional screening assays for Tat-mediated transcriptional inhibitors are based on the biochemical properties of Tat and transactivation-responsive RNA. We developed an inducible system based on two lentiviral expression cassettes for doxycycline (Dox)-inducible Tat and Renilla luciferase (R-Luc) using TZM-bl cells harboring LTR-driven firefly luciferase (F-Luc). The cells simultaneously expressed both Tat-induced F-Luc and R-Luc, so it was possible to recognize off-target effects in the presence of Dox. The system was validated with known inhibitors: CYC202 obtained high sensitivity and specificity, whereas 6Bio and DRB had off-target effects. The MTT-based cytotoxicity test indicated the resistance of the system even at concentrations with off-target effects. The specificity of the system was confirmed using antiretroviral drugs. Our dual reporter system can simply detect Tat inhibitory effects, as well as precisely discriminate between the inhibitory and off-target effects of inhibitors, and may be useful for the development of a therapeutic anti-HIV drug. [ABSTRACT FROM AUTHOR]

Craniosynostosis is the early fusion of one or more sutures of the infant skull and is a common defect occurring in approximately 1 of every 2,500 live births. Nonsyndromic craniosynostosis (NSC) accounts for approximately 80% of all cases and is thought to have strong genetic determinants that are yet to be identified. ALX4 is a homeodomain transcription factor with known involvement in osteoblast regulation. By direct sequencing of the ALX4 coding region in sagittal or sagittal-suture-involved nonsyndromic craniosynostosis probands, we identified novel, nonsynonymous, familial variants in three of 203 individuals with NSC. Using dual-luciferase assay we show that two of these variants (V7F and K211E) confer a significant gain-of-function effect on ALX4. Our results suggest that ALX4 variants may have an impact on the genetic etiology of NSC. Hum Mutat 33:1626-1629, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Hepatic toxicity remains a major concern for drug failure; therefore, a thorough examination of chemically induced liver toxicity is essential for a robust safety evaluation. Current hypotheses suggest that the metabolic activation of a drug to a reactive intermediate is an important process. In this article, we describe a new high-throughput GADD45β reporter assay developed for assessing potential liver toxicity. Most importantly, this assay utilizes a human cell line and incorporates metabolic activation and thus provides significant advantage over other comparable assays used to determine hepatotoxicity. Our assay has low compound requirement and relies upon 2 reporter genes cotransfected into the HepG2 cells. The gene encoding Renilla luciferase is fused to the CMV promoter and provides a control for cell numbers. The firefly luciferase gene is fused to the GADD45β promoter and used to report an increase in DNA damage. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. Results are expressed as the ratio of the 2 luciferase activities; increases over the control are interpreted as evidence of stress responses. This mammalian dual luciferase reporter has been characterized with, and without, metabolic activation using positive and negative control agents. Our data demonstrate that this assay provides for an assessment of potential toxic metabolites, is adaptable to a high-throughput platform, and yields data that accurately and reproducibly detect hepatotoxicants. [ABSTRACT FROM AUTHOR]

Overaccumulation of lignin (a physiological disorder known as granulation) often occurs during fruit ripening and postharvest storage in pomelo (Citrus grandis). It causes an unpleasant fruit texture and taste. Previous studies have shown that lignin metabolism is closely associated with the process of juice sacs granulation. At present, the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate NAC transcription factor, CgNAC043, that is involved in the regulation of lignin biosynthesis in Citrus grandis, which has homologs in Arabidopsis and other plants. We used the fruit juice sacs of 'San hong' as the material, the staining for lignin with HCl−phloroglucinol of fruit juice sacs became dark red from the various developmental stages at 172 to 212 days post anthesis (DPA). The RT-qPCR was used to analyze the gene expression of CgNAC043 and its target gene CgMYB46 in fruit sacs, it was found that the expression trend of CgNAC043 was basically same as CgMYB46, which increased gradually and peaked at 212 DPA. The expression level of CgNAC043 in juice sacs obtained away from the core was the lowest, while those near the core and granulated area were highly expressed. The transcriptional activation activity of CgNAC043 and CgMYB46 was analyzed by a yeast two-hybrid system, with only CgNAC043 showing transcriptional activation activity in Y2H Gold yeast. A transformation vector, p1301- CgNAC043, was transformed into the mesocarp of 'San hong' by Agrobacterium-mediated transformation. Results showed that the expression of transcription factors CgMYB58 and CgMYB46 are all upregulated. Further experiments proved that CgNAC043 not only can directly trans-activate the promoter of CgMYB46 but also trans-activate the promoters for the lignin biosynthesis-related genes CgCCoAOMT and CgC3H by dual luciferase assay. We isolated the CgNAC043 gene in pomelo and found CgNAC043 regulates target genes conferring the regulation of juice sacs granulation. [ABSTRACT FROM AUTHOR]

Abstract: The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds. [Copyright &y& Elsevier]

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of -3,500, -914, -408, and -215 bp promoters increased in response to 1 wk of OL. The smallest -171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the -171 and -408 bp region were performed. The -408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter. [ABSTRACT FROM AUTHOR]