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1 Department of Applied Biology, and
2 Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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It has been established that the CCAAT motif is present in promoters of many mammalian genes, including those expressed in specific cell types during the cell cycle, such as topoisomerase II
, cyclin B1, CDC25C, E2F1, CDC2 and thymidine kinase genes (Kao et al. 1999; Hu et al. 2002). It is reported that NF-Y regulates transcription of Hoxb4, 
-globin, major histocompatibility (MHC) class II, TGF-β receptor II and Sox family genes (Reith et al. 1994; Wiebe et al. 2000; Gilthorpe et al. 2002; Fang et al. 2004; Niimi et al. 2004; Grujicic et al. 2005; Huang et al. 2005). Histone deacetylase 4 (HDAC4) is known to be recruited on NF-Y-dependent repressed promoters and a relationship between p53 and HDAC4 recruitment following DNA damage has also been noted (Basile et al. 2005). While NF-Y activity is clearly present in all mammalian tissues, genes that are actually regulated by NF-Y in vivo have still to be determined in detail. The fact that knockout of mouse NF-YA results in early embryonic lethality indicates essential roles in early development (Bhattacharya et al. 2003).
To study Drosophila NF-Y (dNF-Y) function during Drosophila development, we have focused on the dNF-YA subunit containing a DNA-binding domain with established transgenic fly lines carrying UAS-HA-dNF-YA or the UAS-dNF-YA inverted repeat (IR) (Yoshioka et al. 2007). Utilizing the GAL4-UAS targeted expression system (Brand & Perrimon 1993), we earlier demonstrated over-expression or knockdown of dNF-YA to be lethal at various developmental stages, suggesting that dNF-YA participates in various gene regulatory pathways during Drosophila development (Yoshioka et al. 2007). Expression of dNF-YA with eyeless-GAL4 mainly resulted in lethality with a headless phenotype in pharate-adults. Reduction of the eyeless gene dose enhanced the dNF-YA-induced phenotype, while reduction of the Distal-less gene dose suppressed the phenotype. Furthermore, crossing the dNF-YA over-expressing flies with a Notch mutant resulted in no apparent effect on the phenotype. From these results we conclude that dNF-YA can disturb eye disc specification, but not eye disc growth (Yoshioka et al. 2007).
In the present study, we examined the effect of knockdown of dNF-YA in the notum region of wing disc by a pannir (pnr)-GAL4 driver. The knockdown flies exhibited a thorax disclosed phenotype, very similar to those of mutants with defects in genes involved in the Jun N-terminal kinase (JNK) cascade responsible for notum development (Agnes et al. 1999: Ishimaru et al. 2004). The JNK cascade is an intracellular relay pathway in which the stress-activated kinases Jun N-terminal kinase kinase (JNKK) and JNK (Martin-Blanco et al. 2000) play essential roles. In Drosophila, JNKK and JNK homologues are encoded by the genes hemipterous (hep) and basket (bsk) (Riesgo-Escovar et al. 1996; Sluss et al. 1996; Agnes et al.1999). Mutants of both of these genes show an embryonic dorsal-open phenotype, as a consequence of the lack of elongation of cells of the lateral epidermis (Glise et al. 1995; Riesgo-Escovar et al. 1996; Sluss et al. 1996). During closure, JNK signaling activity is modulated by the product of the gene puckered (puc), which encodes a dual-specificity phosphatase. puc is expressed in the dorsal-most cells of the epidermis (‘leading-edge’ cells), and in its absence, cell recognition at the dorsal midline is impaired (Martin-Blanco et al. 1998). We further examined the effects of reduction of the bsk and hep gene doses and showed the former to enhance the phenotype induced by knockdown of dNF-YA with pnr-GAL4. Based on these results and findings of other cytological and molecular biological studies, possible roles of dNF-Y in regulating the JNK signal transduction pathway during Drosophila thorax development are discussed.
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Results |
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We have established 17 independent UAS-dNF-YAIR transgenic fly strains with targets set between aa 231 and aa 399 (Yoshioka et al. 2007). Using these strains, we revealed that dNF-YA participates in various gene regulatory pathways during Drosophila development (Yoshioka et al. 2007). Furthermore, detailed analyses in eye disc development also revealed that dNF-YA can disturb eye disc specification, but not eye disc growth (Yoshioka et al. 2007).
As noted previously, the pnr-GAL4 driver strain specifically expressed GAL4 in a domain of wing imaginal discs corresponding to the central presumptive notum (Fig. 1A, panel a) (Ishimaru et al. 2004). Specific knockdown of dNF-YA by pnr-GAL4 in the corresponding region of wing discs was confirmed by immunostaining with anti-dNF-YA antibodies (Fig. 1A, panel b). Specific effects of dNF-YA double strand RNA (dsRNA) on dNF-YA expression were further confirmed by a flip-out experiment (Fig. 1B) (Sun & Tower 1999). Cells marked by GFP expressed dNF-YA dsRNA (Fig. 1B, panels b and c). In the RNAi clone area, the level of dNF-YA signals was specifically reduced (Fig. 1B, panels a and c). In addition, Western immunoblot analysis with extracts of Act5C-GAL4>UAS-dNF-YAIR larvae using an antibody against dNF-YA revealed the amount of dNF-YA protein to be reduced to below the level of detection in extracts (Fig. 1C, lane 3), but extensively increased in Act5C-GAL4>UAS-dNF-YA larval extracts (Fig. 1C, lane 1). These results pointed to specific knockdown of dNF-YA by expression of dNF-YA dsRNA.
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Normal thorax development requires the JNK pathway (Agnes et al. 1999; Zeitlinger & Bohmann 1999; Ishimaru et al. 2004). And reduction of Drosophila JNK gene bsk or JNKK gene hep expression results in a thorax disclosed phenotype as observed with pnr-GAL4>UAS-dNF-YAIR flies (Agnes et al. 1999; Ishimaru et al. 2004). Thus, we considered that knockdown of dNF-YA may inhibit JNK signaling through genetic interactions. Genetic crossing of dNF-YA knockdown flies, pnr-GAL4>UAS-dNF-YAIR with bsk1 resulted in severe enhancement of their thorax disclosed phenotypes (Fig. 3C). Crossing with the other allele, bsk2, resulted in similar enhancement (Fig. 3D). However, genetic crossing with hepr75 resulted in only marginal enhancement of the thorax disclosed phenotype (Fig. 3E). Crossing with the other allele hep1, a hypomorphic homo viable allele, exhibited no further effects (Fig. 3F). These results suggest that the transcription factor dNF-Y positively regulates a gene(s) involved in the JNK pathway during thorax closure and the bsk gene is the most likely target.
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The observation that mutations in bsk enhance the thorax disclosed phenotype induced by knockdown of dNF-YA suggests that expression or activation of Bsk is reduced in dNF-YA knockdown flies. Therefore, we measured Bsk activation using an enhancer trap line, pucE69, in which LacZ is inserted into the puc gene intron (Martin-Blanco et al. 1998; Adachi-Yamada 2002) so that puc enhancer activity can be monitored with reference to LacZ expression. It is well known that the puc gene is highly expressed in Bsk-activated cells (Martin-Blanco et al, 1998; Adachi-Yamada 2002) and the puc enhancer trap line has been widely used to monitor Bsk activity in vivo (Adachi-Yamada et al. 1999; Tateno et al. 2000; Igaki et al. 2002). The LacZ expression pattern in the wing disc of puc-LacZ enhancer trap line is shown in Fig. 4A. The puc gene is normally expressed in the stalk region of wing imaginal discs (Fig. 4A) (Adachi-Yamada 2002). We monitored puc expression in the stalk region by immunostaining of the wing discs of dNF-YA knockdown flies with anti-LacZ antibody (Fig. 4B). Cells marked with GFP are expressing dNF-YAdsRNA (Fig. 4B, panels b and c). In the RNAi clone area, at the level of the puc-LacZ signaling was found to be extensively reduced (Fig. 4B, panels a and c), suggesting dNF-YA involvement in activation of Bsk during thorax development.
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Our data can be explained by reduction of either Bsk-activation or bsk-expression in the NF-YA knockdown region. The latter is more likely, since dNF-YA is a transcription factor and the genetic interactions were observed between dNF-YA and bsk but not between dNF-YA and hep. It is also noteworthy that reduction of bsk gene expression by bsk dsRNA also induces a thorax disclosed phenotype similar to that observed with dNF-YA knockdown flies (Ishimaru et al. 2004). Therefore, we examined whether the thorax disclosed phenotype induced by knockdown of dNF-YA can be suppressed by Bsk expression. Although over-expression of wild-type Bsk itself did not exhibit a thorax disclosed phenotype (Fig. 5D), it effectively suppressed that induced by knockdown of dNF-YA (Fig. 5B,C). The results thus suggest that the thorax disclosed phenotype of dNF-YA knockdown fly is caused by reduction of bsk expression and the upstream Bsk-activating signals are not rate-limiting. In addition, co-expression of P35, an inhibitor of apoptosis, exerted no effect on a thorax disclosed phenotype (Fig. 5E) and pnr-GAL4>UAS-p35 flies do not exhibit a thorax disclosed phenotype (Fig. 5F). These results suggest that the thorax disclosed phenotype of dNF-YA knockdown flies are not caused by induction of apoptosis.
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NF-Y is a major CCAAT-binding transcription factor that specifically recognizes consensus sequences, 5'-CTGATTGGYYRR-3' or 5'-YYRRCCAATCAG-3' (Y, pyrimidines and R, purines), present in promoter regions (Matsuoka & Chen 1999). Drosophila NF-Y can also bind to the same consensus sequences in vitro (Yoshioka et al. 2007). A data base search revealed that the 5' flanking region of the bsk gene contains a CCAAT motif at –90 with respect to the transcription initiation site (Fig. 6A). To obtain further insight into dNF-Y-binding to this CCAAT motif in the bsk gene, we performed chromatin immunoprecipitation (ChIP) assays with anti-dNF-YA IgG. Immunoprecipitated samples were subjected to quantitative real-time PCR using primers to amplify the bsk gene promoter region containing the CCAAT box (Fig. 6A, region 1). The 2 kb upstream region from the transcription initiation site of the bsk gene was chosen as a negative control, because it does not contain a CCAAT box (Fig. 6A, region 2).
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dNF-YA is required for the bsk gene promoter activity in S2 cells
To examine the role of CCAAT box in bsk gene promoter activity, we constructed the plasmid carrying the bsk gene promoter (–1000 to +60) fused with the luciferase reporter gene (p5'-1000bskwt-Luc) and the derivative carrying mutations in the CCAAT box (p5'-1000bskmutCCAAT-Luc). These plasmids were transfected into S2 cells, and after 48 h luciferase activities were determined (Fig. 7A). Base-substituted mutations in the CCAAT box reduced bsk gene promoter activity by 20%. Although the extent of reduction was not so large, the value was statistically significant. These results indicate that the CCAAT box plays a positive role in the bsk gene promoter activity.
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To examine the role of dNF-YA in the endogenous bsk gene expression, the level of bsk mRNA in the dNF-YA knockdown flies was quantified by real time PCR (Fig. 7C). In the experiments, the β-tubulin gene was used as a negative control. The dNF-YA mRNA level in flies carrying Act5C-GAL4/UAS-dNF-YAIR was 2% of that of the wild-type Canton S (Fig. 7C, dNF-YA columns), confirming the efficient knockdown of dNF-YA in the transgenic flies. The bsk mRNA level in the dNF-YA knockdown flies was 24% of that of the wild-type Canton S (Fig. 7C, bsk columns). However, no such reduction of bsk mRNA level was observed in the transgenic flies carrying Act5C-GAL4 alone. These results further support that dNF-YA are required for the bsk gene expression.
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Discussion |
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Do JNK signaling pathway genes contain the consensus NF-Y-binding sequence box in their 5'-flanking regions?
The present study revealed that dNF-YA binds to the bsk (JNK) gene promoter region containing a CCAAT box. The luciferase transient expression assay in S2 cells revealed that the CCAAT box plays a positive role in bsk gene promoter activity. Treatment of S2 cells with dNF-YAdsRNA reduced bsk gene promoter activity (Fig. 7A, B), further suggesting the requirement of dNF-YA for the bsk gene expression. Moreover, the level of endogenous bsk mRNA is reduced in dNF-YA knockdown larvae. Based on these observations, we conclude that dNF-Y likely regulates the JNK pathway via control of bsk gene expression (Fig. 8). However, the extent of reduction of bsk promoter activity was not so large. The 5' flanking region of the bsk gene contains putative binding sites for several other transcription factors. The bsk gene is therefore probably regulated by not only dNF-Y, but also some other transcription factors.
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Do mammalian JNK signaling pathway genes contain consensus NF-Y-binding sequences?
In humans, three JNK genes, JNK1, JNK2 and JNK3, have been reported (Davis 2000). Since the 5' flanking region of the JNK2 gene contains a CCAAT motif at –142 with respect to the transcription initiation site, human NF-Y could regulate its expression as in the Drosophila NF-Y case. The other human JNK genes do not share any consensus NF-Y-binding sequence within the 300 bp upstream regions.
The JNK signaling cascade is triggered by a variety of signals, including UV radiation and oxidative stress. At the cellular level, it may serve a protective function (Minamino et al. 1999) and may also promote apoptosis (Tournier et al. 2000). Drosophila puc mutants accumulate less oxidative damage and live dramatically longer than wild-type flies (Wang et al. 2003). Furthermore, it is well known that exposure of flies to endotoxic lipopolysaccharide initiates an insect immune response which leads to Bsk activation (Sluss et al. 1996). It should be noted that mammalian NF-Y also regulates expression of various genes related to immune responses such as -globin, MHC class II.
Additionally, it is reported that Parkin, an E3 ubiquitin ligase, negatively regulates the JNK pathway in dopaminergic neurons of Drosophila (Cha et al. 2005). Thus, loss of Parkin function up-regulates the JNK signaling pathway, which may contribute to the vulnerability of dopaminergic neurons in Drosophila parkin mutants and perhaps autosomal recessive juvenile parkinsonism patients (Cha et al. 2005). It would clearly be of interest to examine possible involvement of dNF-Y in this Parkin–JNK pathway.
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Experimental procedures |
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To construct the plasmid pWIZNF-YAIR63–228, the following oligonucleotides were synthesized:
To construct the plasmid p5'-1000bskwt-Luc and p5'-1000bskmutCCAAT-Luc, the following oligonucleotides were synthesized:
To carry out ChIP, the following PCR primers were chemically synthesized. These primer sets were designed to amplify 150 bp amplicons:
To carry out quantitative real time PCR, the following oligonucleotides were synthesized:
Plasmid construction
A 500-bp DNA fragment containing the N-terminal region (aa 63 to aa 228 ) of dNF-YA was isolated by the polymerase chain reaction (PCR) using pTrc-His-dNF-YA (Yoshioka et al. 2007) and the primers NF-YA185NheI and NF-YA185XbaI. A 500-bp DNA fragment was digested with NheI, then ligated to the NheI sites of pWIZ (Reichhart et al. 2002). Second PCR was performed using this pWIZ-5'-dNF-YA template DNA with primers WIZ-EcoRI and NF-YA185NheI. The obtained DNA fragments were digested with EcoRI and XbaI, and then inserted into EcoRI and XbaI sites of pWIZ to create pWIZ-5'-dNF-YA. Then the 500 bp DNA fragment containing the N-terminal region of dNF-YA (aa 63 to aa 228) was amplified by PCR using pTrc-HisNF-YA (Yoshioka et al. 2007), and the primers NF-YA185BglII and NF-YA185NheI. The amplified 500 bp DNA fragment was then inserted into the BglII and AvrII sites of pWIZ to create pWIZ-dNF-YAIR63–228.
To construct the plasmid p5'-1000bskwt-Luc, PCR was performed using Drosophila genomic DNA as a template and primers bskUS1000MluI and bskUS-60XhoI in combination. PCR products were digested with MluI and XhoI and inserted between the MluI and XhoI sites of the PGVB plasmid (Toyo Ink).
For site-directed mutagenesis, PCR was carried out using a Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Oligonucleotide pairs carrying base-substitutions in the region of interest were used as primers and the p5'-1000bskwt-Luc DNA was used as a template for the PCR. Fully amplified PCR products were digested with DpnI to remove the methylated template DNA and then transformed into Escherichia coli XL-1 blue. The mutated nucleotide sequences were confirmed by nucleotide sequencing and the resultant plasmids were named p5'-1000bskmutCCAAT-Luc.
Anti-dNF-YA antibodies
The anti-dNF-YA antibodies were affinity purified from anti-serum (Yoshioka et al. 2007) using a GST-fused dNF-YA protein coupled Hitrap HP column (GE Healthcare, Little Chalfont, UK) after passage through a GST-coupled Hitrap HP column. The specificity of the affinity-purified anti-dNF-YA IgG was confirmed by Western immunoblot analysis (Fig. 1C).
Western immunoblot analysis
Larvae of transgenic flies carrying Act5C-GAL4/UAS-HA-dNF-YA or Act5C-GAL4/UAS-dNF-YAIR and those of Oregon R were frozen in liquid nitrogen and homogenized in a solution containing 50 mM Tris–Borate (pH 7.6), 400 mM KCl, 0.1% Triton X-100, 1 mMb dithiothreitol, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg each of aprotinin and leupeptin/mL, and 1 µg each of pepstatin, chymostatin and phosphoramidon/mL. Homogenates were centrifuged at 12 000 g at 4 °C for 5 min, and extracts (100 µg of protein per each lane) were electrophoretically separated on SDS-12% polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (BIO-RAD, Herts, UK) in a solution containing 25 mM Tris–HCl, pH 9.5 and 20% methanol for 45 min at 25 °C. Whole cell extracts from S2 cells prepared as described earlier (Kwon et al. 2000) were also applied to SDS-12% polyacrylamide gels and transferred to PVDF membranes. Blotted membranes were blocked with Tris-buffered saline (TBS) (50 mM Tris–HCl, pH 8.3 and 150 mM NaCl) containing 10% skim milk for 1 h at 25 °C and incubated with the anti-dNF-YA antibody at 1 : 500 dilution, or an anti- tubulin monoclonal antibody (Sigma, St. Louis, MO) at 1 : 2000 dilution at 4 °C for 16 h. After washing with TBS, the blots were incubated with a horseradish peroxidase-labeled anti-rabbit IgG and a horseradish peroxidase-labeled anti-mouse IgG (GE healthcare) at a 1 : 5000 dilution for 1 h at 25 °C. Detection was performed with ECL Western blotting detection reagents (GE healthcare), and images were analyzed with a Lumivision Pro HSII image analyzer (Aisin Seiki, Japan).
Fly stocks
Flies were cultured at 25 °C on standard food. The Oregon R or Canton S flies were used as the wild-type strain. The transgenic fly lines carrying UAS-dNF-YAIR and UAS-HA-dNF-YA were described earlier (Yoshioka et al. 2007). The hep1/FM7C, hepr75/FM7C, pucE69/TM3, UAS-bsk(III)/TM3, hs-flp and Act5C>FRT y FRT>GAL4, UAS-GFP lines were kindly provided by Dr T. Adachi-Yamada. The pnr-GAL4 driver, mutant stocks, bsk1/CyO, bsk2/CyO, UAS-bsk and UAS-GFP used in this study were obtained from the Bloomington Drosophila stock center.
Establishment of transgenic flies
P-element-mediated germ line transformation was carried out as described earlier (Spradling 1986) and F1 transformants were selected on the basis of white-eye color rescue (Robertson et al. 1988). Three independent lines were established for pWIZ-dNF-YAIR63–228. Transgenic lines carrying UAS-dNF-YAIR63–228 were crossed with the pnr-GAL4 driver line.
Flip-out experiments
RNAi clones in wing discs were generated with a flip-out system (Sun & Tower 1999). Female flies with hs-flp; Act5C>FRT y FRT>GAL4, UAS-GFP were crossed with male flies with UAS-NF-YAIR and clones were marked by the presence of GFP expressed under control of the Act5C promoter. Flip-out was induced 24–48 h after egg laying with 60 min heat shock at 37 °C.
Scanning electron microscopy
Adult flies were anesthetized, mounted on stages and observed with a VE-7800 (Keyence Inc., Woodcliff Lake, NJ) scanning electron microscope in the low vacuum mode.
Immunohistochemistry
Third instar larvae were dissected in Drosophila Ringer's solution and imaginal discs were collected and fixed in 4% paraformaldehyde in PBS for 10 min at 4 °C or 30 min at 25 °C. After washing with PBS containing 0.3% Triton X-100 (PBS-T), the samples were blocked with PBS-T containing 10% normal goat serum for 20 min at 25 °C and incubated with an anti-β-galactosidase mouse monoclonal antibody (DSHB) (1 : 500) or anti-dNF-YA rabbit polyclonal antibodies (1 : 500) at 4 °C for 16 h. After extensive washing with PBS-T, the imaginal discs were incubated with an anti-mouse IgG conjugated with Alexa 594 (Invitrogen, Carlsbad, CA) (1 : 400) for 16 h at 4 °C. After extensive washing with PBS-T and PBS, samples were mounted in Fluoroguard Antifade Reagent (Bio-Rad) and inspected with an Olympus BX-50 microscope equipped with a cooled CCD camera (Hamamatsu Photo).
Preparation of double stranded RNA (dsRNA) for RNAi experiments
The 355 nucleotides of cDNA spanning DNA-binding domain (aa 282 to aa 399) of dNF-YA were cloned into pBluescript II SK(–) and the plasmid was used for synthesizing dsRNA. The dsRNA was prepared using the RiboMax T7 kit (Promega, Madison, WI) and MEGAscript T3 kit (Ambion, Austin, TX) according to the manufacture's instructions. RNAi analysis was carried out as described earlier (Crevel et al. 2005; Seto et al. 2006; Ida et al. 2007).
Chromatin immunoprecipitation (ChIP)
ChIP was performed using a ChIP-Assay kit as recommended by the manufacturer (Upstate, Lake Placid, NY) with minor modifications (Thao et al. 2006). Approximately 2 x 107 S2 cells were fixed in 1% formaldehyde at 37 °C for 10 min and then quenched in 125 mM glycine for 5 min at 25 °C. Cells were collected and washed twice in PBS containing protease inhibitors (1 mM PMSF, 1 µg/mL aprotinin and 1 µg/mL pepstatin A) and lysed in 2 mL of SDS lysis buffer (Upstate). Lysates were sonicated to break DNA into fragments of < 1 kb and centrifuged at 15 300 g for 10 min at 4 °C. The sonicated cell supernatants were diluted 10-fold in ChIP Dilution Buffer (Upstate) and pre-cleared with 80 µL of salmon sperm DNA/protein G agarose – 50% slurry for 30 min at 4 °C. After a brief centrifugation, supernatants were incubated with 4 µg of normal rabbit IgG (Sigma) or anti-dNF-YA IgG for 16 h at 4 °C. Salmon sperm DNA/protein G agarose –50% slurry was added, and incubated for 1 h at 4 °C. After washing, immunoprecipitated DNA was eluted with the elution buffer containing 1% SDS and 0.1 M NaHCO3. Then the protein–DNA cross-links were reversed by heating at 65 °C for 4 h. After deproteinization with proteinase K, DNA was recovered by phenol–chloroform extraction and ethanol precipitation. Then, the immunoprecipitated DNA fragments were detected by quantitative real time PCR using SYBR Green I (Takara, Tokyo, Japan) and the Applied Biosystems 7500 Real Time PCR system. The 
CT value of each sample was calculated by subtracting the CT value for the input sample from the CT value obtained for the immunoprecipitated sample. Fold differences of each sample relative to control using non-immune IgG were then calculated by raising 2 to the CT power. The CT was calculated by subtracting the CT value for the sample immunoprecipitated with control IgG (Morrison et al. 1998).
For dsRNA interference experiments, 3 x 106 S2 cells were plated in 10 cm dish in the presence of 90 µg of dNF-YAdsRNA and LacZdsRNA in FBS free M3(BF) medium for 1 h. After the incubation, 4 volumes of M3(BF) medium containing 10% FBS were added. Seventy-two hours after the RNAi treatment, the cells were processed for the ChIP assay as described above.
Luciferase transient expression assays
For luciferase transient expression assays, 1 x 105 S2 cells were plated in 24-well dishes. Transfection of various DNA mixtures was performed using Cell-Fectin reagent (Invitrogen). Cells were harvested at 48 h after transfection. Luciferase activity was measured as described earlier (Hayashi et al. 1999; Seto et al. 2006; Ida et al. 2007) and was normalized to the Renilla luciferase activity using pAct5C-seapansy (Sawado et al. 1998) as an internal control. All plasmids for transfection were prepared by using a QIAGEN Plasmid Kit (Hilden, Germany).
For dsRNA interference experiments, 30 µg each of dNF-YAdsRNA and LacZdsRNA were added to 1 x 106 S2 cells plated in each of 6-well dishes. Seventy-two hours after the RNAi treatment, the cells were transfected with various DNA mixtures. Cells were harvested at 48 h after transfection and processed for the luciferase assay as described above.
All transient expression data reported in this paper represent the means from three independent experiments, each performed in triplicate. Average of the relative luciferase activity was graphed and statistically analyzed by Welch's t-test.
Quantitative RT-PCR
Total RNA was isolated from third instar larvae (wandering stage) using Trizol® Reagent (Invitrogen) and 1 µg aliquots were reverse transcribed with oligo dT primer using a Takara high fidelity RNA PCR kit (Takara). Then, real-time PCR was performed with a SYBR Green I kit (Takara) and the Applied Biosystems 7500 Real Time PCR system using 1 µL of reverse transcribed sample per reaction. DNA fragment was amplified by using set of the primers bskRTset2F and bskRTset2R, dNF-YA691NheI and NF-YA1200XbaI (Yoshioka et al. 2007), β-tubulin-F and β-tubulin-R (Ida et al. 2007) and mod-F and mod-R (Thao et al. 2008). Levels of mRNAs in transgenic flies carrying Act5C-GAL4/UAS-dNF-YAIR or Act5C-GAL4/+ and those in Canton S were investigated by the CT comparative method. The β-tubulin gene was chosen as a negative control. The mod gene was used as an endogenous reference gene. Experiments were performed in triplicate for each of three RNA batches isolated separately.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: myamaguc{at}kit.ac.jp
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Accepted: 4 November 2007
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