Transcriptional regulation of the human PNRC promoter by
NFY in HepG2 cells
Yan Zhang, Bin Chen, Yuping Li, Jian Chen, Guiyu Lou, Min Chen, Dujin Zhou *
Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing 400038, China
Running title: NFY regulates transcription of PNRC gene
*To whom correspondence should be addressed. Dujin Zhou, Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing 400038, China. Tel: +86 2368752940, Fax: +86 23 68752940. E-mail: dujinzhou@https://www.doczj.com/doc/bd5678886.html,
? 2008 The Japanese Biochemical Society
Journal of Biochemistry Advance Access published February 14, 2008 at The 3rd Military Medical University on December 3, 2010
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PNRC (Proline-rich Nuclear Receptor Coactivator) was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC has been demonstrated to be a novel coactivator for multiple nuclear receptors. To understand the molecular mechanisms that regulation of the expression of human PNRC gene, in this study, potential transcriptional start site was determined by 5′ RACE analysis. Functional analysis of the 5′ flanking region of the human PNRC gene by deletion mutagenesis, transient transfection and luciferase assays revealed that the -123/+27 region is the minimal promoter of the human PNRC gene. Within this promoter region, there is one putative binding site for the transcription factor NFY (nuclear factor Y). EMSA and ChIP analyses demonstrated the specific binding of NFY
protein to the human PNRC promoter. Transient transfection and luciferase assays further
revealed that over-expression of NFY represses promoter activity of PNRC gene in a
dose-dependent manner. These results indicate that the transcription factor NFY specifically
bind to promoter region of PNRC and negatively regulate the transcription of the human
PNRC gene.
Key words: Nuclear receptor coactivator, PNRC, NFY, Promoter analysis, Transcriptional regulation.
Abbreviations: ChIP, chromatin immunoprecipitaion; EMSA, electrophoresis mobility shift and supershift assay; NFY, nuclear factor Y; PNRC, proline-rich nuclear receptor coactivator ; RACE, rapid amplification of cDNA ends; SF1, steroidogenic factor 1; SH3, Src homology domain-3. at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
The nuclear receptor superfamily comprises a large group of transcription factors that bind to the specific hormone-responsive elements and regulate the target gene expression in response to their cognate ligands (1,2). Transcription controlled by steroid hormone receptor plays a key role in many important physiological processes include cell growth, development, differentiation and metabolism. Besides binding to hormone responsive elements and their ligands, the subsequent recruitment of coregulator proteins is required for nuclear receptor to initiate the transcription of their target genes. These regulatory cofactors include coactivators and corepressors that enhance or inhibit transactivation activity of nuclear receptor, respectively (3,4). The best-studied coactivators are of the steroid receptor coactivator (SRC) family. Most of these coactivators of
nuclear receptors have molecular weights of ~160 kDa and interact with the liganded nuclear receptors using the NR-box or LXXLL-motif (5).
PNRC (Proline-rich nuclear receptor coregulatory protein (6), and PNRC2 (7) were identified as SF-1 (steroidogenic factor 1)-interacting proteins in a yeast two-hybrid screening of a human mammary gland cDNA expression library. These proteins were found to interact with the ligand-binding domains of all the nuclear receptors tested, including ERα, ERβ, PR, GR, TR, RAR, and RXR, in a ligand-dependent manner. They were also found to interact in a ligand-independent manner with orphan receptors such as SF1 and ERR. Furthermore, PNRC2 and PNRC were also repeatedly isolated as multiple nuclear receptor interacting proteins in a large interaction-screening project using an automated version of the yeast two-hybrid system (8). In their screening, PNRC was isolated as the interacting partner of RARβ, RARγ, RORα, RORβ, HNF4α, HNF4γ, ERβ, ERRγ, and LRH1. In the same study, PNRC2 was isolated as a prey with the ligand-binding domains of 10 different nuclear receptors baits, including ERα, ERβ, ERRγ, HNF4α, HNF4γ, at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
LRH1, PXR, RARγ, RORα, and RORβ. Thus, PNRC and PNRC2 appear to be more general cofactors for nuclear receptors than previously thought. These two coactivators are unique in that they are significantly smaller than most of the coregulatory proteins previously identified and are proline-rich. Unlike most of the coactivators that interact with nuclear receptors through its LXXLL motif, these new coactivators interact with nuclear receptors through a proline-rich Src homology domain-3 (SH3)-binding motif, S-D (E)-P-P-S-P-S (9).
In addition to functioning as a nuclear receptor coactivator, PNRC also plays a role in the growth factor/Ras-signaling pathway through its interaction with Grb2, a central adaptor protein in Ras pathway (10). MCF-7 cells that overexpress PNRC protein were found to grow slower than control cells, and the activation of Ras and MAP kinase in these PNRC overexpressing cells were found to be
down regulated. Furthermore, PNRC was recently demonstrated to stimulate RNA pol III transcription through its interaction with the subunit RPC39 of RNA pol III (11). PNRC is a unique coactivator that has profound effects on many aspects of cellular function by directly influencing both RNA pol II- and RNA pol III- dependent transcription.
Significantly reduced PNRC mRNA expression in stomach, colorectal, and hepatocellular carcinomas (12) and in breast cancer (10)was observed in comparison with the corresponding non-cancerous tissues. Furthermore, the reduced expression of PNRC was observed in association with hepatitis B virus infection of HCC patients (12). In addition, time-dependent induction of mRNA expression of PNRC was observed during 5-azacytidine treatment. These data suggest a possibility of PNRC as a tumor-related gene and that the regulation of PNRC expression plays a role in human multistage carcinogenesis. However, very little is known about the regulation of the expression of PNRC genes itself. To better understand the molecular mechanisms that regulate at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
the expression of PNRC gene, in this study, we have cloned and characterized the 5′ flanking region
of the human PNRC gene. Potential transcriptional start site was determined by 5′ RACE analysis. The minimal promoter region of the human PNRC gene was mapped in the region from residue -123 to +27. The transcriptional activity of human PNRC promoter was found to be regulated negatively by transcription factor NFY through binding NFY putative binding site within PNRC promoter region.
MATERIALS AND METHODS
Database Searches—The cDNA sequence of the human PNRC (NM 006813) was obtained from the National Center for Biotechnology Information (NCBI) (website: https://www.doczj.com/doc/bd5678886.html,). The genomic organization of the human PNRC and the 5′-flanking region sequence of PNRC were
obtained from the UCSC human gene browser at https://www.doczj.com/doc/bd5678886.html,/ cgi-bin/hgBlat by Blat research using the PNRC cDNA sequence (NM 006813) against the human genome database. The transcription factor binding sites in the promoter region was analyzed using the TRANSFAC Professional software in the Biobase Biological Database (website: www. biobase. de/ pages / products/ databases.html # t ransfac).
Plasmids Construction — The human PNRC 5′ flanking fragments of different lengths were amplified by PCR using HepG2 cell genomic DNA as template with the oligonucleotide primers, which were designed according to the sequences from human genome database and contained Kpn I and Bgl II restriction site at 5′ and 3′ ends, respectively. The PCR products were digested with Kpn I and Bgl II and cloned into the Kpn I and Bgl II site of the luciferase reporter plasmid, pGL3-Basic (Promega). The orientations and the sequences of the inserts were verified by restriction digestion and sequencing. at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
RNA Ligase-mediated Rapid Amplification of cDNA Ends —Total RNA was isolated from HepG2 cells using TRIzol reagent (Invitrogen). The transcriptional start point was determined using RLM-RACE kit (Ambion) according to manufacturer’s instructions. Briefly, in order to select full-length mRNAs, 10μg total human RNA is treated with Calf Intestine Alkaline Phosphatase (CIP) to remove free 5'-phosphates from molecules such as ribosomal RNA, fragmented mRNA, tRNA, and contaminated genomic DNA. Then, the RNA is treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from the capped mRNAs and leave a terminal 5′ -monophosphate. A 45 base RNA adapter oligonucleotide is ligated to the RNA population using T4 RNA ligase. During the ligation reaction, the majority of the full length decapped mRNA acquires the adapter sequence as its 5′ end. After random-primed reverse transcription reaction, the cDNA was amplified by nested PCR
using two forward primers provided by RLM-RACE kit, 5′ -RACE outer primer and 5′ -inner primer, corresponding to the 5′ RACE adapter sequence and the following two specific reverse primers for human PNRC gene, outer primer, 5′-ACC TTC TTC TTT CGC CGC TTC TT - 3′ and inner primer, 5′-GTC CGG GAT CCG GGG TAA AG -3′, respectively. After the PCR is complete, run 5–10 μl of each sample in a 2% high resolution agarose gel containing 1 μg/ml EtBr and visualize on a UV transilluminator. The PCR products then were cloned into pCRIITOPO TA cloning vector (Invitrogen) and were sequenced.
Cell Culture, Transient Transfections and Luciferase Assays — HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and antibiotics including 100U of penicillin/ml, 100 mg of streptomycin sulfate/ml. Twenty-four hours before transfection, cells were seeded in 12-well plates at 1×105/per well. Transfect cells using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Each well of cells was at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
transfected with 2 μg Lipofectamine 2000 and 1 μg of either pGL3-Basic (as no promoter control) or luciferase reporter containing the 5′ flanking fragments of human PNRC gene. The cells were also transfected with 0.2 μg pSV-β-Galactosidase (Promega) to correct variation in transfection efficiency. In co-transfection experiments, 1 μg of NFY expression vector was used.
Luciferase activity was assayed 24 h after transfection. Cells were washed twice with PBS. Then the cells were lysised with 200 μl of 1×reporter lysis buffer (Promega). The lysate was centrifuged at 13,000 rpm for 20 s to pellet the cell debris. The supernatants were transferred to a fresh tube and their β-Galactosidase activities were determined using the β-Galactosidase enzyme assay reagent according to the manufacturer’s protocol (Promega). The luciferase activities were measured using Luciferase assay substrate (Promega). All transfection data were expressed as the ratios of luciferase
activities / β-Galactosidase activities. Each transfection was performed in triplicate.
Site-directed Mutagenesis — Point mutations of CCAAT box (NFY binding site) was generated using QuikChange Site-Directed Mutagenesis Kit (Stratagene). Mutant constructs were generated by mutating two or five bases indicated by the lower case letters in primers. For mutagenesis of CCAAT box (change ATTGG to AAAGG, CA-Mut), primers were 5′ -GCA CTC TAA aa G GGC TTT CGC AGG-3′ and 5′ -CCT GCG AAA GCC C tt TTA GAG TGC - 3′ ; for inverse CCAAT box (change ATTGG to CCAAT, CA-Inverse), primers were 5′ -GCA CTC T ac caa t GC TTT CGC AGG - 3′ and 5′ -CCT GCG AAA G ca ttg g TA GAG TGC- 3′.
Electrophoresis Mobility Shift and Supershift Assay—Nuclear extracts were prepared from HepG2 cells as previously described (13). Protein concentration was determined using Dye Reagent Concentrate (Bio-Rad). A fragment of 5’ flanking region of the PNRC gene, -123/+27, that contains the NFY binding site, was amplified by PCR and used as a probe in the gel-shift and supershift assays. at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
This probe was labeled with [γ-32P-ATP] in the presence of T4 polynucleotide kinase (New England Biolabs) and purified using a ProbeQuant G-50 column (Amersham).
Binding reactions were performed in a 15 μl reaction mixture containing Gel Shift Binding 5×Buffer (5mM MgCl2, 2.5mM EDTA, 2.5mM DTT, 250mM NaCl, 50mM Tris-HCl (pH7.5), 25mg/ml poly(dl-dc), 20% glycerol) and 5 μg of nuclear extract. In the competition experiments, unlabeled competitors as indicated in Fig. 4 were added to the reaction mixtures. The mixtures were incubated at room temperature for 10 minutes. In the supershift experiments, four μg of a polyclonal antibody against NFYA (Santa Cruz Biotechnology, CA) or 4 μg of control normal IgG was added to the reaction mixtures and incubated on ice for 30 min. After binding reactions, the 6000 cpm of 32P-labeled probe was added to the mixture and the mixtures were incubated at 25℃for another 20
min. After addition of 1μl of gel loading 10× buffer per reaction, the reaction products were analyzed on a 4% nondenaturing polyacrylamide (59 : 1 acrylamide : bisacrylamide) gel. At the end of electrophrosis, the gel was dried in gel dryer and the dried gel was exposed to X-ray film (Kodak) overnight at –70°C with intensifying screen.
Chromatin Immunoprecipitation Assay —ChIP assay was performed using ChIP Assay kit (Upstate) according to the manufacturer’s instructions. Briefly, approximately 106 cells were treated with 1% formaldehyde (final concentration, v/v) in cell culture medium for 10 min at 37 ℃ to cross-link proteins to DNA. The medium was aspirated and the cells were washed twice with ice cold PBS containing protease inhibitor cocktail (Roche Molecular Biochemicals). The cells were harvested by centrifugate for 4 minutes at 2000 rpm at 4°C, and resuspended in 200 μl SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl (pH 8.1)), and protease inhibitor cocktail) and incubated on ice for 10 min. To shear DNA to lengths between 200 and 1000 basepairs, the cell lysate was at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
sonicated with a Digital Sonifier (Dranson Ultrasonics Corporation, CT) at 30% of maximum power 10 times for 15s each, with 2 min of cooling on ice in between sonications.
The sonicated DNA was diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl (pH 8.1), 167 mM NaCl, and protease inhibitor cocktail). A portion of the diluted cell supernatant 1% (~20 μl) was kept as input for PCR. The rest of the supernatant was pre-cleared by incubation with protein A Sepharose beads for 30 minutes at 4°C with agitation to reduce nonspecific background. Agarose was pelleted by brief centrifugation and the supernatant fraction was transferred into a fresh tube. The antibodies against NFYA (2 μg each) was added to the supernatant fraction and incubated overnight at 4°C with rotation. The negative controls, with no antibody immunoprecipitation or normal rabbit IgG immunoprecipitation,
were included. Immunocomplexes were precipitated for 2 h with protein A Sepharose beads. Then, the beads containing specific precipitates were collocted and washed once with 1 ml of low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1), 150 mM NaCl), once with 1 ml of high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.1)), and 500 mM NaCl), once with LiCl wash buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris, pH 8.1), and twice with TE buffer. All washes were for 5 min, rotating at 4 °C. The protein/DNA complexes were eluted from beads with 250 μl of elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature for 15 minutes with rotation. Formaldehyde cross-links were reversed by overnight incubation at 65°C and the proteins subsequently degraded by proteinase K. DNA was recovered by phenol–chloroform extraction and ethanol precipitation in the presence of 0.3 M sodium acetate and 20 μg glycogen. PCR amplification of a 150 bp (-123/+27) of the human PNRC promoter region, at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
which harbors the NFY-binding site, was performed using a sense primer, 5′-CGGAGGCGGAGCGAGG-3′, and an anti-sense primer, 5′ - TGACTCGGGCGGTGGC - 3′.
For additional control of non-specific binding, another PCR amplification reaction of the anti-NFY precipitated chromatin DNA was also performed to detect a 150-bp fragment that is corresponding to Exon II of the human PNRC gene and locates about 2.6 kb downstream from the -123/+27 promoter region. This control PCR reaction was performed using a sense primer, 5′ -TAG TCA CTG GAT GGG AAG CAC-3′, and an anti-sense primer, 5′ -AAG AGT CCA GGG ATA TGG GAA-3′. PCR was performed using Hot StarTaq DNA polymerase (Qiagen) as following conditions: Initial denaturation for 15 min at 95°C followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 57 °C for 30 sec, extension at 72 °C for 30 sec, ending with a final 7 min
extension at 72 °C. The PCR products were resolved electrophoretically on a 2% agarose gel and visualized with EtBr staining.
Western Blot Analysis —Fiftyμg nuclear extract was analysed by 12% SDS–PAGE. Proteins were transferred to PVDF Membrane and the membrane was blocked 1 h with 5% milk in TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20). The membrane was then incubated with the affinity purified rabbit anti-NFY (1:100) IgGs or anti-β-actin antibody (1:2000) at 4 °C over night, and HRP-conjugated goat anti- rabbit antibodies for an additional 1 h at room temperature. After washing three times in TBST, enhanced chemiluminescence reagents (ECL) were used for the final detection.
RESULTS
Genomic Organization of the Human PNRC Gene —Human PNRC mRNA sequence (NM 006813) which was obtained by searching GenBank was used to perform human genome blat at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
searching by using UCSC human genome browser to obtain information regarding the genomic organization of the human PNRC gene. Through alignment of human genomic sequence with human cDNA sequence (NM 006813), the human PNRC gene is defined to be located on chromosome 6 and comprised of 2 exons and 1 intron: 657 bp-exon I, 1048 bp-exon II, and 2319 bp- intron I. The translational start site is located in exon I. The 984 bp coding sequence in exon I and exon II encode 327 amino acids (Fig. 1A). Each intron begins with a GT and ends with an AG, confirming to the GT/AG rule.
Determination of the Transcription Start Site of Human PNRC Gene —Ambion’s First Choice 5′RLM-RACE kit was used to determine the transcription initiation site of human PNRC gene of in HepG2 cells (Fig. 2). The relative positions of all the primers used for 5′ RACE analysis were
shown in Fig. 2A. The results of the analysis of 5′ RACE are shown in Fig. 2B. A 300 bp fragment was obtained from nested PCR reactions using the 5′ RACE adapter inner primer and PNRC reverse inner primer pair and cDNA template derived from tobacco acid pyrophosphatase (TAP) treated HepG2 cells RNA (Fig. 2B Lane 2). No product was obtained when RNA was not treated with TAP (lane 3), indicating that the 300 bp product was derived from full-length mRNA. As a control to validate the assay, the expected 248-bp (lane 4) and 412-bp product (lane 5) were obtained from cDNA template that derived from TAP treated RNA using human PNRC primers, control forward primer 5′ -TCT TCT CAG GCT CTC CTA GCA GCA T- 3′ and reverse inner primer 5′ -ACC TTC TTC TTT CGC CGC TTC TT- 3′ or reverse outer primer 5′ -ACC TTC TTC TTT CGC CGC TTC TT- 3′, respectively. A sample of the outer PCR using 5′ RACE outer primer and PNRC outer primer (lane 1) was also run for evaluation. The 300 bp PCR product was subcloned into pCRIII-TOPO TA cloning vector (Invitrogen) and the resulting clones containing the PCR inserts at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
were sequenced. The result showed that the major human PNRC transcription initiation site was mapped at the nucleotide G, twenty-seven nucleotide more 5′ upstream from the 5′ end of the longest PNRC EST in available databases. This major transcription initiation site is indicated in bold letter and is designed +1 (Fig. 1B).
Identification and Characterization of the Human PNRC Gene Promoter —To map the minimal promoter activity and the regulatory sequences, a series of luciferase reporter constructs containing various 5′ flanking region deletions of PNRC gene were generated (Fig. 3, left panel) by PCR, based on the information of both transcription factor binding site and the PNRC gene transcription start site. Promoter activity for each PNRC gene fragment is shown in the right panel of Fig. 3. Transfection of the longest PNRC fragment in pGL3hPNRC (-1373/+727) reporter construct into HepG2 cells
resulted in a luciferase activity near to that of empty pGL3 Basic reporter vector (no promoter, no enhancer). Deleting the region from –1373 to –724 , –723 to –324 and -323 to –124, (construct pGL3hPNRC (-723/+727) , pGL3hPNRC (-323/+727), pGL3hPNRC (-123/+727)) resulted in 2-fold, 4-fold and 6.3-fold increase in the promoter activity, suggesting that the region from-1373 to -123 harbors negative response elements. Further deletion of the sequence from -123 to –24 (construct pGL3hPNRC2 (-23/+727)) resulted in the complete lose of the promoter activity, suggesting that the region of -123 to -23 contains the core elements necessary for the basal promoter function of the human PNRC gene. Several constructs with the deletion from 3′end were also generated. Deletion of the region +727 to +327 (construct pGL3hPNRC (-123/+327)) resulted in 16.6 -fold increase in the promoter activity. Deletion of the region +327 to +57 (construct pGL3hPNRC (-123/+57)) resulted in 120 -fold increase in the promoter activity repectively, further deleted the sequence from +57 to +27 (construct pGL3hPNRC (-123/+27)) resulted in 86 -fold increase in the promoter activity, repectively. at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
These results suggest that the region from +727 to +27 harbor negative response elements. When further deletion of +27 to -23 (construct pGL3hPNRC (-123/-23)), this deletion almost completely abolished the promoter activity. Taken together, our deletion and transfection results demonstrated that the minimal human promoter activity is located in the region of -123/+27.
Specific Binding of NFY Protein to the Human PNRC Promoter in vitro and in vivo—As shown in Fig. 1B, region spanning -123/+27 harbors one CCAAT box (NFY-binding site). To identify protein interacting with the -123/+27 sequences, EMSA was performed by using the nuclear extract prepared from HepG2 cells and a probe spanning -123/+27 that contains the NFY-binding site. As shown in Fig. 4, at least five DNA/protein complexes, marked as A, B, C, D and E were formed between the probe and the nuclear proteins in HepG2 cells (lane 2). All five of the complexes were
competed by the addition of a 50-fold molar excess of unlabeled probe DNA (lane 3), but not competed by non-specific competitor DNA, poly dI-dC (lane 6), demonstrating that all the complexes are the sequence-specific complexes between –123/+27 DNA and the nuclear proteins. These complexes could also be competed by the oligonucleotide that contains wild type inverted CCAAT box sequence (i.e., NFY-binding site, lane 5), but not competed by the mutated CCAAT oligonucleotide (mutCA: 5′- TCG CAC TCT AAAAGG GCT TTC GCA G-3′) (lane 4), indicating that NFY protein present in HepG2 cells binds to the probe used in the gel shift assay. The binding of NFY protein was further confirmed in a supershift reaction. The addition of anti-NFY antibody to the binding reaction furthermore caused a lose of complex C, a density increase of complexes D and E, and a supershift band F (lane 8). As expected, normal mouse IgG did not compete any of the complexes (lane 7). at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
To determine whether NFY interact with the human PNRC promoter in vivo, we performed ChIP assay (Fig. 5). Proteins were cross-linked to genomic DNA in HepG2 cells using 1% formaldehyde, then the chromatin was sonicated and immunoprecipitated with normal rabbit IgG, or polyclonal antibody NFYA. Before the immunoprecipitations, a small aliquot of chromatin was saved and used as an input control. The precipitated DNA was subjected to PCR utilizing primers designed to amplify a 150 bp fragment (-123/+27) of the 5′ flanking region of the human PNRC gene. As shown in Fig. 5, the 150-bp DNA fragment was only amplified from the precipitates by anti-NFYA (lane 1) and the input chromatin DNA (positive control) (lane 2). In contrast, the PNRC promoter fragment was not amplified from negative control immunoprecipitations using no antibody (lane 3) or normal rabbit IgG (lane 4, very weak band). To further rule out the possibility that the PNRC promoter region
precipitated by anti-NFY antibody is due to a non-specific binding of NFY antibody, an additional PCR amplification of a distinct genomic region 2.6 kb downstream of PNRC promoter was performed on the anti-NFY precipitated chromatin DNA. As shown in Fig. 5, lane 5, this region was not precipitated by anti-NFY antibody, demonstrating that NFY specifically bind to the PNRC promoter region. To validate that the DNA fragment amplified was the expected 150-bp area of the human PNRC promoter region, this fragment was subcloned into TA cloning vector and then sequenced. Sequence data confirmed that the PCR product has the exact sequence of PNRC promoter containing fragment, -123/+27 (data not shown). These results demonstrated the recruitment of NFY transcriptional factor to the human PNRC promoter in vivo.
Regulation of PNRC Transcriptional Activity by NFY —After demonstration of the specific binding in vitro and in vivo of NFY to the promoter region of the human PNRC gene, the function of the binding by this transcription factor was also checked. The NFYs expression vector was at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
co-transfected along with the PNRC promoter luciferase reporter plasmids into HepG2 cells. As shown in Fig. 6A, cotransfection of pGL3hPNRC (-123/+57) reporter plasmid and NFYA expression plasmid decreased the activity of PNRC promoter by 2.0-fold, while NFYB or NFYC did not affect, or only slightly decreased, the promoter activity. When HepG2 cells were cotransfected with the expression plasmids for all three NFY subunits along with the pGL3hPNRC (-123/+57) reporter plasmid, the luciferase activity in these transfected cells was 3.9-fold lower than that in the cells transfected with human PNRC promoter alone. Furthermore, NFY inhibited the luciferase activity driven by the human PNRC promoter in a dose-dependent manner (Fig. 6B). However, cotransfection of the NFY expression plasmids along with reporter plasmid that contains the mutated NFY binding site (CA-Mut and CA-Inverse) did not inhibit, rather slightly stimulated, the luciferase activity (Fig.
6C). These results demonstrated that NFY functions as a transcriptional repressor in PNRC transcription through the binding to the CCAAT sequences within PNRC promoter region.
Over-expression of NFY decreases PNRC expression levels in vivo —To investigate whether over-expression of NFY regulate PNRC expression levels in vivo, HepG2 cells were transiently transfected with the expression plasmid of NFY. The levels of PNRC protein in transfected HepG2 cells were then analyzed by Western blot. Contrast to the group that transfected with the empty vector, pCMV, the level of PNRC expression was decreased in the group that transfected with the expression plasmid of NFY (Fig. 7). These results further demonstrated that NFY negtively regulates the expression of PNRC in HepG2 cells. at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
DISCUSSION
PNRC is a unique nuclear receptor coactivator that interacts with nuclear receptors through its SH3-binding motif. It plays an important role in regulation of gene transcription. Therefore, it is important to understand the mechanisms of PNRC gene expression. In this study, we have identified and characterized the human PNRC promoter and found that one important transcription factor, NFY, regulated the promoter activity of the human PNRC gene.
After the transcriptiona l start site was verified, the 5′ flanking region of the human PNRC gene was amplified and analyzed in detail for minimal promoter localization utilizing a series of 5′ and 3′ deletions of the PNRC 5′ flanking region fused upstream of the firefly luciferase reporter gene. These experiment identified the minimal promoter region, -123/+27, that is
necessary for the expression of human PNRC gene. Sequence analysis of -123/+27 region revealed that there is an inverted CCAAT box sequences (NFY binding site) (Fig. 1B). We therefore determine to examine whether transcriptional factor NFY bind to its consensus sequences in PNRC gene and thereby regulate the transcription of PNRC gene. One technique that is central in studying gene regulation and determining protein/ DNA interactions is the electrophoretic mobility shift assay (EMSA). To verify the specific binding of the NFY transcription factor to the PNRC promoter -123/+27 region, we carried out the EMSA analysis. Our results from EMSA demonstrated that there were five sequence-specific DNA-protein complexes caused by the binding of specific protein to PNRC promoter region as shown in Figure 4, lane 2, and some of these complexes were probably caused by the binding of NFY as that the oligos carrying the mutated NFY -binding site did not compete the binding. We understand that the change in relative mobility does at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
not identify the bound proteins. Therefore, we have carried out the supershift assays using NFY antibody to confirm the binding of this protein to the promoter region. In addition to forming a relative less clear, less sharp supershift band F (Fig. 4), the addition of antibodies repeatedly caused the disappearance of the complex C and density increase of complexes D and E.
ChIP analysis is a powerful assay that define the interaction or the recruitment of transcription factors with specific chromatin sites in living cells, thereby providing a snapshot of the native chromatin structure and factors bound to genes in different functional stages. In our ChIP assays, NFY was demonstrated to associate with the DNA sequence of -123/+27 of the human PNRC gene in HepG2 cells (Fig.5). Taken together the results from gel shift
binding and supershift assay, ChIP analysis, and mutagenesis/ luciferase assay, transcription factor NFY was demonstrated to bind to their binding site in the promoter region of the human PNRC gene.
To address the function of the NFY binding to PNRC promoter region, we utilized a transient cotransfection assay with PNRC promoter region containing NFY binding site along with the expression plasmid for NFYs. NFY was found to suppress the promoter activity of the human PNRC gene (Fig. 6). We also found over expression of NFY in HepG2 cells that decreases the expression of PNRC protein in vivo (Fig. 7).
NFY is a ubiquitous transcription factor which binds with high specificity to CCAAT motifs in the promoters of genes. NFY consists of three subunits, NFYA, NFYB and NFYC, all of which are essential for DNA binding. The NFYB and NFYC subunits form a tight heterodimer that offers a complex surface for NFYA association. The N-terminal part of NFYA is required for association with at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
the NFYB/NFYC heterodimer, while the NFYA C-terminus is required for DNA binding (14). Therefore, NFYA represents the regulatory subunits of the NFY trimer (15). In this study, NFYA was found to be the dominant subunit in repressing the promoter activity of human PNRC gene.
It has been suggested that binding of NFY factor to the promoter alone is not able to exert its effect on transcription (16), unless it interacts with relevant protein factors, including Sp1 (17), c-myb (18), SREBP-1 (19), and GATA-1 (20). NFY functions as both an activator and a repressor, depending on its interacting cofactors. The activation effect of NFY can be mediated through its direct interaction with histone acetylases (21), such as p300 (22), or by facilitating the binding of TFIID to the core promoters (23, 24). NFY also recruits transcriptional repressors to negatively regulate gene expression. For example, NFY directly recruits histone deacetylases (HDACs) to repress the
expression of the von Willebrand factor in non-endothelial cells (25). In our cotransfection experiment, NFY was shown to repress the promoter activity of human PNRC gene.
There is an increasing evidence suggests a possibility of PNRC as a tumor-related gene and that the regulation of PNRC expression plays a role in human multistage carcinogenesis. In our study, the expression of PNRC was regulated by NFY protein, but the biological significance of this finding is not clear at the present time. Future experiments will be aimed at revealing the mechanism of regulation of PNRC expression, and the possible role of PNRC in the cell cycle progression and tumor suppression.
We are grateful to Dr. Shiuan Chen at Beckman Research Institute of City of Hope, Duarte, California, USA for his long term support of our research program, Dr. Roberto Mantovani at Universita di Milano, Milano, Italy for the expression plasmid of NFYA, YB, and YC. This work was supported by grants from National Natural Science Foundation of P.R. China (No.30671056 and 30672055). at The 3rd Military Medical University on December 3, https://www.doczj.com/doc/bd5678886.html, Downloaded from
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高二《甜美纯净的女声独唱》教案 一、基本说明 教学内容 1）教学内容所属模块：歌唱 2）年级：高二 3）所用教材出版单位：湖南文艺出版社 4）所属的章节：第三单元第一节 5）学时数： 45 分钟 二、教学设计 1、教学目标： ①、在欣赏互动中感受女声的音域及演唱风格，体验女声的音色特点。 ②、在欣赏互动中，掌握美声、民族、通俗三种唱法的特点，体验其魅力。 ③、让学生能够尝试用不同演唱风格表现同一首歌。 ④、通过学唱歌曲培养学生热爱祖国、热爱生活的激情。 2、教学重点： ①、掌握女高音、女中音的音域和演唱特点。 ②、掌握美声、民族、通俗三种方法演唱风格。 3、教学难点： ①、学生归纳不同唱法的特点与风格。
②、学生尝试用不同演唱风格表现同一首歌。 3、设计思路 《普通高中音乐课程标准》指出：“音乐课的教学过程就是音乐的艺术实践过程。”《甜美纯净的女声独唱》作为《魅力四射的独唱舞台》单元的第一课，是让学生在丰富多彩的歌唱艺术形式中感受出女声独唱以其优美纯净的声音特点而散发出独特的魅力。为此，本课从身边熟悉的人物和情景入手，激发学生学习兴趣，把教学重心放在艺术实践中，让学生在欣赏、学习不同的歌唱风格中，培养自己的综合欣赏能力及歌唱水平。在教学过程中让学生体会不同风格的甜美纯净女声的内涵，感知优美纯净的声音特点而散发出的独特魅力，学会多听、多唱，掌握一定的歌唱技巧，提高自己的演唱水平。为实现以上目标，本人将新课标“过程与方法”中的“体验、比较、探究、合作”四个具体目标贯穿全课，注重学生的个人感受和独特见解，鼓励学生的自我意识与创新精神，强调探究、强调实践，将教学过程变为整合、转化间接经验为学生直接经验的过程，让学生亲身去感悟、去演唱，并力求改变现在高中学生普遍只关注流行歌曲的现状，让学生自己确定最适合自己演唱的方法，自我发现、自我欣赏，充分展示自己的的声音魅力。 三、教学过程 教学环节及时间教师活动学生活动设计意图
The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法） 举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法： 在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because
适合女生KTV唱的100首好听的歌别吝色你的嗓音很好学 1、偏爱----张芸京 2、阴天----莫文蔚 3、眼泪----范晓萱 4、我要我们在一起---=范晓萱 5、无底洞----蔡健雅 6、呼吸----蔡健雅 7、原点----蔡健雅&孙燕姿 8、我怀念的----孙燕姿 9、不是真的爱我----孙燕姿 10、我也很想他----孙燕姿 11、一直很安静----阿桑 12、让我爱----阿桑 13、错过----梁咏琪 14、爱得起----梁咏琪 15、蓝天----张惠妹 16、记得----张惠妹 17、简爱----张惠妹 18、趁早----张惠妹 19、一念之间----戴佩妮 20、两难----戴佩妮 21、怎样----戴佩妮 22、一颗心的距离----范玮琪 23、我们的纪念日----范玮琪 24、启程----范玮琪 25、最初的梦想----范玮琪 26、是非题----范玮琪 27、你是答案----范玮琪 28、没那么爱他----范玮琪 29、可不可以不勇敢----范玮琪 30、一个像夏天一个像秋天----范玮琪 31、听，是谁在唱歌----刘若英 32、城里的月光----许美静 33、女人何苦为难女人----辛晓琪 34、他不爱我----莫文蔚 35、你是爱我的----张惠妹 36、同类----孙燕姿 37、漩涡----孙燕姿 38、爱上你等于爱上寂寞----那英 39、梦醒了----那英 40、出卖----那英 41、梦一场----那英 42、愿赌服输----那英
43、蔷薇----萧亚轩 44、你是我心中一句惊叹----萧亚轩 45、突然想起你----萧亚轩 46、类似爱情----萧亚轩 47、Honey----萧亚轩 48、他和他的故事----萧亚轩 49、一个人的精彩----萧亚轩 50、最熟悉的陌生人----萧亚轩 51、想你零点零一分----张靓颖 52、如果爱下去----张靓颖 53、我想我是你的女人----尚雯婕 54、爱恨恢恢----周迅 55、不在乎他----张惠妹 56、雪地----张惠妹 57、喜欢两个人----彭佳慧 58、相见恨晚----彭佳慧 59、囚鸟----彭羚 60、听说爱情回来过----彭佳慧 61、我也不想这样----王菲 62、打错了----王菲 63、催眠----王菲 64、执迷不悔----王菲 65、阳宝----王菲 66、我爱你----王菲 67、闷----王菲 68、蝴蝶----王菲 69、其实很爱你----张韶涵 70、爱情旅程----张韶涵 71、舍得----郑秀文 72、值得----郑秀文 73、如果云知道----许茹芸 74、爱我的人和我爱的人----裘海正 75、谢谢你让我这么爱你----柯以敏 76、陪我看日出----蔡淳佳 77、那年夏天----许飞 78、我真的受伤了----王菀之 79、值得一辈子去爱----纪如璟 80、太委屈----陶晶莹 81、那年的情书----江美琪 82、梦醒时分----陈淑桦 83、我很快乐----刘惜君 84、留爱给最相爱的人----倪睿思 85、下一个天亮----郭静 86、心墙----郭静
The way的用法及其含义（二） 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中
定冠词the的用法： 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...
2019-2020年高一音乐甜美纯净的女声独唱教案 一、教学目标 1、认知目标：初步了解民族唱法、美声唱法、通俗唱法三种唱法的风格。 2、能力目标：通过欣赏部分女声独唱作品，学生能归纳总结出她们的演唱 风格和特点，并同时用三种不同风格演唱同一首歌曲。 3、情感目标：通过欣赏比较，对独唱舞台有更多元化的审美意识。 二、教学重点：学生能用三种不同风格演唱形式演唱同一首歌。 三、教学难点：通过欣赏部分女声独唱作品，学生能归纳总结出她们的演唱 风格和特点。 四、教学过程： （一）导入 1、播放第十三界全国青年歌手大奖赛预告片 （师）问：同学们对预告片中的歌手认识吗 （生）答： （师）问：在预告片中提出了几种唱法？ （生）答：有民族、美声、通俗以及原生态四种唱法，今天以女声独唱歌曲重点欣赏民族、美声、通俗唱法，希望通过欣赏同学们能总结出三种唱法的风格和特点。 （二）、音乐欣赏
1、通俗唱法 ①(师)问：同学们平常最喜欢唱那些女歌手的歌呢？能唱唱吗？ （可让学生演唱几句喜欢的歌，并鼓励） ②欣赏几首通俗音乐 视频一：毛阿敏《绿叶对根的情谊》片段、谭晶《在那东山顶上》片段、韩红《天路》片段、刘若英《后来》片段 视频二：超女《想唱就唱唱得响亮》 ①由学生总结出通俗音乐的特点 ②师总结并板书通俗音乐的特点：通俗唱法是在演唱通俗歌曲的基础上发展起来的，又称“流行唱法”。通俗歌曲是以通俗易懂、易唱易记、娱乐性强、便于流行而见长，它没有统一的规格和演唱技法的要求，比较强调歌唱者本人的自然嗓音和情绪的渲染，重视歌曲感情的表达。演唱上要求吐字清晰，音调流畅，表情真挚，带有口语化。 ③指出通俗音乐尚未形成系统的发声训练体系。其中用沙哑、干枯的音色“狂唱”和用娇柔、做作的姿态“嗲唱”，不属于声乐艺术的正道之物，应予以摒弃。 2、民族唱法 ①俗话说民族的才是世界的那么民族唱法的特点是什么呢？ ②欣赏彭丽媛《万里春色满人间》片段 鉴赏提示：这首歌是剧种女主角田玉梅即将走上刑场时的一段难度较大的咏叹调。
“tｈｅwａｙ+从句”结构的意义及用法 首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。 下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句 例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法
不对。 Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.
表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。 如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如： 我不喜欢他说话的态度。 正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。 一、in a way的用法 表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。 二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。 三、by the way的用法
女生唱的歌曲欢快甜美 美妙的歌曲能令我们陶醉其中而无法自拨，最激烈的歌曲能令我们的身体不由自主的跟着手舞足蹈起来，下面是小编整理的欢快甜美的歌曲的内容，希望能够帮到您。 欢快甜美的歌曲 1. Talking - 2. 羽毛- 劲歌金曲 3. 为你- 黑龙 4. 我的小时候- 罗艺达 5. 听说爱情回来过 6. 那个男人 7. 夫妻观灯_韩再芬、李迎春- 中国民歌宝典二 8. 往生- 镀飞爱在阳光空气中- 区瑞强- 音乐合辑 9. 说中国- 班- 华语群星 10. 第十八封信- Kent王健 11. 那一夜你喝了酒- 傅薇 12. 最近比较烦- 周华健/李宗盛/品冠- 滚石群星 13. 告白- 张娜拉 14. Talking VIII - 15. my love - 网友精选曲 16. 音乐人民- 音乐合辑 17. 深深深深- 徐誉滕 18. Honkytonk U - Toby Keith 19. 征服- 阿强 20. 我总会感动你- 沙宝亮欢快甜美的歌曲 1. 一千步的距离- 高桐 2. fleeing star - 音乐合辑 3. My Life - 李威杰 4. 小妹听我说- 金久哲 5. 上海滩- 梁玉嵘- 华语群星 6. 爱我多爱一些- 黎姿 7. 恋人未满- 8. 玛奇朵飘浮- 音乐听吧 9. 風- 音乐听吧 10. 七月- 小鸣 11. 滚滚红尘- 罗大佑 12. 张震岳—想要- 华语群星 13. 有梦有朋友- 14. 童年- 拜尔娜 15. 洪湖水，浪打浪- 宋祖英 16. 只爱到一半- 魏晨 17. 风雨人生路- 何静 18. 居家男人- 回音哥如果当时- 许嵩 19. 那个男人的谎言Tae In - 非主流音乐
The way的用法及其含义（一） 有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析： 一、the way作先行词，后接定语从句 以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.
【适合女生唱的各种难度的歌】以后点歌的时候记得挑战一下自己（哈哈，今天心情高兴，在微博整理下的小东西和大家分享） 1.我不知道--唐笑（特别喜欢的一首歌） 2.那个--文筱芮（特别伤的歌，真的可以听到心里去） 3.一半--丁当（喜欢喜欢，但没能力唱） 4.指望--郁可唯（本来不喜欢她，但她唱歌挺有水平） 5.路人--江美琪（推荐，好听又挺好唱的） 6.过敏--杨丞琳（听听就知道了） 7.大女人--张亚飞（没什么名气的超级女生，这歌挺棒的） 8.一个人的星光--许静岚（绿光森林的主题曲） 9.不要说爱我--许紫涵（高潮真的挺好听，我爱单曲循环，但这歌还没腻） 10.为你我受冷风吹--林忆莲（没那么简单，都是很喜欢的老歌，偶尔听听老歌感觉特别好） 11.一秒也好--卓文萱（她的（爱我好吗）也不错，最近挺喜欢她的歌） 12.你在哪里--张婧（不被太多人知道的歌手） 13.你的背包--莫艳琳（在校内看到一个女孩唱的，觉得挺好听的） 14.原来爱情那么难--泳儿（好听好听，没什么难度，就是在ktv不太好找） 15.在你眼里--同恩（也是到副歌特别吸引人的一首）
16.很久很久以后--梁文音（爱她的歌，她的很多歌都特别好听） 17.知道我们不会有结果--金莎（听着特别有感觉，那些喜欢听悲伤歌的都是因为这种感觉吧） 18.指尖的星光--钟汶（不太好唱的，我就只有听的份了） 19.放不下--龚诗嘉（挺简单的一首，调调挺平的，她的（远远在一起）也不错） 20.灰色的彩虹--范玮琪 21.现在才明白--萧贺硕（不被太多人知道的歌手，有些歌真的很好听，只是需要慢慢挖掘） 22.终点--关心妍（这首歌大多都听过，自己感觉吧） 23.遇到--王蓝茵（旋律让人感觉特舒服的，很爱的一首） 24.一个人--蔡依（她的毅力不是一般人能做到的） 25.婴儿--陈倩倩（这首歌真的凄凉到有点甚人的感觉。“喜欢一个人的心情”--江语晨，因为这歌的词）26.那又怎么样呢--张玉华（我爱听音乐，但一定要是伤感的，虽然不会听到泪流满面，但是那种感觉真的很好） 27.还爱你--景甜（像这样好听，又不被大家熟悉的歌还有很多吧）
way 的用法 【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗？ 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。 【归纳总结】 ●way作“方法，方式”讲时，如表示“以……方式”，前面常加介词in。如例1； ●way作“方法，方式”讲时，其后可接不定式to do sth.，也可接of doing sth. 作定语，表示做某事的方法。如例2，例3；
分享适合女生KTV唱的100首好听的歌别吝色你的嗓音很好学 1、偏爱----张芸京 2、阴天----莫文蔚 3、眼泪----范晓萱 4、我要我们在一起---=范晓萱 5、无底洞----蔡健雅 6、呼吸----蔡健雅 7、原点----蔡健雅&孙燕姿 8、我怀念的----孙燕姿 9、不是真的爱我----孙燕姿 10、我也很想他----孙燕姿 11、一直很安静----阿桑 12、让我爱----阿桑 13、错过----梁咏琪 14、爱得起----梁咏琪 15、蓝天----张惠妹 16、记得----张惠妹 17、简爱----张惠妹 18、趁早----张惠妹 19、一念之间----戴佩妮 20、两难----戴佩妮 21、怎样----戴佩妮 22、一颗心的距离----范玮琪 23、我们的纪念日----范玮琪 24、启程----范玮琪 25、最初的梦想----范玮琪 26、是非题----范玮琪 27、你是答案----范玮琪 28、没那么爱他----范玮琪 29、可不可以不勇敢----范玮琪 30、一个像夏天一个像秋天----范玮琪 31、听，是谁在唱歌----刘若英 32、城里的月光----许美静 33、女人何苦为难女人----辛晓琪 34、他不爱我----莫文蔚 35、你是爱我的----张惠妹 36、同类----孙燕姿 37、漩涡----孙燕姿 38、爱上你等于爱上寂寞----那英 39、梦醒了----那英 40、出卖----那英 41、梦一场----那英 42、愿赌服输----那英
43、蔷薇----萧亚轩 44、你是我心中一句惊叹----萧亚轩 45、突然想起你----萧亚轩 46、类似爱情----萧亚轩 47、Honey----萧亚轩 48、他和他的故事----萧亚轩 49、一个人的精彩----萧亚轩 50、最熟悉的陌生人----萧亚轩 51、想你零点零一分----张靓颖 52、如果爱下去----张靓颖 53、我想我是你的女人----尚雯婕 54、爱恨恢恢----周迅 55、不在乎他----张惠妹 56、雪地----张惠妹 57、喜欢两个人----彭佳慧 58、相见恨晚----彭佳慧 59、囚鸟----彭羚 60、听说爱情回来过----彭佳慧 61、我也不想这样----王菲 62、打错了----王菲 63、催眠----王菲 64、执迷不悔----王菲 65、阳宝----王菲 66、我爱你----王菲 67、闷----王菲 68、蝴蝶----王菲 69、其实很爱你----张韶涵 70、爱情旅程----张韶涵 71、舍得----郑秀文 72、值得----郑秀文 73、如果云知道----许茹芸 74、爱我的人和我爱的人----裘海正 75、谢谢你让我这么爱你----柯以敏 76、陪我看日出----蔡淳佳 77、那年夏天----许飞 78、我真的受伤了----王菀之 79、值得一辈子去爱----纪如璟 80、太委屈----陶晶莹 81、那年的情书----江美琪 82、梦醒时分----陈淑桦 83、我很快乐----刘惜君 84、留爱给最相爱的人----倪睿思 85、下一个天亮----郭静 86、心墙----郭静
1、偏爱----张芸京 2、阴天----莫文蔚 3、眼泪----范晓萱 4、我要我们在一起---=范晓萱 5、无底洞----蔡健雅 6、呼吸----蔡健雅 7、原点----蔡健雅&孙燕姿 8、我怀念的----孙燕姿 9、不是真的爱我----孙燕姿 10、我也很想他----孙燕姿 11、一直很安静----阿桑 12、让我爱----阿桑 13、错过----梁咏琪 14、爱得起----梁咏琪 15、蓝天----张惠妹 16、记得----张惠妹 17、简爱----张惠妹 18、趁早----张惠妹 19、一念之间----戴佩妮 20、两难----戴佩妮 21、怎样----戴佩妮 22、一颗心的距离----范玮琪 23、我们的纪念日----范玮琪 24、启程----范玮琪 25、最初的梦想----范玮琪 26、是非题----范玮琪 27、你是答案----范玮琪 28、没那么爱他----范玮琪 29、可不可以不勇敢----范玮琪 30、一个像夏天一个像秋天----范玮琪 31、听，是谁在唱歌----刘若英 32、城里的月光----许美静 33、女人何苦为难女人----辛晓琪 34、他不爱我----莫文蔚 35、你是爱我的----张惠妹 36、同类----孙燕姿 37、漩涡----孙燕姿 38、爱上你等于爱上寂寞----那英 39、梦醒了----那英 40、出卖----那英 41、梦一场----那英 42、愿赌服输----那英 43、蔷薇----萧亚轩 44、你是我心中一句惊叹----萧亚轩
45、突然想起你----萧亚轩 46、类似爱情----萧亚轩 47、Honey----萧亚轩 48、他和他的故事----萧亚轩 49、一个人的精彩----萧亚轩 50、最熟悉的陌生人----萧亚轩 51、想你零点零一分----张靓颖 52、如果爱下去----张靓颖 53、我想我是你的女人----尚雯婕 54、爱恨恢恢----周迅 55、不在乎他----张惠妹 56、雪地----张惠妹 57、喜欢两个人----彭佳慧 58、相见恨晚----彭佳慧 59、囚鸟----彭羚 60、听说爱情回来过----彭佳慧 61、我也不想这样----王菲 62、打错了----王菲 63、催眠----王菲 64、执迷不悔----王菲 65、阳宝----王菲 66、我爱你----王菲 67、闷----王菲 68、蝴蝶----王菲 69、其实很爱你----张韶涵 70、爱情旅程----张韶涵 71、舍得----郑秀文 72、值得----郑秀文 73、如果云知道----许茹芸 74、爱我的人和我爱的人----裘海正 75、谢谢你让我这么爱你----柯以敏 76、陪我看日出----蔡淳佳 77、那年夏天----许飞 78、我真的受伤了----王菀之 79、值得一辈子去爱----纪如璟 80、太委屈----陶晶莹 81、那年的情书----江美琪 82、梦醒时分----陈淑桦 83、我很快乐----刘惜君 84、留爱给最相爱的人----倪睿思 85、下一个天亮----郭静 86、心墙----郭静 87、那片海----韩红 88、美丽心情----RURU
t h e-w a y-的用法
The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.
婴儿——陈倩倩 这首歌真的凄凉到有点儿甚人的感觉。“喜欢一个人的心情”——江语晨，因为这歌的词。 那又怎么样呢——张玉华 我爱听音乐，但一定要是伤感的，虽然不会听到泪流满面，但是那种感觉真的很好 还爱你——景甜 你可以爱我很久吗——游艾迪 夜夜夜夜——原唱齐秦 爱一直存在——梁文音 有人想找男生唱的，真不常听男生的歌，不过有几首觉得挺不错的。 初雪的忧伤——赵子浩 爱你，离开你——南拳妈妈 说谎——林宥嘉 三人游、爱爱爱——方大同 分开以后——唐禹哲 突然好想你——五月天 还是男生的， 寂寞的季节、暗恋——陶喆 情歌两三首——郭顶 掌纹——曹格 需要人陪——王力宏 王妃——箫敬腾（除了这首，其他几首都是比较安静抒情的。） 挥之不去——殷悦 别再哭了——罗忆诗 前段时间特别喜欢这首歌，听的快吐了，真的挺好听。 热气球——黄淑惠 很特别，超级好听，强烈推荐。 你是爱我的——张惠妹 她的嗓音让我着迷，超级喜欢。 问——粱静茹 老歌了，不过很喜欢。
忽略——萧萧 握不住的他，看到萧萧还是会第一个想起这首。 趁早——张惠妹 她有点儿沙哑的声音让我着迷。 幸运草——丁当 早点儿的歌了，喜欢丁当。 哭了——范晓萱 越听越喜欢，。 氧气——范晓萱 小时候喜欢听她的歌，不过随着年龄的增长，喜欢的类型也变了。 温柔的慈悲——阿桑 喜欢她的歌，只是她的声音不能再更新了。 礼物——刘力扬 罗美玲的也还好。。 洋葱——丁当杨宗纬（两个不一样的感觉） 挺难的唱不好，不过喜欢听。 眼泪知道——温岚 喜欢，唱出来特别有激情哈。
类似爱情——萧亚轩 不难又有感觉。 第三者——梁静茹 还好，喜欢这首歌的歌词。 心墙——郭静 “我不想忘记你”，“不药而愈”，“每一天都不同”，都好听喜欢她的歌。 我知道你很难过——蔡依林 唱起来有感觉也不难唱，推荐。 夏伤——SARA 感觉很特别，喜欢。 那天——蓝又时 喜欢她的歌，她的调调，强烈推荐。“秘密”也不错。 礼物——罗美玲 好听有感觉，不过不太好唱。 我比想象中爱你——JS 老歌了一直很喜欢，唱起来有感觉。
魅力四射的独唱舞台 ——甜美纯净的女声独唱 一、教学目标 1、认知目标：初步了解民族唱法、美声唱法、通俗唱法三种唱法的风格。 2、能力目标：通过欣赏部分女声独唱作品，学生能归纳总结出她们的演唱 风格和特点，并同时用三种不同风格演唱同一首歌曲。 3、情感目标：通过欣赏比较，对独唱舞台有更多元化的审美意识。 二、教学重点：学生能用三种不同风格演唱形式演唱同一首歌。 三、教学难点：通过欣赏部分女声独唱作品，学生能归纳总结出她们的演唱 风格和特点。 四、教学过程： （一）导入 1、播放第十三界全国青年歌手大奖赛预告片 （师）问：同学们对预告片中的歌手认识吗 （生）答： （师）问：在预告片中提出了几种唱法？ （生）答：有民族、美声、通俗以及原生态四种唱法，今天以女声独唱歌曲重点欣赏民族、美声、通俗唱法，希望通过欣赏同学们能总结出三种唱法的风格和特点。
（二）、音乐欣赏 1、通俗唱法 ①(师)问：同学们平常最喜欢唱那些女歌手的歌呢？能唱唱吗？ （可让学生演唱几句喜欢的歌，并鼓励） ②欣赏几首通俗音乐 视频一：毛阿敏《绿叶对根的情谊》片段、谭晶《在那东山顶上》片段、韩红《天路》片段、刘若英《后来》片段 视频二：超女《想唱就唱唱得响亮》 ①由学生总结出通俗音乐的特点 ②师总结并板书通俗音乐的特点：通俗唱法是在演唱通俗歌曲的基础上发展起来的，又称“流行唱法”。通俗歌曲是以通俗易懂、易唱易记、娱乐性强、便于流行而见长，它没有统一的规格和演唱技法的要求，比较强调歌唱者本人的自然嗓音和情绪的渲染，重视歌曲感情的表达。演唱上要求吐字清晰，音调流畅，表情真挚，带有口语化。 ③指出通俗音乐尚未形成系统的发声训练体系。其中用沙哑、干枯的音色“狂唱”和用娇柔、做作的姿态“嗲唱”，不属于声乐艺术的正道之物，应予以摒弃。 2、民族唱法 ①俗话说民族的才是世界的那么民族唱法的特点是什么呢？ ②欣赏彭丽媛《万里春色满人间》片段 鉴赏提示：这首歌是剧种女主角田玉梅即将走上刑场时的一段难度较大的咏叹调。
way的用法总结大全 way的用法你知道多少，今天给大家带来way的用法，希望能够帮助到大家，下面就和大家分享，来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地，大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。
way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句
适合女生唱的100首好听的歌 1、偏爱----张芸京 2、阴天----莫文蔚味道很难把握 3、眼泪----范晓萱还好，张学友版本适合男生 4、我要我们在一起---范晓萱A段超级难唱的，拍子和口气都不好抓 5、无底洞----蔡健雅听起来简单，唱起来很难——这首是典型的例子 6、呼吸----蔡健雅 7、原点----蔡健雅&孙燕姿到哪里找两个好听的女中音呢？ 8、我怀念的----孙燕姿 9、不是真的爱我----孙燕姿 10、我也很想他----孙燕姿 11、一直很安静----阿桑 12、让我爱----阿桑 13、错过----梁咏琪 14、爱得起----梁咏琪为什么没有洗脸？剪发？胆小鬼？ 15、蓝天----张惠妹 16、记得----张惠妹 17、简爱----张惠妹是剪爱吧？ 18、趁早----张惠妹男生可以试试张宇的版本 19、一念之间----戴佩妮 20、两难----戴佩妮 21、怎样----戴佩妮 22、一颗心的距离----范玮琪 23、我们的纪念日----范玮琪 24、启程----范玮琪 25、最初的梦想----范玮琪 26、是非题----范玮琪 27、你是答案----范玮琪 28、没那么爱他----范玮琪 29、可不可以不勇敢----范玮琪 30、一个像夏天一个像秋天----范玮琪范范的歌这么多啊。。。 31、听，是谁在唱歌----刘若英 32、城里的月光----许美静 33、女人何苦为难女人----辛晓琪我一直在找这个歌曲的粤语版，却找不到
34、他不爱我----莫文蔚 35、你是爱我的----张惠妹 36、同类----孙燕姿 37、漩涡----孙燕姿 38、爱上你等于爱上寂寞----那英在KTV点这个歌曲的MTV不是会很尴尬吗？ 39、梦醒了----那英 40、出卖----那英 41、梦一场----那英 42、愿赌服输----那英没有征服？也许是太大众了吧。 43、蔷薇----萧亚轩 44、你是我心中一句惊叹----萧亚轩 45、突然想起你----萧亚轩 46、类似爱情----萧亚轩 47、Honey----萧亚轩 48、他和他的故事----萧亚轩 49、一个人的精彩----萧亚轩 50、最熟悉的陌生人----萧亚轩还记得她第一张专籍里面最喜欢那首《没有人》 51、想你零点零一分----张靓颖 52、如果爱下去----张靓颖 53、我想我是你的女人----尚雯婕 54、爱恨恢恢----周迅 55、不在乎他----张惠妹 56、雪地----张惠妹 57、喜欢两个人----彭佳慧 58、相见恨晚----彭佳慧 59、囚鸟----彭羚 60、听说爱情回来过----彭佳慧 61、我也不想这样----王菲 62、打错了----王菲 63、催眠----王菲 64、执迷不悔----王菲 65、阳宝----王菲 66、我爱你----王菲 67、闷----王菲