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研究生:徐敏娟
研究生(外文):Min-Chuan Hsu
論文名稱:睪丸及著床前發育專一性鋅指蛋白基因Rnf33轉錄調控機制之研究
論文名稱(外文):Transcriptional regulation of the testis-specific and developmentally regulated Rnf33 RING finger protein gene
指導教授:朱廣邦
指導教授(外文):Kong-Bung Choo
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:67
中文關鍵詞:轉錄
外文關鍵詞:Rnf33RING finger proteinNF-kBHRE
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RNF33屬於TRIM/RBCC蛋白家族中的一員,而Rnf33基因只在小鼠未受精卵、著床前早期胚胎時期和小鼠睪丸中表現,因此Rnf33對小鼠著床前胚及小鼠睪丸細胞中可能扮演重要生物功能。在本研究中,我們探討Rnf33基因的轉錄調控機制。
首先利用螢火蟲冷光酶分析法(luciferase assay)探討Rnf33促進子的功能,結果顯示,非典型TATA box和轉錄起始子(transcription initiator)是Rnf33的核心促進啟動子(core promoter element)。另一方面,我們也發現移除Rnf33 intron中距離轉錄起始點1,187 bp位置的305 bp序列後(我們稱此片段為R4),轉錄活性下降80%,顯示此區域含有重要的cis element。利用插入不同位置、方向及數目的R4,更進一步證明R4是一個正向調控子(positive regulator)。另外,我們將R4分為三個部份進行轉錄因子預測資料庫分析:R4-1含NF-�羠蛋白結合位、缺氧反應因子(hypoxia response element;HRE) 和GATA 蛋白結合位;R4-2 富含AT序列;R4-3含一個N-box 及兩個E-boxes。利用螢火蟲冷光酶分析法分析R4的三個片段,結果顯示其中R4-1是調控Rnf33表現的必要片段。進一步利用定點突變的方式分別突變三個轉錄因子結合位,分析結果顯示突變NF-�羠結合位會明顯影響Rnf33轉錄活性。
利用電泳遷移率改變實驗(electrophoresis mobility shift assay) 競爭及超級遷移分析(supershift assay)進行分析,證明Rnf33 R4-1中的NF-�羠結合位在活體外(in vitro)會被NF-�羠 家族中的成員 p65 and p50所辨識進而結合。再利用染色質免疫沈澱(chromatin immunoprecipitation)更進一步證明在小鼠睪丸及小鼠睪丸細胞株TM3及TM4中,Rnf33和NF-�羠會於活體內(in vivo)結合。此外,我們發現小鼠睪丸細胞株TM3及TM4並不會因為加入TNF���n而活化NF-�羠。
根據以上實驗結果我們推論,在小鼠睪丸中,Rnf33基因的表現是經由NF-�羠 家族中的成員 p65 and p50所辨識進而結合到 Rnf33 intron中cis element 所調控。我們的研究也進一步將NF-�羠的基因調控功能延伸到睪丸基因甚至早期胚胎發育時期基因。
Rnf33 is a mouse TRIM/RBCC protein gene expressed in the unfertilized egg, the pre-implantation embryo and in the testis, and may play an important biological role in early development and in the testis. In this study, we aim to investigate transcriptional regulation of Rnf33.
In order to analyze transcriptional regulation, we used luciferase assays to dissect the Rnf33 promoter functions. We showed that a putative atypical TATA box and a transcription initiator (Inr) element act as the core promoter elements of Rnf33. Our data also indicate that removal of a 305-bp sequence, designated as R4, located at the 3’-end of the 1,187-bp intron sequence resulted in 80% loss of transcription activity indicating the presence of a crucial transcriptional cis element in this region. Further, we demonstrated the intronic R4 sequence acts as a positive regulator. In addition, we searched for putative transcription factor binding sites (TFBS) in the R4 fragment using TFBS’s databases, R4 could be divided into three parts: R4-1 contains a NF-kB, a hypoxia response element (HRE) and a GATA protein binding site; R4-2 contains an AT-rich fragment; R4-3 contains one N-box and two E-boxes. Luciferase assays indicated that R4-1 is crucial to Rnf33 regulation. Mutation and deletion analysis of these TFBS in R4-1 indicated that NF-�羠 could affect Rnf33 transcription.
To demonstrate Rnf33 is targeted by the NF-kB subunits p65 and p50, EMSA were performed using a R4-1 NF-kB element probe; supershift assays were also performed using anti-p65 and anti-p50 antibodies. The results of EMSA indicated that R4-1 sequence is targeted by the p65 and p50 in vitro. Chromatin immunoprecipitation experiments (ChIP) further demonstrated in vivo Rnf33-NF-kB association in the testis, TM3 and TM4 mouse testicular cell lines. We further demonstrate TNF�� would not induce NF-kB activation in the TM3 and TM4 mouse testicular cell lines. In conclusion, our results show that the Rnf33 expression in the testis is regulated by the ubiquitous NF-kB subunits p65 & p50 binding at an intronic cis-element and further understanding of transcriptional regulation of an active retrogene via a cis sequence present in the intron. My work also expands the biological roles of NF-kB to regulation of gene expression in the testis and maybe in pre-implantation development.
Signature Page ………………………………………………………………………. i
Thesis Approval Form……………………………………………………………. …. ii
Acknowledgments…………………………………………………………………… iii
Chinese Abstract……………………………………………………………………... iv
English Abstract……………………………………………………………………… v
Table of Contents…………………………………………………………………….. vii
List of Figures………………………………………………………………………... ix
List of Tables ………………………………………………………………………… x
1. Introduction
1.1. Retrogenes…………………………………………………………………… 1
1.2. Rnf33 gene is a RING-finger protein (RNF) gene........................................... 1
1.3. Transcriptional regulation of the Rnf33 homologous gene, Rnf35 gene……..4
1.4. The P1-exon 1 flanking sequence of the Rnf33 gene………………….. …... 7
1.5. Biological functions of RNF33……………………………………………… 7
1.6. Aim and scope of the present study…………………………………………. 9
2. Materials and methods
2.1. Cell lines and mice used…………………………………………………….. 11
2.2. Plasmids construction……………………………………………………….. 11
2.2.1. BAC45 BAC clone extraction…………………………………………. 11
2.2.2. Preparative PCR………………………………………………………... 12
2.2.3. DNA purification using Gel/PCR DNA Fragment Extraction Kit……... 13
2.2.4. Restriction enzyme digestion…………………………………………... 14
2.2.5. Ligation of DNA fragments…………………………………………..... 14
2.2.6. Preparation of bacterial competent cells and transformation………….. 15
2.2.7. Cracking analysis of clones with inserts………………………………. 16
2.2.8. Plasmid extraction using the High-Speed Plasmid Mini Kit………….. 16
2.3. QuikChange® Site-directed mutagenesis kit for mutation…………………. 17
2.3.1. Mutant strand synthesis reaction………………………………………. 17
2.3.2. Dpn I digestion of the amplification products…………………………. 18
2.4. Phusion™ Site-Directed Mutagenesis Kit for deletion…………………….... 18
2.4.1. 5’-Phosphorylation of oligonucleotides……………………………….. 18
2.4.2. PCR......................................................................................................... 18
2.4.3. Ligation & transformation....................................................................... 19
2.5. Reverse transcription-PCR............................................................................... 19
2.5.1. First-Strand cDNA Synthesis Using SuperScript™ II………………......... ….. 19
2.5.2. PCR……………………………………………………………………….. 20
2.6. Transient transfection………………………………………………………... 24
2.7. Luciferase activity assay……………………………………………………... 24
2.8. Electrophoretic mobility shift assay (EMSA) and supershift assay………….. 25
2.8.1. Preparation of nuclear extracts and total protein lysate………………... 25
2.8.2. EMSA and supershift assays………………………………………….. 25
2.9. Chromatin immunoprecipitation (ChIP)……………………………………. 26
3. Results
3.1. Identification of the Rnf33 core promoter elements and a putative
positive regulator (R4) in the intron………………………………………….29
3.2. A sub-fragment of R4, R4-1 (94 bp), is a positive regulator and
contains for putative binding sites HIF-1, NF-�羠 and GATA. ………………. 31
3.3. NF-�羠 binding site is an important cis-element of the Rnf33 promoter……. 39
3.4. R4-1 HRE may not directly participate in transcriptional regulation
of Rnf33……………………………………………………………………... 41
3.5. The NF-�羠 subunits p65 & p50 bind to the NF-�羠 binding site in R4-1….. 43
3.6. NF-�羠 activation of the Rnf33 promoter is TNF�� independent……………. 52
4. Discussion
4.1. Intron-mediated regulation of gene expression…………………………….. 55
4.2. The role of the putative HRE in Rnf33 promoter…………………………… 56
4.3. Rnf33 and NF-�羠 co-exist in the developmental stage and in the testis……. 58
4.4. NF-�羠 activation pathways…………………………………………….…… 60
5. References……………………………………………………………...……….... 63
Atchley, W.R. and Fitch, W.M. (1997) A natural classification of the basic helix-loop-helix class of transcription factors. Proc. Natl. Acad. Sci. USA 94, 5172-5176.

Borden, K.L. (1998) RING fingers and B-boxes: zinc-binding protein-protein interaction domains. Biochem. Cell Biol. 76, 351-358.

Borden, K.L. and Freemont, P.S. (1998) The RING finger domain: a recent example of a sequence-structure family. Curr. Op. Struct. Biol. 6, 395-401.

Boulouvala, S., Price, N., Plant, K.E. and Sim, E. (2003) Structure and transcriptional regulation of the Nat2 gene encoding for the drug- metabolizing enzyme arylamine N-acetyltransferase type 2 in mice. Biochem. J. 375, 593-602.

Buchman, A.R. and Berg, P. (1988) Comparison of intron-dependent and intron- independent gene expression. Mol. Cell Biol. 8, 4395-4405.

Bui, N.T., Livolsi, A., Peyron, J.F. and Prehn, J.H. (2001) Activation of nuclear factor �羠 and Bcl-x survival gene expression by nerve growth factor requires tyrosine phosphorylation of I�羠a. J. Cell Biol. 152, 753-764.

Callis, J., Fromm, M. and Walbot, V. (1987) Introns increase gene expression in cultured maize cells. Genes Dev. 1, 1183-1200.

Chen, H.H., Liu, T.Y.C. and Choo, K.B. (2002a) Utilization of a common promoter by two juxtaposed and intronless mouse early embryonic genes, Rnf33 /2czf45 and Rnf35: implications in zygotic gene expression. Genomics 80, 140-143.

Chen, H.H., Liu, T.Y.C., Huang, C.J. and Choo, K.B. (2002b) Generation of two homologous and intronless zinc-finger protein genes, Zfp352 and Zfp353, with different expression patterns by retrotransposition. Genomics 79, 18-23.

Choi, T., Huang, M., Gorman, C. and Jaenisch, R. (1991) A generic intron increases gene expression in transgenic mice. Mol. Cell Biol. 11, 3070-3074.

Choo, K.B., Chen, H.H., Cheng, W.T., Chang, H.S. and Wang, M. (2001) In silico mining of EST databases for novel pre-implantation embryo-specific zinc finger protein genes. Mol. Reprod. Dev. 59, 249-255.

Choo, K.B., Chen, H.H., Liu, T.Y.C. and Chang, C.P. (2002) Different modes of regulation of transcription and pre-mRNA processing of the structurally juxtaposed homologs, Rnf33 and Rnf35, in eggs and in pre-implantation embryos. Nucl. Acids Res. 30 (224), 4836-4844.

D´ery, M.A.C., Michaud, M.D. and Richard, D.E. (2005) Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators. Int. J. Biochem. Cell Biol. 37, 535-540.

Davis, W.J. and Schultz, R.M. (2000) Developmental change in TATA-box utilization during preimplantation mouse development. Dev. Biol. 218, 275-283.

Duncker, B.P., Davies, P.L. and Walker, V.K. (1997) Introns boost transgene expression in Drosophila melanogaster. Mol. Gen. Genet. 254, 291-296.

Etkin, L.D., el-Hodiri, H.M., Nakamura, H., Wu, C.F., Shou, W. and Gong, S.G. (1997) Characterization and function of Xnf7 during early development of Xenopus. J. Cell Physiol. 173, 144-146.

Freemont, P.S. (1993) The RING finger. A novel protein sequence motif related to the zinc finger. Ann. N.Y. Acad. Sci. 684, 174-192.

Fong, Y.W. and Zhou, Q. (2001) Stimulatory effect of splicing factors on transcriptional elongation. Nature 414, 929-933.

Furger, A., O‘Sullivan, J.M., Binnie, A., Lee, B.A. and Proudfoot, N.J. (2002) Promoter proximal splice sites enhance transcription. Genes Dev. 16, 2792-2799.

Hayden, M. S. and Ghosh, S. (2008) Shared principles in NF-�羠 signaling. Cell 132, 344-362

Hayden, M.S. and Ghosh, S. (2004) Signaling to NF-κB. Genes Dev. 18, 2195-2224.

Le Hir, H., Nott, A. and Moore, M.J. (2003) How introns influence and enhance eukaryotic gene expression. Trends Biochem. Sci. 28 (4) 215-220.

Huang, C.J. and Choo, K.B. (2009) Retrogenes in preimplantation embryo development: a unique mode of transcriptional regulation. J. Chin. Med. Assoc. (in press)

Huang, C.J., Wu, S.C. and Choo, K.B. (2005a) Transcriptional modulation of the pre-implantation embryo-specific Rnf35 gene by the Y-box protein NF-Y/CBF. Biochem. J. 387, 367-375.

Huang, C.J., Chang, J.G., Wu, S.C. and Choo, K.B. (2005b) Negative transcriptional modulation and silencing of the bi-exonic Rnf35 gene in the preimplantation embryo: binding of the CCAAT-displacement protein/Cux to the untranslated exon 1 sequence. J. Biol. Chem. 280, 30681-30688.

Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L., Traenckner, E.B., Mueller- Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P.A. and Peyron, J. (1996) Tyrosine phosphorylation of I�羠-�� activates NF-�羠 without proteolytic degradation of I�羠-��. Cell 86, 787-798.

Jones, S. (2004) An overview of the basic helix-loop-helix proteins. Genome Biol. 5, 226.

Kandasamy, R.A. and Orlowski, J. (1996) Genomic organization and glucocorticoid transcriptional activation of the rat Na1/H1 exchanger Nhe3 gene. J. Biol. Chem. 271(18), 10551-10559.

Kato, T., Delhase, M., Hoffmann, A. and Karin, M. (2003) CK2 is a C-terminal I�羠 kinase responsible for NF-�羠 activation during the UV response. Mol. Cell 12, 829-839.

Kitamura, K., Tanaka, H. and Nishimune, Y. (2005) The RING-finger protein haprin: domains and function in the acrosome reaction. Curr. Protein Pept. Sci. 6, 567-574.

Kwek, K.Y., Murphy, S., Furger, A., Thomas, B., O'Gorman, W., Kimura, H., Proudfoot, N.J. and Akoulitchev, A. (2002) U1 snRNA associates with TFIIH and regulates transcriptional initiation. Nat. Struct. Biol. 9, 800-805

Lagrange, T., Kapanidis, A.N., Tang, H., Reinberg, D. and Ebright, R.H. (1998) New core promoter element in RNA polymerase II-dependent transcription: sequence- specific DNA binding by transcription factor IIB. Genes Dev. 12, 34-44.

Lu, J., Sivamani, E., Azhakanandam, K., Samadder, P., Li, X. and Qu, R. (2008) Gene expression enhancement mediated by the 5’ UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants. Mol. Genet. Genomics 279, 563-572.

Majumder, S. and DePamphilis, M.L. (1994) TATA-dependent enhancer stimulation of promoter activity in mice is developmentally acquired. Mol. Cell. Biol. 14, 4258- 4268.

Maniatis, T. and Reed, R. (2002) An extensive network of coupling among gene expression machines. Nature 416, 499-506.

Matfier, J.P. (1980) Establishment and characterization of two distinct mouse testicular epithelial cell lines. Biol. Reprod. 23, 243-252.

Meroni, G. and Diez-Roux, G. (2005) TRIM/RBCC, a novel class of ‘single protein RING finger’ E3 ubiquitin ligases. BioEssays 27, 1147-1157.

Mukhopadhyay, A., Manna, S.K. and Aggarwal, B.B. (2000) Pervanadate-induced nuclear factor-�羠 activation requires tyrosine phosphorylation and degradation of IkB��. Comparison with tumor necrosis factor-��. J. Biol. Chem. 275, 8549-8555.

Natoli, G., Saccani, S., Bosisio, D. and Marazzi, I. (2005) Interactions of NF-kappaB with chromatin: the art of being at the right place at the right time. Nat. Immunol. 6, 439-445.

Ogawa, S., Goto, W., Orimo, A., Hosoi, T., Ouchi, Y., Muramatsu, M. and Inoue, S. (1998) Molecular cloning of a novel RING finger-B box-coiled coil (RBCC) protein, terf, expressed in the testis. Biochem. Biophys. Res. Commu. 251, 515-519.

Perkins, N.D. and Gilmore, T.D. (2006) Good cop, bad cop: the different faces of NF-�羠. Cell Death Differ. 13, 759-772.

Perkins, N.D. (2006) Post-translational modifications regulating the activity and function of the nuclear factor κB pathway. Oncogene 25, 6717-6730.

Perkins, N.D. (2007) Integrating cell-signalling pathways with NF-κB and IKK function. Nat. Rev. Mol. Cell Biol. 8, 49-62.

Ravasi, T., Huber, T., Zavolan, M., Forrest, A., Gaasterland, T., Grimmond, S., RIKEN GER Group, GSL Members and Hume, D.A. (2003) Systematic characterization of the zinc-finger-containing proteins in the mouse transcriptome. Genome Res. 13, 1430-1442.

Reddy, B.A., Etkin, L.D. and Freemont, P.S. (1992) A novel zinc finger coiled-coil domain in a family of nuclear proteins. Trends Biochem. Sci. 17, 344-345.

Rocha, S. (2007) Gene regulation under low oxygen: holding your breath for transcription. Trends Biochem. Sci. 32, 389-397.

Romieu-Mourez, R., Landesman-Bollag, E., Seldin, D.C. and Sonenshein, G.E. (2002) Protein kinase CK2 promotes aberrant activation of nuclear factor-�羠, transformed phenotype, and survival of breast cancer cells. Cancer Res. 62, 6770-6778.

Rose, A.B. (2004) The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. Plant J. 40, 744-751.

Saurin, A.J., Borden, K.L., Boddy, M.N. and Freemont, P.S. (1996) Does this have a familiar RING? Trends Biochem. Sci. 21, 208-214.

Vinckenbosch, N., Dupanloup, I. and Kaessmann, H. (2006) Evolutionary fate of retroposed gene copies in the human genome. Proc. Natl. Acad. Sci. USA 103, 3220-3225.

Zhang, X.H. and Chasin, L.A. (2004) Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev. 18, 1241-1250.
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