황상준, 전상영, 이경아 大韓不妊學會誌 : 第 33 卷第 2 號 2006 Kor. J. Fertil. Steril., Vol. 33, No. 2, 2006, 6 포천중문의과대학교생명과학전문대학원 1, 전남대학교생명과학기술학부 2, 차병원여성의학연구소 3 황상준 1,2 전상영 2 이경아 1,3 * In silico Analysis of Downstream Target Genes of Transcription Factors Sang-Joon Hwang 1,2, Sang-Young Chun 2, Kyung-Ah Lee 1,3* 1 Graduate School of Life Science and Biotechnology, Pochon CHA University College of Medicine, Seoul, Korea, 2 School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea, 3 CHA Research Institute, Fertility Center, CHA General Hospital, Seoul, Korea Objective: In the previous study, we compiled the differentially expressed genes during early folliculogenesis. 1 Objective of the present study was to identify downstream target genes of transcription factors (TFs) using bioinformatics for selecting the target TFs among the gene lists for further functional analysis. Materials & Methods: By using bioinformatics tools, constituent domains were identified from database searches using Gene Ontology, MGI, and Entrez Gene. Downstream target proteins/genes of each TF were identified from database searches using TF database (TRANSFAC 6.0) and eukaryotic promoter database (EPD). Results: DNA binding and trans-activation domains of all TFs listed previously were identified, and the list of downstream target proteins/genes was obtained from searche of TF database and promoter database. Based on the known function of identified downstream genes and the domains, 3 (HNF4, PPARg, and TBX2) out of 26 TFs were selected for further functional analysis. The genes of wee1-like protein kinase and p21waf1 (cdk inhibitor) were identified as potential downstream target genes of HNF4 and TBX2, respectively. PPARg, through protein-protein interaction with other protein partners, acts as a transcription regulator of genes of EGFR, p21waf1, cycd1, p53, and VEGF. Among the selected 3 TFs, further study is in progress for HNF4 and TBX2, since wee1-like protein kinase and cdk inhibitor may involved in regulating maturation promoting factor (MPF) activity during early folliculogenesis. Conclusions: Approach used in the present study, in silico analysis of downstream target genes, was useful for analyzing list of TFs obtained from high-throughput cdna microarray study. To verify its binding and functions of the selected TFs in early folliculogenesis, EMSA and further relevant characterizations are under investigation. Key Words: Folliculogenesis, Bioinformatics, Transcription factor, Binding element, Downstream target gene 포유동물의난소에존재하는난포는태어날때에대부분원시난포 (primordial follicle) 로성장이멈춰있으며이때난자는제 1 감수분열전기상태로존 재한다. 포유류의난포발달과정은난소내외의다양한요인에의해조절되고, 2 사춘기를지나면서성장이억제되어있는원시난포중일부만이선택적 주관책임자 : 이경아, 우 ) 135-081 서울특별시강남구역삼 1 동 606-13, 차병원기초의학연구소분자생식생리학연구실 Tel: 822-557-3937, Fax: 822-563-2028, e-mail: leeka@ovary.co.kr * 이논문은 2004 년도한국학술진흥재단의지원에의하여연구되었음 (KRF-2004-C00135). - 125 -
으로 1차난포 (primary follicle) 로의성장을개시하게된다. 이와같이원시난포에서 1차난포로성장이다시시작되는기전은여성의생식능, 난소의노화, 폐경의시기및불임등에대한정보를제공함으로써여성생식을좀더정확히이해하는데필수적이나그기전에대한연구는매우미약하다. 3,4 본연구진은난소내에서원시난포의초기발달에관여하는특이적인유전자를찾아내기위해 DNA Chip 을이용하여원시난포, 1차난포및 2차난포사이에서차이나게발현하는유전자를발굴하는연구를수행하였고, 1 이렇게선행연구로부터얻어진유전자들의목록중에서전사인자 (transcription factors) 유전자에관심을갖고본연구를시작하였다. 이를통하여초기난포발달과정에연관되어있는유전자발현조절네트워크 (transcriptional regulatory network) 를분석할수있을것으로사료되기때문이다. 전사인자는 sequence-specific DNA-단백질복합체를형성함으로써유전자발현조절에중심적인역할을수행한다. 5 진핵생물에서의전사개시는 RNA 중합효소 II 복합체 (RNA polymerase II complex) 와전사개시부위와의결합뿐만아니라염색질변형효소 (chromatin-modifying enzyme) 나전사인자와프로모터부위 (promoter region) 와의결합에도영향을받는복잡한과정이다. 6 최근생명과학분야에서는대량분석 (High- ThroughPut Screening; HTS) 기술의발전에힘입어새로운분자생물학실험기법이발달하고이로인하여엄청난양의데이터들이쏟아져나오고있다. 예를들면, 많은종 (species) 에대한대규모의 genome 서열정보해독작업뿐만아니라 DNA microarray 및 proteomics, Yeast-Two-Hybrid (Y2H) 실험방법역시 HTS 기술로서기존의분자생물학실험스케일과는다른차원의데이터를제공하고있다. 그중에서도기능적인유전자발굴을위해, 내외부의자극에대한 transcriptome의반응을확인함으로써전체유전자의발현양상 (gene expression profiling) 을분석하는 DNA microarray와같은기능유전체학접근방법은다양한생명현상과관련된다수유전자들의발현양의변화에대한정보를제공함으로써미지유전자의기능에대한실마리를제시할수있다. 또한특정병리학적, 생리학적현상에대 한분자수준에서의조절기전을이해하는데많은도움을주고있다. 그러나이런기능유전체학실험기법은방대한규모의접근방식에내재되어있는 false data의생산이라는약점을가지고있다. 따라서, 이런방대한양의데이터중에서특정생명현상과관련하여의미있는데이터를선별해내기위해서는생명정보학 (Bioinformatics) 이라는 computer science 기술및통계학적접근방법을통한분류, 가공처리의과정이필요하게되었다. 생명정보학적연구는 computer science 및통계학적접근을통한원료데이터 (raw data) 의분석과정을거쳐유용한정보를발굴해내는 data-driven discovery로써, 최근 Human Genome Project의성공적인진행, DNA microarray 실험기술의발달, 단일염기변이 (Single Nucleotide Polymorphism; SNP) 연구의발달등으로인하여생명정보학분야는더욱더그중요성이부각되고있으며, 그연구성과들은다양한생명현상에대한분자수준에서의이해를위해많은정보들을제공하고있다. 따라서본연구는선행된 cdna microarray 연구로부터얻은유전자목록중에서생명정보학적분석을통해초기난포발달과정에긴밀하게관여하고있을것으로추정되는전사인자를선별해냄으로써이후해당전사인자에대한심도있는연구를진행하기위하여이루어졌다. 본연구의결과는유전체학적대량분석기술로얻어진방대한양의데이터를연구함에있어의미있는정보를선별해내는새로운접근방식을제시하여생명정보학작업의유용성을보여줄것이며, 이후실험적인검증결과와더불어초기난포발달과정에서의유전자발현조절네트워크를이해하는데중요한초석이될것이다. 연구대상및방법 1. 전사인자유전자의 1차구조분석 Figure 1은선행연구결과 DNA Chip을이용한유전자발현분석연구로부터얻어진초기난포발달단계에따라차이나게발현하는양상을보이는전사인자의유전자목록이다. 각전사인자의구성 - 126 -
황상준, 전상영, 이경아 질데이터베이스검색을수행하여각전사인자가가지고있는도메인을확인하였다. 2. 전사인자의도메인분석전사인자의 1차구조분석에서확인된각구성도메인의분석을위해서는 Web-based Gene Ontology (http://www.geneontology.org/, http://www.godatabase. org/cgi-bin/amigo/go.cgi) 데이터베이스와 InterPro (http://www.ebi.ac.uk/interpro/), Pfam (http://www.sanger. ac.uk/software/pfam/) 도메인데이터베이스를참조하였다. 3. 전사인자의 cis-acting 및 trans-acting 하위표적유전자분석 Figure 1. Heat map of the transcription factors obtained from previous DNA microarray analysis. The numbers 1, 2 and 3 indicate the primordial, primary, and secondary follicles, respectively. Green color indicates down-regulation of gene expression from primordial to primary (numbers 1 and 2) or primary to secondary (numbers 2 and 3) follicles. Red color indicates upregulation, and black color indicates no change in gene expression. 도메인을확인하기위해데이터베이스검색을수행하였고, 연구에사용된방법론의전체적인흐름도는 Figure 2에요약하였다. 전사인자의 1차구조분석을위해서는 MGI (http://www.informatics.jax.org/), Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene), UniGene (http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=unigene) 유전자데이터베이스, 그리고 Swiss-Prot (http://www.expasy.org/sprot/) 단백 cis-acting DNA 결합부위를통해각각의전사인자와결합하여유전자발현이조절되는것으로알려진하위표적유전자와그 DNA 결합부위에대한정보를얻기위하여 TRANSFAC 6.0 (http://www. biobase.de/pages/products/transfac.html) 전사인자데이터베이스검색을수행하였다. DNA 결합부위의염기서열정보를이용하여 binding element를가지고있는잠재적인하위표적유전자를동정하기위해 EPD (http://www.epd.isb-sib.ch/) 프로모터데이터베이스검색을수행하였다. 또전사인자와상호작용하는다른작용단백질 (protein partner) 과단백질- 단백질상호작용을통해발현이조절되는 transacting 하위표적유전자검색도동일한전사인자데이터베이스를통해수행하였다. 결과 1. 전사인자의 1차구조분석 26개의각전사인자들의 1차구조에대한생명정보학적분석을위해 MGI 데이터베이스를기본플랫폼으로해서 Entrez Gene, UniGene 유전자데이터베이스와 Swiss-Prot 단백질데이터베이스검색을수행하였다. 분석된 1차구조에대한정보를바탕으로각전사인자를구성하는도메인을확인하였다. 26개의전사인자에대하여모두구조분석을실행하였으나, 그결과는양적으로너무나방대하여본논문에다발표하지않고그중에서가장관심을갖 - 127 -
Figure 2. Flow chart of in silico analysis of downstream target genes of the transcription factors. Table 1. Constituent domains of the selected 3 TFs TF MGI ID Protein Domains (InterPro ID) HNF4 (HNF4α, HNF4α1, TCF4) MGI : 109128 Retinoid X receptor (IPR000003) Vitamin D receptor (IPR000324) NHR, ligand-binding (IPR000536) NHR, DNA-binding (IPR001628) STR (IPR001723) PPARg MGI : 97747 NHR, ligand-binding (IPR000536) NHR, DNA-binding (IPR001628) STR (IPR001723) PPAR (IPR003074) PPAR, gamma (IPR003077) TBX2 MGI : 98494 TF, T-box (IPR001699) TF, Brachyury (IPR002070) TF (transcription factor), NHR (nuclear hormone receptor), STR (steroid hormone receptor), PPAR (peroxisome proliferator-activated receptor) 고연구하고자축약된세개의유전자즉, 목적하는바하위표적유전자의분석까지이루어진후에하위 표적유전자의종류에의해채택된세개의전사인자 (HNF4, PPARg, TBX2) 에대해서만구조분석의결 - 128 -
황상준, 전상영, 이경아 과를정리하였다 (Table 1). HNF4와 PPARg는스테로이드수용체의리간드결합도메인 (IPR000536) 과 DNA 결합도메인 (IPR- 001628) 으로구성되어있는데, 스테로이드수용체는배아발생이나세포분화의조절을포함하여다양한생리적인기능의전사조절자로알려져있다. 리간드결합도메인은리간드와결합한후구조변화를통해 nuclear translocation, oligomerisation, cofactor/ kinase/transcription factor association, 그리고 DNA binding 등의현상을유도하여전사활성을조절하는분자스위치로서작용한다. 7 DNA 결합도메인은 hormone-response element로불리는 DNA의특이적부위를인식한후결합을통해스테로이드수용체의 위치를지정함으로써수용체가전사조절자로서발현을활성화시키거나억제시키도록유도한다. 8,9 TBX2는특정염기서열에결합하는 DNA 결합도메인인 T-box (IPR001699) 를가지고있으며, T-box family의전사인자들은특정조직의세포에서만발현되어초기배발생과정에서세포의운명을결정하는데중요한역할을담당하는것으로알려져있다. 10 2. 전사인자의하위표적유전자분석전사인자데이터베이스와프로모터데이터베이스검색을통해 26개각각의전사인자가유전자의 responsive element에결합하여그유전자의발현을 TFs * Table 2. Downstream target genes of the selected 3 TFs Cis-acting downstream target gene Protein partner Trans-acting downstream target gene HNF4 (Wee1-like protein kinase) N/A N/A PPARg aqp 7 RXR-α β-catenin SMAD3 HNF4 RXR- α RAR- α1/ RAR-γ1 cyclin H p53 (RXR-α ) EGFR / p21waf1 / H1(0) / CYP7A (RXR-α β-catenin LEF-1 )CAD1 / cycd1 (RXR-α β-catenin TCF-4E )cycd1 (RXR-α COUP-TF2 )HNF4α (RXR-α PPAR-γ2 p300 )cycd1 (RXR-α RAR-α1/RAR-beta/RAR-γ )H1(0) / HOXA1 (RXR-α T3R-α /T3R-β1/T3R-β )GH (RXR-α T3R- β1 )EGFR (RXR-α TIF1 ER-α )p53/lh β /VEGF (RXR-α VDR )GH/p21WAF1 TBX2 trp-1 (p21waf1) N/A N/A * Transcription Factors: HNF4 (hepatocyte nuclear factor 4), PPARg (peroxisome proliferator-activated receptor gamma), TBX2 (T-box protein 2). Protein Partners RXR-alpha (retinoid X receptor alpha), SMAD3 (Similar to mothers against decapentaplegic 3), RAR-alpha1/RARgamma1 (retinoic acid receptor alpha1/-gamma1), p53 (tumor protein p53), LEF-1 (lymphocyte enhancer binding factor 1), TCF-4E (= TCF7L2; transcription factor-7-like-2), COUP-TF2 (chicken ovalbumin upstream promoter transcription factor 2), p300 (transcriptional co-activator p300), T3R-alpha/ T3R-beta1/ T3R-beta (thyroid hormone receptor-alpha/ beta1/beta), TIF1 (transcriptional intermediary factor 1), ER-alpha (estrogen receptor alpha), VDR (Vitamin D3 Receptor). Downstream target genes aqp7 (aquaporin 7), trp-1 (tyrosinase-related protein 1), p21waf1 (cdk inhibitor p21), EGFR (epidermal growth factor receptor), H1 (0) (histone 1 zero), CYP7A (cholesterol 7alpha-hydroxylase), CAD1 (E-cadherin), cycd1 (cyclin D1), HOXA1 (homeobox gene 1), GH (growth hormone), LHbeta (luteinizing hormone beta), VEGF (vascular endothelial growth factor). N/A: Not applicable - 129 -
직접조절하는하위표적유전자들에대한목록을작성한후 (data not shown), 표적유전자의종류에따라원시난포에서 1차난포로의선택적성장개시과정조절에중요한역할을할것으로생각되는 3개의전사인자를일차적으로선택하였다. 세개의전사인자에대한하위표적유전자는이외에도데이터베이스검색으로부터결합부위 (binding element) 의염기서열, 상호작용하는단백질에관한정보를얻었다. cis-acting element의염기서열정보를바탕으로진핵생물의프로모터데이터베이스검색을통해그결합부위를가지고있는잠재적인하위표적유전자도동정하여구성된하위표적유전자목록에추가하였고이는 Table 2에서괄호로묶어표시하였다. 또한전사인자의상호작용단백질에관한정보를바탕으로전사인자데이터베이스검색을실시하여, 단백질 partner와상호작용함으로써간접적으로발현을조절하는하위표적유전자도동정후목록에추가하였다. Table 2에서 는단백질상호작용을나타내며 ( 괄호로묶음 ), 는전사인자가하위표적유전자의발현을조절함을의미한다. Wee1-like protein kinase와 p21waf1 (cdk inhibitor) 유전자는각각 HNF4와 TBX2에대한 binding element를그프로모터부위에가지고있는잠재적인하위표적유전자로동정되었으며, PPARg 는 RXR-α 를비롯한여러다른단백질과의상호작용을통해그들과복합체를형성하여 EGFR, p21waf1, cycd1, p53, VEGF 등기능이많이알려진여러가지중요한유전자들의발현에영향을미치는것으로나타났다. 고찰및결론본연구진은초기난포발달에관여하는인자들을밝혀내고그들을통해조절기전을연구하고자 cdna microarray를이용하여초기난포발달단계에서차이나게발현하는유전자목록을획득하였고, 1,11 그유전자목록을중심으로중요한유전자에대한연구를하나하나진행하고있다. 12~14 본연구에서는그중에서도특히, 전사조절인자 (transcription factor) 군에대하여초점을맞추어연구를진행 하였다. 대량으로얻어진유전자목록중에서어떤유전자를먼저연구대상으로택할것인지를결정하는일은쉬운일은아니다. 특히전사인자와같이자기자신만이아니라해당전사인자가영향을미치는하위표적유전자가무엇인가에따라서그기능의중요성이달라지는경우는목록에작성된유전자만으로연구대상을결정하기어려운점이있겠다. 따라서 cdna microarray 결과얻어진 26개의전사인자목록중, 각전사인자에대한하위표적유전자를생명정보학적방법론을이용하여찾아낸하위표적유전자의종류및기능에따라중요하다고생각되어지는전사인자를결정하여, 그다음기전연구를진행하기로계획을세우고본연구를진행하였다. 생명정보학적방법을이용하여 26개의전사인자에대한하위표적유전자분석한결과, 매우다양한기능을갖는유전자의목록이얻어졌는데, 그중에서특별히관심을갖게된유전자는 HNF4와 TBX2, 그리고 PPARg였다. HNF4의 cis-acting 하위표적유전자로 wee1-like protein kinase가분석되었는데이미본연구실에서는 wee1 protein kinase가일반적으로잘알려진체세포의 mitosis 억제뿐아니라동일한기전으로난자의 meiosis arrest에도관여하고있을것으로보고한바있다. 13 따라서아직까지그염기서열이나기능이확실하게보고된바없이, 염기서열의유사성으로이름붙여진 wee1-like protein kinase가 HNF4의하위표적유전자로검색되었다는것은매우고무적인결과로서, wee1과의유사성및관련성을연구해볼필요가있다고사료되었다. 또한 TBX2가 cyclin-dependent kinases (cdk) inhibitor 유전자의발현을억제한다는것이보고되어있음을알게되었다. 15 Cdk는 mitosis 조절과정에관련되어있는세포분열의중요한조절인자이므로 TBX2가 cdk inhibitor의발현을조절한다는사실은 HNF4와함께초기난포발달을조절함에있어난자및과립세포의 meiosis 및 mitosis 조절에매우긴밀하게연결되어있는전사인자를찾아내었음을시사하는일이라할수있다. 따라서이두전사인자자체의발현과더불어하위표적유전자와의관계등을조사분석함으로써초기난포발달에관여하는역할을밝혀낼수있을것으로기대된다. - 130 -
황상준, 전상영, 이경아 이외에도전사인자의 cis-acting 또는 trans-acting downstream 유전자에대한결과와유전자발현의패턴이매우흥미로운 PPARg를선택할수있었다. PPARg 의경우는 EGFR, p21waf1, cycd1, p53, VEGF와같이매우중요한역할을하는것으로잘알려져있는여러가지하위유전자의기능을조절하는데관련되어있는것으로초기난포발달과정에도복잡하고긴밀하게관련되어있을것으로추정할수있다. 따라서앞으로이세전사인자및하위표적유전자의유전자발현및상호작용에대한심도있는후속연구가진행되어야할것이다. 전사인자와프로모터와의결합은특정유전자의발현조절을위한필요조건이지만충분조건이라할수는없다. 결과적으로, 단순히전사인자와프로모터지역의작은 DNA 조각과의결합여부를확인하는 electrophoretic mobility shift assay (EMSA) 실험은어떤유전자의발현이특정전사인자에의해서조절될것이라는가능성만을 in vitro 수준에서보여주는것이다. 염색체상에서전사인자의표적유전자와그프로모터부분의 binding element를동정하는것은유기적인유전자발현조절을위한전사단계에서의조절네트워크를연구하는데매우유용한도구이다. 따라서어떤유전자가특정전사인자의실제적인표적 (functional target) 임을증명하여생명현상과관련된전사단계에서의조절네트워크를구성하기위해서는 in vivo 수준에서전사인자의모든 DNA 결합부위를동정하여 genomewide transcription factor binding pattern을확인하는 Chromatinimmunoprecipitation (ChIP)-microarray나 Sequence Tag Analysis of Genomic Enrichment (STAGE) 등의실험방법을이용하여연구할수있을것이다. ChIP은세포내에서특정전사인자가어떠한프로모터부위에직접결합하는지를실험적으로규명하는방법이다. ChIP 방법으로얻어진게놈 sequence들을분석하는방법으로는 DNA microarray를이용한전사체분석방법이주로사용되어왔으나, 16 최근에는 Serial Analysis of Gene Expression (SAGE) 를이용한분석방법이제안되었다. 17 SAGE는원래 transcriptome 수준에서유전자발현분석을위해고안된기능유전체학적방법이며, 18 ChIP과연결하여얻어진각각의게놈 sequence들의짧은 tag로이루 어진연속접합체 (concatemer) 를클로닝한후, 그염기서열을분석하는과정으로사용될수있다. 참고문헌 1. Yoon SJ, Kim KH, Chung HM, Choi DH, Lee WS, Cha KY, Lee KA. Gene expression profiling of early follicular development in primordial, primary, and secondary follicles. Fertil Steril 2006; 85: 193-203. 2. Richards JS. Perspective: The ovarian follicle-a perspective in 2001. Endocrinol 2001; 142: 2184-93. 3. Fortune JE, Cushman RA, Kito WS. The primordial to primary follicle transition. Mol Cell Endocrinol 2000; 163: 53-60. 4. Fortune JE. The early stages of follicular development: activation of primordial follicles and growth of preantral follicles. Anim Reprod Sci 2003; 78: 135-63. 5. Wingender E, Dietze P, Karas H, Knüppel R. TRANSFAC: A database on transcription factors and their DNA binding sites. Nucleic Acids Res 1996; 24: 238-41. 6. Pilpel Y, Sudarsanam P, Church GM. Identifying regulatory networks by combinatorial analysis of promoter elements. Nat Genet 2001; 29: 153-9. 7. Edwards DP. The role of coactivators and corepressors in the biology and mechanism of action of steroid hormone receptors. J Mammary Gland Biol Neoplasia 2000; 5: 307-24. 8. Moehren U, Eckey M, Baniahmad A. Gene repression by nuclear hormone receptors. Essays Biochem 2004; 40: 89-104. 9. Claessens F, Gewirth DT. DNA recognition by nuclear receptors. Essays Biochem 2004; 40: 59-72. 10. Wilson V, Conlon FL. The T-box family. Genome Biol 2002; 3: REVIEWS3008. 11. Park CE, Cha KY, Kim K, Lee KA. Expression of cell cycle regulatory genes during primordial-primary follicle transition in the mouse ovary. Fertil Steril 2005; 83: 410-8. 12. Park CE, Shin MR, Jeon EH, Cha KY, Lee SH, Kim - 131 -
K, Kim NH, Lee KA. Oocyte-selective expression of MTi7 and finding its role in oocyte maturation and cell division in zygote by RNA interference. Mol Reprod Dev 2004; 69: 365-74. 13. Park CE, Kim YH, Jeon EH, Cha KY, Lee SH, Lee KA. Expression of weel and its related cell cycle components in mouse early stage follicles. Cells Tissues Organs 2004; 177: 221-8. 14. Kim KH, Park CE, Yoon SJ, Lee KA. Characterization of genes related to the cell size growth and CCN family according to the early folliculogenesis in the mouse. Kor J Fertil Steril 2005; 32: 269-77. 15. Harrelson Z, Kelly RG, Goldin SN, Gibson-Brown JJ, Bollag RJ, Silver LM, Papaioannou VE. Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development. Development 2004; 131: 5041-52. 16. Saha S, Sparks AB, Rago C, Akmaev V, Wang CJ, Vogelstein B, et al. Characterization of the yeast transcriptome. Cell 1997; 88: 243-51. 17. Jonghwan K, Akshay AB, Xochitl CM, Vishwanath RI. Mapping DNA-protein interactions in large genomes by sequence tag analysis of genomic enrichment. Nature Methods 2005; 2: 47-53. 18. Velculescu VE, Zhang L, Vogelstein B, Kinzler KW. Serial analysis of gene expression. Science 1995; 270: 484-7. = 국문초록 = 목적 : 본연구진은초기난포발달과정의각발달단계별난포를분리하여 cdna microarray를이용한유전자발현목록을보유하고있다. 1 본연구는이들유전자중에서전사인자들의목록에주목하여이들의하위표적유전자를생명정보학적기법을이용해동정함으로써이후초기난포발달의조절기전연구를위한중요한전사인자를결정하고자실시하였다. 연구방법 : 26개의전사인자들에대해서 Gene Ontology, MGI, 그리고 Entrez Gene 등의유전자데이터베이스검색을통해전사인자들을구성하는도메인을확인하였고, 전사인자데이터베이스 (TRANSFAC 6.0) 와진핵세포프로모터데이터베이스검색을실시하여, 전사인자의 cis-acting 및 trans-acting 하위표적유전자를분석하였다. 결과 : 26개전사인자들에대해서 DNA 결합도메인과단백질상호작용도메인을확인하였다. 또전사인자데이터베이스와프로모터데이터베이스검색으로부터하위표적유전자에대한정보를얻었다. 위와같은생명정보학적분석결과로부터흥미로운하위표적유전자를갖는 3개의전사인자로목표를압축할수있었다. 그중에서 HNF4는 MPF 억제조절자로알려져있는 Wee1 단백질인산화효소의유사유전자프로모터부위에결합하는전사인자며, TBX2는 cdk 억제자유전자의발현을억제하는전사인자로알려져있어, 초기난포발달과정의 MPF 기능조절에매우중요한역할을할것으로사료된다. 결론 : 본연구는생명정보학적분석을통하여전사인자의하위표적인자를알아내고, 이를이용하여 26개전사인자중에서다음연구를위한목표를결정하는접근방법을제시했다는데의미가있다고사료된다. 실제로이렇게결정된전사인자들이초기난포발달을조절하는분자생물학적기전에어떻게관여하는지를연구하기위해서는 EMSA 등과같은실험적증명을통한확인과보충연구가필요할것으로사료된다. 중심단어 : 난포발달, 생명정보학, 전사인자, 결합요소, 하위표적유전자 - 132 -