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研究生：	張衣萍 Chang, Yi-Ping
論文名稱：	人類DAZL 基因於轉錄過程中分子調控機制之特性研究 Characterization of transcriptional regulation mechanisms of DAZL gene
指導教授：	曾大千 Tseng, T. Joseph 郭保麟 Kuo, Pao-Lin
學位類別：	碩士 Master
系所名稱：	醫學院 - 醫學檢驗生物技術學系 Department of Medical Laboratory Science and Biotechnology
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	72
中文關鍵詞：	DAZL 、甲基化、啟動子區域、不孕症
外文關鍵詞：	methylation, SNP, infertile, EMSA, CpG island
相關次數：	點閱：91 下載：1
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中文摘要
全世界大約有百分之十至十五比例的夫婦受到不孕症的影響，其中男性與女性的因素各占了一半。精子發育過程的缺陷是造成男性不孕症的主要因素。精子發育是一個很複雜的過程,主要受到一連串位於體染色體及性染色體的基因所調控。“Deleted in azoospermia” gene (DAZ)被認為是和人類不孕症相關的主要基因。DAZ 基因家族主要包含有三個同源基因,分別是BOULE,DAZL及DAZ 。至目前為止,許多相關的動物研究皆指出DAZL (DAZ-like) 是生殖細胞的特異性基因,且被認為是和人類精子發育過程缺失相關的基因。目前的報告顯示,位於DAZL編碼區的單一核苷酸多型性和精子發育過程缺失有很大的相關性,然而仍然沒有任何相關的研究去探討位於DAZL啟動子區域的單一核苷酸多型性和精子發育過程缺失的關聯性。因此本研究便是想試著去探討位於DAZL 啟動子區域的基因變異是否會導致男性不孕症的產生。我們首先使用luciferase reporter assay 找出DAZL的functional promoter的區域。我們也在生物資訊的資料庫找到9個位於DAZL啟動子區域的SNPs;另外利用定序的方式,在生殖能力正常的台灣男性中發現3個SNPs是生物資料庫所沒有的。我們並發現SNP -844G/A在男性不孕症發生率較高。於是,我們試著探討包含-844G/A在內的單一核苷酸多型性在功能上的意義,利用reporter assays 及DNA-EMSA 的實驗證實一些SNPs確實會降低轉錄調控活性及影響轉錄調控因子的結合能力。進一步的利用supershift及overexpression的實驗,認為SNP -844G/A會干擾轉錄因子NRF-1 的結合能力進而影響DAZL基因的轉錄活性。另外一方面,我們利用bisulfite DNA sequencing 的實驗去觀察體細胞及精子DNA位於DAZL啟動子區域CpG 島的甲基化程度。由實驗結果,我們部份解答epigenetic的機制如何調控生殖細胞DAZL基因的表現，以及DAZL啟動子區域的基因變異在男性造精障礙所扮演之角色。

ABSTRACT
It is estimated that 10-15% of couples are infertile and male factors account for about half of the cases. The causes of male infertility are largely unknown. Of male-factor infertility, defects in spermatogenesis or spermiogenesis account for the majority of cases. Spermatogenesis is a complex process regulated by a network of genes or gene family located on autosomes and on sex chromosomes. In humans, the “deleted in azoospermia” gene (DAZ) is considered a critical sterility-associated gene. The DAZ gene family consists of three orthologs: BOULE, DAZL and DAZ. In all animals studied thus far, DAZL (DAZ-like) is a germ cell–specific autosomal gene and a strong candidate gene for human spermatogenic failure. Polymorphisms located within the DAZL coding region have been implicated in human spermatogenic failure. However, there has been no study which was carried to investigate the association between the polymorphisms of regulatory region in DAZL gene and human spermatogenic failure. In this study, we sought to identify genetic variants located within the DAZL promoter region which may confer susceptibility to male infertility. First, we used reporter assay to define the human DAZL functional promoter elements. Using dual luciferase reporter assay, we found the region about 1Kb located upstream of DAZL harbors the functional elements for regulation of human DAZL gene. By searching the SNP databases, 9 SNPs were found to be located within the DAZL promoter region. We sequenced the promoter region of DAZL in men with proven sterility and infertility and three novel SNPs were identified in the Taiwanese men. Mutagenesis and reporter assays were used to evaluate the functional significance of SNPs. We found some of these SNPs were significantly associated with decreased luciferase expression level. We used electrophoretic mobility shift assays (EMSA) to evaluate transcriptional factor binding status for some of these SNPs. We found one of these SNPs, -844G/A was associated with decreased binding of the transcriptional factors (NRF1). By using bisulfite DNA sequencing, we also identified the differentially methylated regions of CpG islands of the DAZL promoter region. The results provide evidence that (1) epigenetic mechanism is involved in the regulation of DAZL gene expression in germ cell; (2) regulatory SNPs of DAZL may confer susceptibility to human spermatogenic defect.

TABLE OF CONTENTS

ABSTRACT IN CHINESE………………………………………………i
ABSTRACT IN ENGLISH……………………………………………iii
ACKNOWLEDGMENTS…………………………………………………v
TABLE OF CONTENTS………………………………………………vi
LIST OF TABLES……………………………………………………xi
LIST OF FIGURES…………………………………………………xii

INTRODUCTION…………………………………………………………1
1 DAZ (Deleted in AZoospermia) gene family…………………1
2 Evolution of DAZ family………………………………………1
3 Pathogenesis of male infertility………………………1
4 Process of mammalian spermatogenesis…………………2
5 Expression patterns and functions of DAZ in the 
    spermatogenesis…………………………………………………3
6 Y chromosome and male infertility…………………………4
7 Role of the DAZ genes in male fertility…………………5
8 Polymorphisms of DAZL may confer susceptibility to 
    spermatogenic failure…………………………………………5
9 Regulatory SNP……………………………………………………6
9.1Cis-acting rSNPs………………………………………………7
10 Epigenetic modification of the genome :DNA
methylation……………………………………………………………7
10.1 CpG island……………………………………………………8
10.2 Cytosine methylation………………………………………8
10.3 DNA methylation function…………………………………8
11 Objectives of this study……………………………………9
MATERIALS AND METHODS……………………………………………10
1 Use of online bioinformatics tools………………………10
2 Direct Sequence for controls and infertile men………10
2.1 Subjects……………………………………………………10
2.2 Polymorphism screening…………………………………10
3 Statistical analysis…………………………………………11
4 Predictive the transcriptional factors binding  
    site………………………………………………………………11
5 Construction of luciferase reporter assay  
    plasmids…………………………………………………………11
5.1 Primer design………………………………………………11
6 Plasmid DNA preparation and DNA purification…………12
6.1 PCR clean up………………………………………………12
6.2 Gel Extraction……………………………………………13
6.3 Midi/Maxipreparation of plasmid DNA…………………14
7 Cell culture……………………………………………………16
7.1 Maintenance and Subculture……………………………16
8 Luciferase Reporter Assay………………………………… 16
8.1 Transient Transfection…………………………………16
8.2 Dual luciferase assay……………………………………17
8.2.1 Preparation of Cell Lysates Using Passive 
             Lysis Buffer…………………………………………17
8.2.2 Dual-Luciferase® Reporter Assay Protocol……17

9 Site-directed mutagenesis……………………………………18
9.1 Primer designs……………………………………………19
10 Statistical analysis…………………………………………20
10.1 Materials…………………………………………………20
10.2 Method………………………………………………………20
11 Computational analysis………………………………………20
11.1 Restriction enzyme site prediction………………20
11.2 Multiple sequence alignment…………………………20
12 DNA -electrophoretic mobility shift assay……………21
12.1Prepare the nuclear protein extract………………21
12.2 32P isotope label DNA probe…………………………22
12.2.1 Label DNA probe with T4 kinase……………………22
12.2.2 purification of labeled probe with G50 micro 
         column………………………………………………………22
12.3 Prepare reaction mixture for gel shift assay………23
13 SDS-Polyacrylamide Gel Electrophoresis and Western  
     Blot……………………………………………………………24
13.1 Materials………………………………………………24
13.2 Method……………………………………………………26
14 Analysis of DNA methylation………………………………27
14.1 CpG island prediction of DAZL………………………27
14.2 Sperm preparation by density gradients…………28
14.3 Mini-preparation of semen DNA………………………28
14.4 Genomic DNA treated with sodium bisulfite………29
14.5 Bisulfite sequencing…………………………………31

RESULTS……………………………………………………………32
1 Define the human DAZL promoter elements…………………32
2 Identify regulatory SNPs which may be associated with male infertility……………………………………………………32
2.1 Database search for polymorphisms located at the DAZL promoter area…………………………………………………32
2.2 Prediction the transcription factor binding sites…32
2.3 Reporter assay for the regulatory SNPs………………33
3 Identify regulatory SNPs which may confer
    susceptibility to spermatogenic defect in the   
    Taiwanese men…………………………………………………33
3.1 Polymorphism screening by sequence analysis of DAZL 
      promoter area………………………………………………33
3.2 Novel SNPs……………………………………………………33
3.3 Allelic and Genotypic Frequencies in Fertile and 
      Infertile Men…………………………………………………34
3.4 Linkage Disequilibrium (LD) Between Different   
      SNPs……………………………………………………………34
3.5 Haplotype Analysis…………………………………………35
4 Confirm transcriptional factors binding by the   
    supershift assay………………………………………………35
5 Identify CpG islands located within the DAZL promoter
     …………………………………………………………………36

DISCUSSIONS…………………………………………………………37
1 The molecular mechanisms which are involved in 
    transcriptional regulation of DAZL………………………37
2 Epigenetic regulation of gene expression………………37
3 How genetic factors affect DAZL gene expression………38
4 Role for DNA methylation in the control of tissue-
    specific expression……………………………………………38
5 Testis-specific transcriptional factors in male germ-
    cell differentiation…………………………………………39
6 Epigenetic factors which may affect DAZL 
    expression………………………………………………………39

CONCLUSION………………………………………………………41
REFERENCES……………………………………………………………42
FIGURE…………………………………………50-52;54-56;62-70
TABLE………………………………………………………53;57-61
APPENDIX………………………………………………………………71
CURRICULUM VITAE…………………………………………………72
                                    

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校內：2010-08-22公開
校外：2010-08-22公開

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