- 學位論文
SNP rs311103於紅血球Xg及CD99抗原表現型 形成之分子機制研究
Study of the molecule mechanism connecting SNP rs311103 and the blood group Xg /CD99 phenotype formation
摘要
Xga是表現於紅血球上的血型抗原，此抗原是否表現會因個人不同，分成Xg (a+)及Xg (a-)；CD99是表現於細胞表面的抗原，而CD99及Xga兩種抗原的表現性具有性別上的差異性。過去的研究已知，CD99的表現量在紅血球上呈現高低的差異，且其在紅血球上表現量的高低跟Xga是否表現有著共同調控的關係。研究指出，當Xg(a+)表現型者時，CD99表現量高；Xg(a-)表現型者，CD99表現量低；但在男性的紅血球上會呈現第三種表現型；特定比例的Xg(a-)男性，CD99卻呈現高表現量。然而Xga及CD99抗原特殊表現型之分子遺傳機制尚未明瞭。 本研究室首先取不同個體之血液的gDNA進行研究，依據其Xga及CD99表現型將其分類成上述三種類型，接著取16人的gDNA以NGS進行性染色體大區域定序，範圍包含XG及CD99兩個基因，並排列出此區域之單一核苷酸多型性(single nucleotide polymorphism,簡稱 SNP)。經由分析結果發現，位於CD99及XG 兩個基因間的SNP rs311103 (G/C) 的基因型吻合三種表現型的分佈，可能對兩者間的調控有很大的關聯性。我們先利用了報導基因法確認了SNP rs311103區域，扮演著enhancer的角色。之後進一步使用程式預測可能與此SNP區域結合呈現親和力差異的轉錄因子，得到GATA family (GATA1~6)、LEF1等轉錄因子的預測。 我們利用K-562細胞可進一步被誘導分化為紅血球性狀做為模型，以及real-time PCR去分析這些轉錄因子在細胞中本身表現量的差異，發現GATA1於K-562細胞分化時表現量增加；接著利用報導基因分析法去做進一步的檢驗，經由分析我們推測GATA1最有可能扮演刺激此SNP的角色。從EMSA實驗中，我們觀察到 GATA1與SNP rs311103基因型為G時可能具有專一性結合。後續實驗我們想利用ChIP進一步證實兩者間的結合作用，並利用報導基因法觀察是否影響兩基因之啟動子以及3C (Chromosome conformation capture) 等實驗更進一步去證實GATA1在SNP rs311103異合子型的細胞株中，對於不同對偶基因型的XG及CD99基因間的表現是否有影響，來證實GATA1轉錄因子扮演差異性調控，因而造成Xga及CD99於紅血球上呈現表現差異之分子機制。
並列摘要
Xga is a blood group antigen present on red blood cells (RBCs). The expression of the Xga blood group antigen on red blood cells is different among individuals, subdivided into Xg (a+) and Xg (a-). CD99 molecule is a cell surface glycoprotein. The expression of these two antigens are different in gender. According to previous studies, CD99 is expressed on all human cells, but on red cells, the level of expression shows individual variation. Xg (a+) RBCs all belong to the group of CD99 high expressors and Xg (a-) RBCs belong to the group of CD99 low expressors. Interestingly, some Xg (a-) RBCs from males express high level expression of CD99. However, the molecule genetic mechanism of the special phenotypic correlation between Xga and CD99 antigens is still unclear. In our investigation, we collected the gDNA from different people, and divided the samples into three groups according to the expression levels of XG and CD99 antigens. We took 16 gDNA samples and sequencing the XG and CD99 gene region by Next Generation Sequencing (NGS). Then we arranged all single-nucleotide polymorphisms (SNPs). Through the data analyzed, we found the SNP rs311103 (G/C), located between the XG gene and the CD99 gene, is associated with the three phenotype groups. In the present study, we aim to demonstrate the transcription factor leading to the different expression levels of the XG and CD99 genes. Several transcription factors, including GATA family (GATA1 to GATA6) and LEF1, were predicted in silico to have different binding affinity to the SNP rs311103 regions with the different genotypes of G and C nucleotides. In further investigation using K-562 erythroleukemia cells as a study model, we found that the expression of the GATA1 gene increased after the K-562 cells differentiated into erythrocytic cells. Ectopic expression of the GATA1 transcription factor also lead to a significant induction of the transcriptional activity in the reporter construct with the SNP rs311103-G form genotype. These result highly suggested that GATA1 may play an important role in activating the XG gene and CD99 gene with the G form genotype at the SNP rs311103 position. In future investigations, we plan to demonstrate whether GATA1 will differentially stimulate the expression the XG gene and CD99 gene with the G form and C form genotype in a SNP heterozygous cell line to substantiate functional role of GATA1 transcription factor in the co-regulation of CD99 and XG blood group phenotypes.
參考文獻
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