A4GALT基因轉錄調控機制的研究

Abstract

P1醣抗原是屬於紅血球抗原系統中的一員，在族群中可區分為P1表現型與P1非表現型兩種型別。P1醣抗原由Galα1-4Galβ1-4GlcNAc的結構所決定，由過去的研究已推斷紅血球表面P1醣抗原的表現極有可能是由α1,4-galactosyltransferase (A4GALT)所負責；而個體間P1抗原表現型與非表現型可能是由A4GALT基因表現量的差異所造成，因此我們推測A4GALT是受到了不同的轉錄調控，造成了P1抗原表現或缺失。 為了要證明這個推測，首先必須要證明A4GALT在紅血球中的轉錄調控機制，找出表現A4GALT所需的轉錄因子。在實驗室之前的研究中，已證明P1抗原表現型與非表現型和A4GALT基因intron 1區域的8個single-nucleotide polymorphism (SNP)有強烈的關聯。我們由這些SNP位點推測出8個可能結合的轉錄因子。我們利用表現載體將這些轉錄因子大量表現於K562細胞中，而後進行qPCR，偵測這些轉錄因子對A4GALT基因表現的影響。 大量表現轉錄因子Ikaros或Aiolos後，利用qPCR測出A4GALT transcript約上升5倍。接著利用reporter assay測試Ikaros或Aiolos對P1抗原表現型與非表現型對偶基因(allele)作用後的差異，進一步確認轉錄因子Ikaros或Aiolos是否造成A4GALT基因表現差異，但實驗結果無法看出Ikaros和Aiolos對P1抗原表現型與非表現型對偶基因有明顯影響。我們推測可能需要多種轉錄因子的合作，使P1抗原表現型與非表現型對偶基因的轉錄活性有明顯差異，此研究還需要未來更多的實驗進一步探討，以了解A4GALT的轉錄調控和紅血球P1抗原表現型與非表現型的分子遺傳機制。

P1 antigen is glycosphingolipid in human erythrocyte membrane. Erythrocytes are classified according to the expression of P1 antigen: P1-positive (P1+) phenotype and P1-negative (P1-) phenotype. P1 antigenic structure is Galα1-4Galβ1-4GlcNAc and the enzyme responsible for synthesis P1 antigen would be α1,4-galactosyltransferase (A4GALT). The difference between P1+/- erythrocytes might result from the difference of A4GALT transcript levels. Hence, the different transcriptional regulation of the A4GALT gene might be a major factor determining P1+/- phenotypes. In order to prove this speculation, we try to prove the transcriptional regulatory mechanisms of the A4GALT and identify transcription factors which are required for A4GALT expression. In previous studies, 8 single-nucleotide polymorphisms (SNP) in A4GALT intron 1 were found with correlation to the P1+/- phenotypes. We speculated that those different eight transcription factors might recognize and bind to these eight SNPs. We used the expression vector to overexpress these transcription factors in K562 cells, and then quantify the expression level of A4GALT by qPCR. Overexpression of the transcription factor Ikaros or Aiolos increased the abundance of A4GALT transcripts (5 times). Then, we used the reporter assay to observe the difference caused by Ikaros or Aiolos between the P1+/- alleles in order to know whether Ikaros or Aiolos could modulate A4GALT expression. We found that Ikaros and Aiolos might not involve in the allele-specific modulation of A4GALT. We suggest that the significant difference between P1+ and P1- allele requires the cooperation of various transcription factors. We hope to understand the transcriptional regulatory mechanisms of A4GALT expression and the relation between A4GALT and P1 antigen polymorphism.