轉錄因子EGR1在紅血球分化過程中的基因調控機制

Abstract

人類的P1PK血型系統，主要包含在紅血球表面的P1及Pk醣類抗原，此兩種抗原的生成主要皆由醣轉移酶α4Gal-T負責。編碼α4Gal-T的A4GALT基因，其表現量高低與P1抗原的含量高低成正相關，因此為決定P1/P2表現型的關鍵基因。 單核苷酸多型性 (single nucleotide polymorphism, SNP) 是由單一核苷酸的不同形成DNA序列上的差異，影響同一物種之間基因組的多樣性。在我們實驗室過去的研究發現，位於A4GALT基因1號內含子的SNP rs5751348 (SNP 6)，能夠決定A4GALT基因表現量的高低，進而影響P1/P2表型的差異。SNP造成的單一核苷酸差異使得特定轉錄因子在該序列上有不同程度的結合能力。我們發現，轉錄因子EGR1能夠結合在SNP 6的P1基因型上，造成A4GALT基因型P1及P2的差異性表現。 同時，我們還觀察到在紅白血病 (erythroleukemia) 細胞株中，EGR1的基因表現量比起EGR家族的其他轉錄因子高出許多，且在K-562細胞中，加入sodium butyrate使K-562分化為紅血球細胞，EGR1的表現也大幅提升，因此我們想探討在紅血球細胞中，EGR1的基因調控機制。 我們利用報導基因分析法 (reporter assay) 檢驗EGR1基因組的5'端調控區域，篩選出EGR1可能的調控區域，進而使用電腦軟體TRANSFAC®、MatInspector及PROMO，預測該序列可能高度結合的轉錄因子，候選出與血球系統有關的轉錄因子：KLF4、KLF6、ETS1、ETS2、GATA1、GATA2、Ikaros及SRF，再將其大量表現於K-562細胞中，觀察EGR1的基因表現量是否因此大幅提高，並確認此基因調控機制，與血球細胞分化之間的關聯。

The antigens in the P1PK blood group system includes glycosphingolipids P1 and Pk. The A4GALT-encoded galactosyltransferase α4Gal-T synthesizes both P1 and Pk antigens. The quantity of A4GALT expression has been shown to be positively correlated with the amount of P1 antigens and quantitative difference of P1 antigen expression corresponds to P1/P2 phenotype. Single nucleotide polymorphism (SNP) is a variation of single nucleotide that occurs at a specific position in the genome and the variation results in the polymorphic diversity within a population. Based on the previous studies in our lab, SNP 6, which locates on the intron 1 of A4GALT, has been demonstrated to correlate with the expression levels of A4GALT and consequently determines the P1/P2 phenotype. SNP in the regulatory region of a gene may affect the binding ability of a specific transcription factor on the located sequences. Our results revealed that the transcription factor EGR1 differentially binds to the SNP6 region with the different genotypes. The expression profiles of the EGR transcription factor family, EGR1~EGR4, were analyzed in the various erythroid-lineage cells. The results showed that EGR1 had higher expression level than the other EGR family members, EGR2-EGR4. Moreover, after treated with sodium butyrate to induce K-562 cells to differentiate into erythrocytic cells, the gene expression of EGR1 increased significantly. This evoked our curiosity about how EGR1 was regulated during erythroid differentiation. In this research, we aimed to elucidate the molecular details of the transcriptional regulation of EGR1 during erythroid differentiation. The 5’-region of EGR1 gene were analyzed by reporter assay and determined the region that showed higher transcriptional activity. The potential transcription factor binding motifs in the region were predicted using online databases, KLF4, KLF6, ETS1, ETS2, GATA1, GATA2, Ikaros and SRF were implicated to be the candidate transcription factors responsible for transcription activation. These candidate transcription factors had been reported to be associated with hematopoietic system. In the present study, these transcription factors were ectopically expressed in K-562 cells in order to analyze their ability to induce the transcriptional level of EGR1. However, none of these transcription factors showed the ability to induce the transcriptional activity of the specific 5’-region of EGR1. Further studies are needed to elucidate the regulatory mechanism for the EGR1 expression during erythroid differentiation.