人β-簇珠蛋白基因LCR调控元件中HS3与反式调控因子相互作用的研究

人β-簇珠蛋白基因LCR调控元件位于ε-珠蛋白基因5’上游20Kb内,由四个DNase-Ⅰ超敏感点(HS1-HS4)组成。已有转基因实验表明,HS3可能与发育早期的胚胎型ε-珠蛋白基因表达调控相关。基因调控是通过调控元件与调控因子的相互作用而完成的。为了揭示HS3的调控作用,我们选用了发育不同时期的小鼠胚胎造血组织作为材料,用凝胶电泳阻抑法,分析了调控因子与HS3的结合。我们的实验结果表明,怀孕13天和18天的小鼠胚胎造血组织的核蛋白因子与HS3的结合模式完全不同;同时我们用Southwestern Blot的方法进一步证明了这种差异性的存在。现已知HS3中有GA-TA和CACCC两类转录因子结合的motif,采用竞争实验分析,发现CACCC motif对结合区带没有竞争作用,而GATA有竞争作用;另外还存在没有被竞争的条带,我们推测它们可能是一类新的、发育时期专一性的核蛋白因子。Western Blot的实验结果表明,在基因调控过程中起到重要作用的红系转录因子GATA家族中的GATA-1和GATA-2的表达也具有时期专一性:即怀孕13天的小鼠造血组织中表达GATA-2,不表达GATA-1;而怀孕18天的小鼠造血组织中则表达GATA-1,不表达GA-TA-2。因此,我们推测,HS3很可能参与时期专一性的基因表达调控,其中时期专一性的蛋白因子可能起到了重要作用。     

人β—簇珠蛋白基因LCR调控元件中HS3与反式调控因子相互作用的研究

Upstream of the human epsilon-globin gene is the Locus Control Region (LCR) of the human beta-globin cluster, which consists of four DNase-I hypersensitive sites(HS1-HS4). It has been reported in transgenic experiments that HS3 preferentially regulates epsilon-globin gene expression. In order to elucidate the regulatory function of HS3 in the expression of globin gene, nuclear extracts from mouse hematopoietic tissues at several developmental stages were prepared and the binding of the nuclear factors to HS3 was analysed by using electrophoresis mobility shift assay(EMSA). Our results showed that the binding patterns of HS3 with nuclear extracts of mouse hematopoietic tissues at day 13 and day 18 of gestation were completely different; furthermore, by Southwestern Blot, the distinction between both stages was also demonstrated. It has been known that GATA and CACCC binding motifs are contained within HS3 core region. Using competitive gel-retardation assay, we found that no shift bands could be competed by using CACCC motif as a competitor. However one shift band at day 13 and day 18 of gestation could be competed respectively by using GATA motif as a competitor. We suggested that the shift bands, which could not be competed by both motifs, might be novel and stage-specific factors. In addition, by using Western Blot, we demonstrated that the two shift bands at day 13 and day 18 of gestation, competed by GATA motif, were GATA-2 and GATA-1 respectively: GATA-1 was expressed in mouse hematopoietic tissues at day 18 of gestation and not expressed at day 13 of gestation; however, GATA-2 was only expressed in mouse hematopoietic tissues at day 13 of gestation. According to these results, we speculated that HS3 might play an important role in regulation of stage-specific expression of globin genes through interaction between stage-specific nuclear factors and HS3.