Regulation of human pyruvate kinase gene in red cells

为了确定人红细胞型丙酮酸激酶（R-PK）在红细胞特异性表达的调控基因序列，我们制备了2个人PK迷你基因构建体，并建立了19个转基因小鼠。虽然我们已经证明含有4个GATA和2个CACCC基序的人R-PK启动子在K562和MEL细胞中都具有突出的转录活性，但该启动子不足以在PK转基因小鼠中进行体内转录。我们在人类PK lr基因上游发现了两个DNaseI敏感位点（HS），加上近端HS （HS- i）使连接的PK转基因以红系特异性方式表达，表明HS- i是人类PK lr基因的红系特异性增强子。hs - 1含有1个GATA、1个CACCC和1个富嘌呤基序（AGGGAGAAG），其结构与在大鼠PK lr基因中发现的红细胞特异性增强子相似。我们建立了3个在红细胞中过表达人R-PK的PK转基因小鼠系。将人β样珠蛋白位点调控序列mu’LCR与R-PK启动子和编码序列连接，并用于显微注射。PK转基因小鼠的红细胞PK活性约为正常对照（83-122 IU/gHb，正常范围41.6*5.8）的2-3倍，PK酶图显示人类R-PK活性。由于mu' LCR不具有位置无关、拷贝数依赖的PK转基因表达，因此LCR可能受到位置效应的影响，并发挥红细胞特异性增强子的作用。

To identify regulatory gene sequence(s) responsible for the erythroid-specific expression of human erythroid-type pyruvate kinase (R-PK), we prepared two human PK mini-gene constructs and established 19 lines of transgenic mice.Although we have shown that the human R-PK promoter containing 4 GATA and 2 CACCC motifs had prominent transcription activity in both K562 and MEL cells, the promoter was not sufficient for in vivo transcription in the PK transgenic mice.We identified two DNaseI hypersensitive sites (HS) upstream of the human PK LR-gene, and an addition of the proximal HS (HS-I) enabled the linked PK transgene to express in an erythroid-specific manner, suggesting that the HS-I is an erythroid-specific enhancer of the human PK LR-gene.The HS-I contained one GATA, one CACCC and one purine rich motif (AGGGAGAAG), and the organization was similar to the erythroid-specific enhancer which had been identified in rat PK LR-gene.We have established three PK transgenic mice lines which overexpress human R-PK in red cells.The mu' LCR, the regulatory sequence of human beta-like globin locus was linked to the R-PK promoter and coding sequences, and used for microinjection.Red cell PK activities of the PK transgenic mice were about 2-3 fold of normal controls (83-122 IU/gHb, normal range 41.6*5.8), and human R-PK activities were demonstrated in the PK zymograms.Since the mu' LCR did not confer the position-independent, copy number-dependent expression of the PK transgene, the LCR might be influenced by position effect, and functioned as an erythroid-specific enhancer.