Semenza G L, Dureza R C, Traystman M D, Gearhart J D, Antonarakis S E
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Mol Cell Biol. 1990 Mar;10(3):930-8. doi: 10.1128/mcb.10.3.930-938.1990.
Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.
促红细胞生成素（EPO）是哺乳动物红细胞生成的主要体液调节因子。单拷贝的EPO基因通常在肝脏和肾脏中表达，贫血或给予氯化钴可诱导其转录增加。为了鉴定负责调控表达的顺式作用DNA序列，通过显微注射分别包含人类EPO基因、0.7 kb的3'侧翼序列以及0.4或6 kb的5'侧翼序列的4千碱基对（kb）（tgEPO4）或10 kb（tgEPO10）克隆DNA片段，培育出了转基因小鼠。tgEPO4小鼠在肝脏中表达转基因，其表达可被贫血或氯化钴诱导，在肾脏中也有表达，但该表达不可被诱导，此外在其他正常情况下不表达EPO的组织中也有表达。在tgEPO10小鼠中，仅在贫血或经钴处理的小鼠肝脏中检测到人类EPO RNA。tgEPO4和tgEPO10小鼠均出现红细胞增多症，表明在肝脏中转录的人类EPO RNA具有功能。这些结果表明：（i）一个肝脏诱导元件定位在包含该基因、0.4 kb的5'侧翼序列和0.7 kb的3'侧翼序列的4 kb范围内；（ii）一个负调控元件位于基因5'端的0.4至6 kb之间；（iii）诱导性肾脏表达所需的序列位于基因5'端大于6 kb或3'端大于0.7 kb处。核糖核酸酶保护分析显示，贫血转基因小鼠肝脏和缺氧人类肝癌细胞中的人类EPO RNA起始于多个位点，其中只有一部分位点在非贫血转基因肝脏和人类胎儿肝脏中被利用。
