Identification of γ-blobin activation factor

为了检测ZFF29蛋白在非红系细胞中对β样珠蛋白基因转录的影响，构建了ZFF29基因的表达载体，并将其导入非红系HL60和KG-1。RT-PCR分析G418耐药细胞珠蛋白基因的表达。强制表达ZFF29基因不能增加或诱导两种细胞系中珠蛋白基因的转录。然而，有趣的是，ε-珠蛋白基因在KG-1细胞中有结构性表达，而在HL60细胞中不表达。首先，通过反向聚合酶链式反应克隆了FKLF-2基因的上游区域，并通过引物延伸分析确定了转录起始点。限制性内切酶序列缺失突变表明，转录起始点的370个碱基的序列是完整启动子活性所必需的。FKLF-2启动子在K562细胞中的活性是在COS7细胞中的2倍，GATA-1是该启动子的潜在反式激活剂。为了检测FKLf(KLF11)能否激活γ-珠蛋白基因在成年红系细胞中的表达，将FKLf基因作为转基因基因导入MEL细胞和携带人γ-珠蛋白基因的小鼠体内。FKLf转基因小鼠或MEL细胞中γ珠蛋白基因的表达频率和数量均增加，而在成体红细胞生成环境中，FKLf基因不能激活沉默的γ珠蛋白基因。这些结果提示，逆转录病毒载体转导FKLf可用于治疗镰状细胞病或β地中海贫血。

To test the activity of ZFF29 proteins on the β-like globin gene transcription in non-erythroid cells, expression vectors of ZFF29 genes were constructed and transduced into non-erythroid lines, HL60 and KG-1. mRNA expression of globin genes in G418-resistant cells were analyzed by RT-PCR. Forced expression of ZFF29 genes failed to increase or induce globin gene transcription in neither type of cell lines. It was interesting, however, that ε-globin gene was constitutively expressed in KG-1 cells but not in HL60 cells.Molecular mechanisms underlying transcriptional control of FKLF-2 (KLF13) gene expression in erythroid cells were analyzed by using erythroid (K562) cells and non-erythroid (COS7) cells. First of all we cloned upstream region of FKLF-2 gene by inverse PCR, and determined transcription initiation site by primer extension analysis. Sequential deletion mutants by restriction enzymes revealed that a 370 bp sequence to the transcription initiation site was necessary for the full promoter activity. Features of the FKLF-2 promoter were that 1)the promoter activity was two times more active in K562 cells than in COS7 cells ; 2)GATA-1 was potential trans activator of the promoter. Our results may shed lights on the molecular control of the erythroid cell development.To test whether FKLF (KLF11) can activate γ-globin gene expression in adult erythroid cells, FKLF gene was introduced into MEL cells and mice carrying human γ-globin gene as a trans-gene. Frequency and amount of γ globin gene expressed in the MEL cells or the transgenic mouse were enhanced upon induction of FKLF trans-gene, while FKLF failed to activate the silent γ globin gene in the environment of adult erythropoiesis. These results suggest that transduction of FKLF by retrovirus vector may be used for the purpose of treatment of patients with sickle cell disease or β-thalassemia.