Reddy P M, Stamatoyannopoulos G, Papayannopoulou T, Shen C K
Section of Molecular and Cell Biology, University of California, Davis 95616.
J Biol Chem. 1994 Mar 18;269(11):8287-95.
In order to gain further insights of the regulatory mechanisms of human beta-like globin gene switch during erythroid development, we have studied protein-DNA interaction in vivo at the human adult beta and fetal gamma globin promoters and their upstream enhancer, 5'HS-2, in purified human adult erythroblasts, in which the beta, but not gamma or epsilon, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with those of different non-erythroid human tissues, an embryonic/fetal erythroid cell line K562, and several non-erythroid human cell lines. Protein-DNA binding in the beta globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the gamma globin promoters were bound with specific nuclear factors in the expressing K562 cells, but not in non-erythroid tissues or cell lines. Relatively weak protein binding could also be detected in the vicinities of the two CCAAT boxes of the inactive gamma globin promoters in the adult erythroblasts. Although the patterns of nuclear factor-DNA interaction in vivo at the NF-E2/AP1, GATA-1, and GT-I motifs of 5'HS-2 enhancer in adult erythroblasts are similar to those in K562 cells, we have identified a previously undetected factor-binding motif of 5'HS-2 that is protected only in the adult erythroblasts. This motif is identical in sequence to the 3'-CACC box of the human beta globin promoter, and it is well conserved at the same location among all mammalian 5'HS-2 enhancers, suggesting an important regulatory role of this element in human beta globin gene transcription in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor binding in non-erythroid tissues, but two of them, NF-E2/AP1 and GT-I, are bound with factors in some non-erythroid cell lines but not in others. The functional implications of these genomic footprinting data and the tissue-specific CpG methylation patterns of the beta-like globin promoters we obtained by genomic sequencing are discussed in terms of positive and negative regulation of the human beta-like globin switch during erythroid development.
为了进一步深入了解人类β样珠蛋白基因在红细胞发育过程中的调控机制，我们研究了在纯化的成人红细胞中，人类成人β珠蛋白和胎儿γ珠蛋白启动子及其上游增强子5'HS-2处的体内蛋白质-DNA相互作用，其中β珠蛋白基因而非γ或ε珠蛋白基因正在活跃转录。对成人红细胞进行的这种基因组足迹分析是与不同的非红细胞人类组织、胚胎/胎儿红细胞系K562以及几种非红细胞人类细胞系的分析一起进行的。β珠蛋白启动子中的蛋白质-DNA结合，特别是在两个CACC启动子框和CCAAT框处，仅在成人红细胞中可检测到。正如预期的那样，γ珠蛋白启动子在表达的K562细胞中与特定的核因子结合，但在非红细胞组织或细胞系中则不然。在成人红细胞中，也可在无活性的γ珠蛋白启动子的两个CCAAT框附近检测到相对较弱的蛋白质结合。尽管成人红细胞中5'HS-2增强子的NF-E2/AP1、GATA-1和GT-I基序处的体内核因子-DNA相互作用模式与K562细胞中的相似，但我们发现了一个以前未检测到的5'HS-2因子结合基序，该基序仅在成人红细胞中受到保护。该基序的序列与人类β珠蛋白启动子的3'-CACC框相同，并且在所有哺乳动物的5'HS-2增强子的相同位置都高度保守，这表明该元件在成人红细胞中人类β珠蛋白基因转录中具有重要的调控作用。5'HS-2的上述所有四个基序在非红细胞组织中都没有核因子结合，但其中两个，NF-E2/AP1和GT-I，在一些非红细胞细胞系中与因子结合，而在其他细胞系中则不然。我们通过基因组测序获得的这些基因组足迹数据以及β样珠蛋白启动子的组织特异性CpG甲基化模式的功能意义，将根据红细胞发育过程中人类β样珠蛋白开关的正负调控进行讨论。
