Lloyd J A, Lee R F, Menon A G, Lingrel J B
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Ohio 45267-0524.
Prog Clin Biol Res. 1989;316A:139-48.
Five broad regions in and near the A gamma globin gene specifically bind proteins in K562 nuclear extracts. Two are located 5' of the gene, one between -1349 and -1094, and the other between -384 and +52. Each of the two introns has a binding region, +184 to +284 and +930 to +1209. The fifth binding region is within the A gamma globin gene enhancer, a 750 bp Hind III fragment located 3' to the gene. The nuclear factors which bind to any or all of these regions may be important for control of regulation of the A gamma globin gene. Since HPFH point mutations occur at -175, -196 and -202, we investigated K562 nuclear factor binding to this region more closely. Footprints were obtained for two binding sites, one from -168 to -185 (site II) and the other from -267 to -293 (site I). Site II contains an octamer sequence (ATGCAAAT) important for protein binding and expression of the histone H2b and immunoglobulin genes. The HPFH mutation at -175 changes the T in the octamer sequence to a C. Site I is upstream of the bases affected by HPFH mutations. A A gamma globin gene promoter - CAT reporter fusion gene was constructed with a clustered - base substitution of site I or a T----C point mutation at -175. The mutation in site I decreases CAT expression 20X compared to wild-type in erythroid and non-erythroid cells. Site I probably binds a positive regulator. The T----C change at -175 increases expression 2-3X over wild-type in erythroid cells, but not in non-erythroid cells. This increase correlates with the effect of the naturally occurring HPFH, and may result from decreased binding of a negative effector, and/or increased positive factor binding.
γ珠蛋白基因及其附近的五个广泛区域特异性结合K562细胞核提取物中的蛋白质。其中两个位于基因的5'端，一个在-1349至-1094之间，另一个在-384至+52之间。两个内含子各有一个结合区域，分别为+184至+284和+930至+1209。第五个结合区域位于γ珠蛋白基因增强子内，是位于基因3'端的一个750bp的Hind III片段。与这些区域中的任何一个或所有区域结合的核因子可能对γ珠蛋白基因的调控控制很重要。由于遗传性胎儿血红蛋白持续存在（HPFH）点突变发生在-175、-196和-202位点，我们更深入地研究了K562核因子与该区域的结合情况。获得了两个结合位点的足迹，一个从-168至-185（位点II），另一个从-267至-293（位点I）。位点II包含一个对组蛋白H2b和免疫球蛋白基因的蛋白质结合及表达很重要的八聚体序列（ATGCAAAT）。-175位点的HPFH突变将八聚体序列中的T变为C。位点I位于受HPFH突变影响的碱基上游。构建了一个γ珠蛋白基因启动子-CAT报告融合基因，该基因在-175位点有一个位点I的簇状碱基替换或T→C点突变。与野生型相比，位点I的突变在红细胞和非红细胞中使CAT表达降低了20倍。位点I可能结合一种正调节因子。-175位点的T→C变化在红细胞中使表达比野生型增加了2至3倍，但在非红细胞中没有增加。这种增加与天然存在的HPFH的作用相关，可能是由于负效应物结合减少和/或正因子结合增加所致。
