Caterina J J, Ryan T M, Pawlik K M, Palmiter R D, Brinster R L, Behringer R R, Townes T M
Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294.
Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1626-30. doi: 10.1073/pnas.88.5.1626.
The human beta-globin locus control region (LCR) is essential for high-level expression of human epsilon-, gamma-, and beta-globin genes. Developmentally stable DNase I hypersensitive sites (designated HS) mark sequences within this region that are important for LCR activity. A 1.9-kilobase (kb) fragment containing the 5' HS 2 site enhances human beta-globin gene expression 100-fold in transgenic mice and also confers position-independent expression. To further define important sequences within this region, deletion mutations of the 1.9-kb fragment were introduced upstream of the human beta-globin gene, and the constructs were tested for activity in transgenic mice. Although enhancer activity was gradually lost with deletions of both 5' and 3' sequences, a 373-base-pair (bp) fragment retained the ability to confer relative position-independent expression. Three prominent DNase I footprints were observed in this region with extracts from the human erythroleukemia cell line K-562, one of which contained duplicated binding sites for transcription factor AP-1 (activator protein 1). When the 1.9-kb fragment containing an 18-bp deletion of the AP-1 binding sites was tested in transgenic mice, enhancer activity decreased 20-fold but position-independent expression was retained.
人类β-珠蛋白基因座控制区（LCR）对于人类ε-、γ-和β-珠蛋白基因的高水平表达至关重要。发育稳定的DNA酶I超敏位点（称为HS）标记了该区域内对LCR活性重要的序列。一个包含5' HS 2位点的1.9千碱基（kb）片段在转基因小鼠中可将人类β-珠蛋白基因表达增强100倍，并且还赋予位置非依赖性表达。为了进一步确定该区域内的重要序列，在人类β-珠蛋白基因上游引入了1.9 kb片段的缺失突变，并在转基因小鼠中测试了构建体的活性。尽管随着5'和3'序列的缺失，增强子活性逐渐丧失，但一个373碱基对（bp）的片段仍保留了赋予相对位置非依赖性表达的能力。用人红白血病细胞系K-562的提取物在该区域观察到三个明显的DNA酶I足迹，其中一个包含转录因子AP-1（激活蛋白1）的重复结合位点。当在转基因小鼠中测试包含AP-1结合位点18 bp缺失的1.9 kb片段时，增强子活性降低了20倍，但位置非依赖性表达得以保留。
