Lee J S, Lee C H, Chung J H
Molecular Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10051-5. doi: 10.1073/pnas.96.18.10051.
Erythroid Krüppel-like factor (EKLF), which binds to the CACCC box in the beta-globin promoter, is required for the expression of the beta-globin gene in adult erythroid cells. It was recently demonstrated that EKLF is also required for the activity of the beta-globin locus control region (LCR) 5'HS3. Some evidence suggests that the LCR and the beta-globin promoter interact in adult erythroid cells, and the network of protein-protein interactions that exists between these two elements may regulate how EKLF is recruited to the LCR. In this report, we use the PIN*POINT assay to study the role of the promoter on the recruitment of EKLF to 5'HS2 and 5'HS3 of the LCR. We find that recruitment of EKLF to 5'HS2 requires the TATA box, but recruitment to 5'HS3 depends on the CACCC and TATA boxes of the beta-globin promoter. Furthermore, recruitment of EKLF to 5'HS3 only occurred in beta-globin-expressing murine erythroid leukemia cells, whereas recruitment of EKLF to 5'HS2 occurred in both gamma-globin-expressing K562 cells and murine erythroid leukemia cells. Unlike EKLF, Sp1, which also binds to CACCC boxes, is not recruited to 5'HS3. We have also examined how one 5'HS affects the recruitment of EKLF to another 5'HS. We have found that the recruitment of EKLF to 5'HS3 depends on the presence of 5'HS2 in cis, but the recruitment to 5'HS2 does not depend on 5'HS3. Based on these results, we present a model that illustrates how EKLF may be recruited to the beta-globin locus.
红系Krüppel样因子（EKLF）可与β-珠蛋白启动子中的CACCC盒结合，是成年红系细胞中β-珠蛋白基因表达所必需的。最近有研究表明，EKLF也是β-珠蛋白基因座控制区（LCR）5'HS3活性所必需的。一些证据表明，LCR和β-珠蛋白启动子在成年红系细胞中相互作用，这两个元件之间存在的蛋白质-蛋白质相互作用网络可能调节EKLF如何被招募到LCR。在本报告中，我们使用PIN*POINT分析来研究启动子在将EKLF招募到LCR的5'HS2和5'HS3中的作用。我们发现，将EKLF招募到5'HS2需要TATA盒，但招募到5'HS3则取决于β-珠蛋白启动子的CACCC盒和TATA盒。此外，EKLF仅在表达β-珠蛋白的小鼠红白血病细胞中被招募到5'HS3，而在表达γ-珠蛋白的K562细胞和小鼠红白血病细胞中均可将EKLF招募到5'HS2。与EKLF不同，同样与CACCC盒结合的Sp1不会被招募到5'HS3。我们还研究了一个5'HS如何影响EKLF对另一个5'HS的招募。我们发现，将EKLF招募到5'HS3取决于顺式存在的5'HS2，但招募到5'HS2则不依赖于5'HS3。基于这些结果，我们提出了一个模型，阐明了EKLF可能如何被招募到β-珠蛋白基因座。
