Wada-Kiyama Y, Peters B, Noguchi C T
Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Jun 5;267(16):11532-8.
K562 human erythroleukemia cells constitutively express epsilon- and gamma- but not beta-globin genes. We have previously shown that the differential expression of globin genes observed in intact K562 cells could be simulated in vitro as K562 nuclear extract (NE) actively transcribes the epsilon-globin (with 2 kilobases of 5'-flanking sequence) and gamma-globin gene DNA templates but not beta-globin gene templates. We have now used the K562 in vitro transcription system to examine a silencer transcriptional control element which has been reported to be localized between -177 and -392 base pairs (bp) 5' of the canonical cap site for the epsilon-globin gene. We find that K562 NE has markedly reduced synthesis of RNA in vitro from epsilon-globin gene DNA deletion templates which contain the silencer sequence, or part thereof, but not the adjacent 5'-positive regulatory region (-453 to -535 bp). Furthermore, those transcripts generated in vitro from DNA templates extending to -453 bp or less of the epsilon-globin gene were not correctly initiated at the canonical cap site. Separating the K562 NE by ion exchange chromatography, we isolated a fraction (F175) transcriptionally active for all tested globin genes including the epsilon-globin gene containing the silencer sequence and a fraction (F50) which contains the trans-acting factors associated with the silencer activity. F50 showed a strong dose-dependent inhibitory effect on correctly initiated epsilon-globin gene transcription directed by either unfractionated K562 NE or F175. This suppression by F50 was not observed on transcriptional activity of the permissive adenovirus 2 major late promoter. In electrophoretic mobility shift assays using the epsilon-globin gene silencer region as probe, F50 and F175 exhibited different DNA binding protein patterns; a specific protein band in F50 appears to be associated with the silencer activity. These studies suggest that this protein may be specifically responsible for the activity of the silencer element of the epsilon-globin gene. The expression and silencing of the epsilon-globin gene during development may be modulated by the interactions of this protein with the cis-acting DNA silencer.
K562人红白血病细胞组成性表达ε-珠蛋白基因和γ-珠蛋白基因，但不表达β-珠蛋白基因。我们之前已经表明，在完整的K562细胞中观察到的珠蛋白基因的差异表达可以在体外模拟，因为K562核提取物（NE）能积极转录ε-珠蛋白（带有2千碱基的5'-侧翼序列）和γ-珠蛋白基因DNA模板，但不能转录β-珠蛋白基因模板。我们现在利用K562体外转录系统来研究一种沉默子转录控制元件，据报道该元件位于ε-珠蛋白基因经典帽位点5'端的-177至-392碱基对（bp）之间。我们发现，K562 NE从包含沉默子序列或其部分但不包含相邻5'-正调控区域（-453至-535 bp）的ε-珠蛋白基因DNA缺失模板体外合成RNA的能力明显降低。此外，从延伸至ε-珠蛋白基因-453 bp或更少的DNA模板体外产生的那些转录本在经典帽位点处没有正确起始。通过离子交换色谱法分离K562 NE，我们分离出了对所有测试的珠蛋白基因具有转录活性的一个组分（F175），包括含有沉默子序列的ε-珠蛋白基因，以及一个含有与沉默子活性相关的反式作用因子的组分（F50）。F50对由未分级的K562 NE或F175指导的正确起始的ε-珠蛋白基因转录表现出强烈的剂量依赖性抑制作用。在允许的腺病毒2主要晚期启动子的转录活性上未观察到F50的这种抑制作用。在使用ε-珠蛋白基因沉默子区域作为探针的电泳迁移率变动分析中，F50和F175表现出不同的DNA结合蛋白模式；F50中的一条特定蛋白带似乎与沉默子活性相关。这些研究表明，这种蛋白可能特异性地负责ε-珠蛋白基因沉默子元件的活性。ε-珠蛋白基因在发育过程中的表达和沉默可能通过这种蛋白与顺式作用DNA沉默子的相互作用来调节。
