Gutman P D, Cao S X, Dave H P, Mittelman M, Schechter A N
National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Gene. 1992 Jan 15;110(2):197-203. doi: 10.1016/0378-1119(92)90648-9.
To clarify the molecular mechanisms involved in the developmental control of hemoglobin-encoding genes we have been studying the expression of these genes in human cells in continuous culture. We have previously reported the presence of a transcriptional control element with the properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin-encoding gene [Cao et al., Proc. Natl. Acad. Sci. USA 86 (1989) 5306-5309]. We also showed that this silencer has stronger inhibitory activity in HeLa cells, as compared to K562 human erythroleukemia cells. Using deletion mutants and cis-cloned synthetic oligodeoxyribonucleotides in transient expression assays, nucleotide sequences responsible for this effect have now been further delimited to 44 bp located from -294 to -251 bp. Gel electrophoresis mobility shift assays and DNaseI footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. Two binding proteins are detected in K562 nuclear extracts, while only one is found in extracts from HeLa cells. Possible mechanisms by which these proteins may regulate transcription of the epsilon-globin-encoding gene in erythroid and non-erythroid cells are discussed.
为了阐明参与血红蛋白编码基因发育调控的分子机制，我们一直在研究这些基因在连续培养的人类细胞中的表达。我们先前报道过存在一种具有沉默子特性的转录控制元件，它相对于人类ε-珠蛋白编码基因的帽位点从-392延伸至-177 bp [曹等人，《美国国家科学院院刊》86 (1989) 5306 - 5309]。我们还表明，与K562人红白血病细胞相比，这种沉默子在HeLa细胞中具有更强的抑制活性。在瞬时表达试验中使用缺失突变体和顺式克隆的合成寡脱氧核糖核苷酸，现在已将造成这种效应的核苷酸序列进一步限定为位于-294至-251 bp的44 bp。凝胶电泳迁移率变动分析和DNaseI足迹分析表明，从HeLa和K562细胞获得的核提取物中存在的蛋白质对这些负调控序列的识别方式不同。在K562核提取物中检测到两种结合蛋白，而在HeLa细胞提取物中仅发现一种。本文讨论了这些蛋白质可能在红系和非红系细胞中调节ε-珠蛋白编码基因转录的机制。
