Enhancement of β-Globin Locus Control Region-Mediated Transactivation by Mitogen-Activated Protein Kinases through Stochastic and Graded Mechanisms

Activation of the mitogen-activated protein kinase (MAPK) pathway enhances long-range transactivation by the β-globin locus control region (LCR) (W. K. Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sci. USA 95:8756–8760, 1998). The enhancement requires tandem recognition sites for the hematopoietic transcription factor NF-E2 within the hypersensitive site 2 (HS2) subregion of the LCR. To distinguish between mechanisms of induction involving the activation of silent promoters or the increased efficacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhancer activity in single, living cells. K562 erythroleukemia cells stably transfected with constructs containing the human Aγ-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without HS2, were analyzed for EGFP expression by flow cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoters. However, under conditions of silencing, in which cells reverted to a state with no measurable EGFP expression, MAPK activated silent promoters. Furthermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted nonexpressing cells into expressing cells and increased expression in expressing cells. These results support a model in which MAPK elicits both graded and stochastic responses to increase HS2-mediated transactivation from single chromatin templates.

丝裂原活化蛋白激酶 (mitogen-activated protein kinase, MAPK) 通路的激活可增强β-珠蛋白基因座控制区 (β-globin locus control region, LCR) 的远程反式激活作用（W. K. Versaw、V. Blank、N. M. Andrews及E. H. Bresnick, 《美国国家科学院院刊》, 1998, 95:8756–8760）。该增强作用依赖于LCR的超敏位点2 (hypersensitive site 2, HS2) 亚区内造血转录因子NF-E2 (hematopoietic transcription factor NF-E2) 的串联识别位点。为区分激活沉默启动子与增强活跃启动子效能这两种诱导机制，我们在单个活细胞中分析了基础状态及MAPK刺激下的HS2增强子活性。将携带人类Aγ-珠蛋白启动子与增强型绿色荧光蛋白 (enhanced green fluorescent protein, EGFP) 报告基因相连的构建体（是否带有HS2序列）稳定转染的K562红白血病细胞，通过流式细胞术 (flow cytometry) 分析其EGFP表达情况。当群体中多数细胞表达EGFP时，MAPK可增强活跃启动子的活性。然而在沉默状态下，即细胞恢复至无可检测EGFP表达的状态时，MAPK可激活沉默启动子。此外，对通过流式细胞术分离得到的EGFP表达阳性与阴性细胞群体的研究显示，MAPK激活可将非表达细胞转化为表达细胞，并提升表达细胞的EGFP表达水平。上述结果支持下述模型：MAPK可引发分级应答与随机应答，以增强单染色质模板 (chromatin templates) 上HS2介导的反式激活作用。