Enhancement of beta-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 P-globin locus control region (LCR) (W. K . Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sc i. USA 95:8756-8760, 1998). The enhancement requires tandem recognition sit es for the hematopoietic transcription factor NF-E2 within the hypersensiti ve site 2 (HS2) subregion of the LCR To distinguish between mechanisms of i nduction involving the activation of silent promoters or the increased effi cacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhance r activity in single, living cells. K562 erythroleukemia cells stably trans fected,vith constructs containing the human A gamma-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without H S2, were analyzed for EGFP expression by how cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoter s. However, under conditions of silencing, in which cells reverted to a sta te with no measurable EGFP expression, MAPK activated silent promoters. Fur thermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted non expressing cells into expressing cells and increased expression in expressi ng cells. These results support a model in which MAPK elicits both graded a nd stochastic responses to increase HS2-mediated transactivation from singl e chromatin templates.