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 beta-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 Agamma-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.
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
E C
EC
Forsberg
T N
TN
Zaboikina
W K
WK
Versaw
N G
NG
Ahn
E H
EH
Bresnick
D020121:5' Untranslated Regions / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D004920:Erythropoiesis; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005914:Globins / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000502:physiology*; D013269:Stochastic Processes; D013698:Templates, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D014157:Transcription Factors; D005914:Globins; D013937:Thymidine Kinase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate