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Molecular and Cellular Biology, August 1999, p. 5565-5575, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

E. Camilla Forsberg,1 Tatiana N. Zaboikina,1 Wayne K. Versaw,1 Natalie G. Ahn,2 and Emery H. Bresnick1,*

Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706,1 and Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 803092

Received 23 March 1999/Accepted 30 April 1999

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.

* Corresponding author. Mailing address: Department of Pharmacology, University of Wisconsin Medical School, 387 Medical Science Building, 1300 University Ave., Madison, WI 53706. Phone: (608) 265-6446. Fax: (608) 262-1257. E-mail: ehbresni{at}facstaff.wisc.edu.

Molecular and Cellular Biology, August 1999, p. 5565-5575, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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