Enhancement of beta-globin locus control region-mediated transactivation by mitogen-activated protein kinases through stochastic and graded mechanisms - PubMed (original) (raw)

Enhancement of beta-globin locus control region-mediated transactivation by mitogen-activated protein kinases through stochastic and graded mechanisms

E C Forsberg et al. Mol Cell Biol. 1999 Aug.

Abstract

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.

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Figures

FIG. 1

FIG. 1

TPA induction of HS2 enhancer activity analyzed in single cells. Cultures were treated with 5 nM TPA (shaded) or vehicle (unshaded) for 20 h before each analysis. Histograms show representative flow cytometric analyses of stably transfected K562 clonal cell lines containing γEGFP, HS2γEGFP, or HS3γEGFP constructs. The number of integrated copies of the reporter plasmid is shown. Nonexpressing cells had a relative fluorescence of no more than 1 on this scale.

FIG. 2

FIG. 2

Distance independence of TPA-induced HS2 enhancer activity. Cultures were treated with 5 nM TPA (shaded) or vehicle (unshaded) for 20 h before each analysis. Histograms show representative flow cytometric analyses of stably transfected K562 clonal cell lines containing HS2(2.2)γEGFP or HS2(7.5)γEGFP constructs. The number of integrated copies of the reporter plasmid is shown. Nonexpressing cells had a relative fluorescence of no more than 1 on this scale.

FIG. 3

FIG. 3

Silencing of EGFP expression reveals stochastic and graded responses to TPA treatment. Clone HS2γEGFP-1 was grown for the indicated times with or without hygromycin (hygro) and then treated with 5 nM TPA (shaded) or vehicle (unshaded) for 20 h before each analysis. (A) Flow cytometric analysis was used to measure the percentage of EGFP-positive cells in the viable cell population. (B) The percentage of EGFP-positive cells determined in panel A is expressed as a function of culture time. (C) EGFP was quantitated in cell lysates by fluorometry and normalized by protein concentration and copy number of the integrated reporter construct.

FIG. 4

FIG. 4

Silencing of EGFP expression in clone HS2(2.2)γEGFP-3. The clone was grown for 26 days with or without hygromycin (hygro) and then treated with 5 nM TPA (shaded) or vehicle (unshaded) for 20 h before each analysis. The percentage of EGFP-positive cells in the viable cell population was measured by flow cytometric analysis. EGFP was quantitated in cell lysates by fluorometry and normalized by protein concentration and the copy number of the integrated reporter construct.

FIG. 5

FIG. 5

Silencing of EGFP expression in single-copy clone HS2(7.5)γEGFP-4. The clone was grown for 26 or 36 days with or without hygromycin (hygro) and then treated with 5 nM TPA (shaded) or vehicle (unshaded) for 20 h before each analysis. The percentage of EGFP-positive cells in the viable cell population was measured by flow cytometric analysis. EGFP was quantitated in cell lysates by fluorometry and normalized by protein concentration and the copy number of the integrated reporter construct.

FIG. 6

FIG. 6

Physical separation of EGFP-expressing and non-EGFP-expressing cells revealed both stochastic and graded responses to TPA treatment. Clone HS2γEGFP-1 was grown without hygromycin for 133 or 135 days and then sorted by FACS into EGFP-expressing and non-EGFP-expressing pools. Pools were treated with 5 nM TPA (shaded) or vehicle (unshaded) for 20 h, and then the percentage of EGFP-positive cells and the mean fluorescence of viable cells were measured by flow cytometric analysis. Representative flow cytometric profiles are shown. The horizontal line represents the EGFP-expressing cells. The values represent the means of two independent experiments in which cells were grown for either 133 or 135 days without hygromycin.

FIG. 7

FIG. 7

Expression of cMEK1 elicits graded and stochastic effects on gene expression. K562 cells were transiently transfected with either constitutive EGFP or EBFP expression vectors to establish conditions for discriminating between green and blue fluorescence. Clone HS2γEGFP-1, grown in media containing hygromycin, was transiently transfected with either EBFP and cMEK1 expression vectors or EBFP and pcDNA3. EGFP expression was analyzed in EBFP-positive cells (Table 3). Regions containing green, blue, and green plus blue cells are delineated. The high level of EGFP expression in clone HS2γEGFP-1 resulted in an upward extension of the green signal. Note that the flow cytometric data for cells transfected with cMEK1 and EBFP shows more green and blue cells than data for cells transfected with pcDNA3 and EBFP; this was not seen in all experiments and did not influence the quantitation of green fluorescence in the cell population.

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