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

Transcription of the β-globin genes is regulated by four erythroid-cell-specific DNaseI-hypersensitive sites (HSs) at the 5′ end of the β-globin locus (12, 46), termed the locus control region (LCR) (16). Although the HSs function together to confer long-range transactivation in transfection assays (5) and transgenic mice (7), HS2 and HS3, individually, have strong erythroid-cell-specific enhancer activity when positioned near a promoter (9, 18, 20, 27, 29, 38, 41, 44). Recently, we reported that LCR-mediated transactivation in stable transfection assays increased upon activation of the mitogen-activated protein kinase (MAPK) signaling pathway (48). The MAPK-dependent stimulation required HS2 and was independent of the distance between HS2 and the promoter. Tandem recognition sites within HS2 for the erythroid-cell- and megakaryocyte-specific transcription factor NF-E2 (1, 33, 37), which are critical for strong enhancer activity, were necessary for induction. Besides the requirement of NF-E2 sites, expression of tethered NF-E2, with covalently linked p45 and p18 subunits, conferred induction in CB3 erythroleukemia cells (30) that lack NF-E2. We postulated that phosphorylation of NF-E2 facilitates the recruitment of a coactivator that partially overcomes a limitation to long-range activation (48). Enhancement of NF-E2-mediated transactivation by MAPK with synthetic promoter constructs in transient expression assays has also been described (36). Two simple mechanisms could explain the MAPK-dependent stimulation of HS2 enhancer activity. First, MAPK may enhance the formation of transcription complexes on inactive promoters. Alternatively, MAPK may augment the activity of preassembled complexes. To distinguish between these possibilities, we established a system for assaying gene expression in single, living cells using cell lines containing integrated constructs with the enhanced green fluorescent protein (EGFP) reporter gene. Gene expression can be quantitatively assayed in single cells with β-galactosidase or GFP reporter genes and flow cytometric analysis. Studies on the mechanism of enhancer action in single cells have found that enhancers act via a stochastic or binary mechanism to increase the probability of gene expression from a given template (11, 15, 24, 32, 40, 43, 50, 53). Accordingly, the enhancer determines whether the promoter will be active or inactive rather than augmenting the efficacy of an active promoter. Thus, there are three possible activity states in a diploid cell, each characterized by either two inactive alleles, one active allele, or two active alleles. Stochastic behavior can be explained by the enhancer-dependent, all-or-none recruitment of the RNA polymerase II holoenzyme (45) to an inactive promoter. As the positioning of a nucleosome on a promoter can occlude cis-acting elements (26, 49), the chromatin could prevent initiation complex assembly and thus maintain the silent state. A signaling pathway that targets an enhancer binding factor could regulate its ability to engage in protein-protein interactions necessary for the assembly of the enhancer complex. Only when the complete complex is assembled, which can be an all-or-none event (4), would the chromatin be disrupted, allowing for holoenzyme recruitment. The alternative graded mechanism (23), involving the augmented efficacy of active promoters, would result from a factor modification that modulates the activity of the enhancer complex rather than the all-or-none complex assembly. Based on the importance of coactivators in transactivation (19), the modification may increase the efficiency of coactivator recruitment and thus increase promoter activity. Although activation may involve exclusively stochastic or graded responses, certain mechanisms may elicit both responses. For example, coactivators that modify chromatin structure to control DNA accessibility could influence both initiation complex assembly and the activity of preassembled complexes. Here, we describe the influence of MAPK on HS2 enhancer activity in single, living cells. Increased enhancer activity is manifested as both graded and stochastic responses, and the implications of this are discussed with respect to a model for HS2-mediated long-range transactivation.