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

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.

Histone acetylation beyond promoters: long‐range acetylation patterns in the chromatin world*

“…(74±76) Activation of the mitogen-activated protein kinase pathway has already been shown to stimulate longrange transactivation mediated by the b-globin LCR in stable transfection assays (77,78) and we proposed that the mechanism involved facilitated recruitment or utilization of limiting HATs. Considering the``histone code'' hypothesis (40) it may be necessary to define all histone modifications within domains to understand how broad acetylation patterns are established.…”

Regulation of human fetal hemoglobin: new players, new complexities

“…In chromatin immunoprecipitation (ChIP) analyses, after chromatin fragmentation, antibodies to specific proteins are used to isolate the DNA sequences associated with these proteins in vivo, for example, antibodies to acetylated histones or specific transcription factors. 24, [45][46][47][48]52,[72][73][74][75][76][77][78][79] In so-called chromosome conformation capture (3C) experiments, specific DNA sequences in contact with each other in vivo are identified by restriction digestion and controlled religation of DNA fragments after chromatin cross-linking. [20][21][22][23]80 In another procedure, tagging and recovery of associated proteins (TRAP) technology, biotinlabeled probes are used to uniquely extract newly synthesized RNA associated with protein sequences after cross-linking.…”

Developmental control via GATA factor interplay at chromatin domains

“…Stochastic transcriptional activity, in which a gene is actively transcribed in only certain cells in a population, has been observed in multiple systems (Stamatoyannopoulos et al, 1981;Ko, 1992;Walters et al, 1995;Graubert et al, 1998;Forsberg et al, 1999;Biggar and Crabtree, 2001), but has not been investigated in the context of GATA-1 and GATA-2 transcriptional regulation.…”