title: Erythropoietin-induced transcription at the murine beta maj-globin promoter. A central role for GATA-1.
abstract: Using J2E cells and the murine beta maj-globin promoter as a model, we have performed the first direct analyses of erythropoietin (EPO)-activated transcription from defined templates. The -346 to +26 beta maj promoter was shown to comprise a target for maximal activation. This included a positive role for a -346 to -107-base pair (bp) domain in J2E cells, but not in F-MEL cells. Mutagenesis of a -215-bp AGATAA element within this domain showed that this effect did not require GATA-1 binding. In contrast, a critical role for GATA-1 at a -60-bp (G)GATAG element was defined by mutagenesis (GGg-TAG and TGATAG), complementation with a synthetic TGATAA element, and the demonstrated specific binding of GATA-1. Proximal CCAAT (-75) and CACCC (-90) elements also were shown to contribute to transcriptional activation in J2E cells, yet exerted quantitatively distinct effects in the F-MEL system. Based on these results, minimal [TGATAA]4-TATA and TGATAA-CACCC-TATA promoters were constructed and assayed in each system. Remarkably, the [TGATAA]4-TATA promoter, but not the TGATAA-CACCC-TATA promoter, was induced efficiently by EPO in J2E cells, whereas the TGATAA-CACCC-TATA promoter was highly induced by Me2SO in F-MEL cells. These findings suggest that mechanisms of EPO-induced transcription in J2E cells involve GATA-1 and differ from chemically activated mechanisms studied previously in F-MEL cells. Globin induction in J2E cells was not associated with effects of EPO on levels or nuclear translocation of GATA-1. However, hemoglobinization was induced by okadaic acid, 8-Br-cAMP, and forskolin, a finding consistent with induction mechanisms that may involve modulated serine/threonine phosphorylation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802.
authors.forename: D J
authors.initials: DJ
authors.lastname: Taxman
authors.forename: D M
authors.initials: DM
authors.lastname: Wojchowski
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors; D004921:Erythropoietin / Q000494:pharmacology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D050982:GATA1 Transcription Factor; D005914:Globins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D004988:Ethers, Cyclic; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; D014157:Transcription Factors; D004921:Erythropoietin; D005576:Colforsin; D019319:Okadaic Acid; D005914:Globins