ABSTRACT

Using J2E cells and the murine -globin promoter as a model, we have performed the first direct analyses of erythropoietin (EPO)-activated transcription from defined templates. The -346 to +26 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 (GGgTAG 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]-TATA and TGATAA-CACCC-TATA promoters were constructed and assayed in each system. Remarkably, the [TGATAA]-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 MeSO 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.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants HL44491 and KO4HL203042 (to D. M. W.) and by a Sigma Xi grant-in-aid of research (to D. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: 115 Henning Laboratory, University Park, PA 16802. Tel.: 814-865-0657; Fax: 814-863-6140.

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The abbreviations used are: EPO, erythropoietin; bp, base pair(s); kb, kilobase(s); DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; mAb, monoclonal antibody.

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D. J. Taxman and D. M. Wojchowski, unpublished results.

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D. J. Taxman and D. M. Wojchowski, unpublished observations.

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