cJun modulates Gγ-globin gene expression via an upstream cAMP response element

Sirisha Kodeboyina, Parimaladevi Balamurugan, Li Liu, Betty S. Pace

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

The upstream Gγ-globin gene cAMP response element (G-CRE) was previously shown to play a role in drug-mediated fetal hemoglobin induction. This effect is achieved via p38 mitogen activated protein kinase (MAPK)-dependent CREB1 and ATF-2 phosphorylation and G-CRE transactivation. Since this motif is also a predicted consensus binding site for cJun we extended our analysis to determine the ability of cJun to transactivate γ-globin through the G-CRE. Using chromatin immunoprecipitation assays we showed comparable in vivo cJun and CREB1 binding to the G-CRE region. Protein-protein interactions were confirmed between cJun/ATF-2 and CREB1/ATF-2 but not between CREB1 and cJun. However, we observed cJun and CREB1 binding to the G-CRE in vitro by electrophoretic mobility shift assay. Promoter pull-down assay followed by sequential western blot analysis confirmed co-localization of cJun, CREB1, and ATF-2 on the G-CRE. To show functional relevance, enforced expression studies with pLen-cJun and a Gγ-promoter (- 1500 to + 36) luciferase reporter were completed; we observed a concentration-dependent increase in luciferase activity with pLen-cJun similar to that produced by CREB1 enforced expression. Moreover, the G/A mutation at -1225 in the G-CRE abolished cJun transactivation. Finally, enforced cJun expression in K562 cells and normal primary erythroid progenitors enhanced endogenous γ-globin gene expression. We conclude that these data indicate that cJun activates the Gγ-globin promoter via the G-CRE in a manner comparable with CREB1 and propose a model for γ-globin activation based on DNA-protein interactions in the G-CRE.

Original languageEnglish (US)
Pages (from-to)7-15
Number of pages9
JournalBlood Cells, Molecules, and Diseases
Volume44
Issue number1
DOIs
Publication statusPublished - Jan 1 2010
Externally publishedYes

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Keywords

  • ATF-2
  • CREB1
  • cAMP response element
  • cJun
  • γ-globin

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Hematology
  • Cell Biology

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