cJun modulates Ggamma-globin gene expression via an upstream cAMP response element.
The upstream Ggamma-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 gamma-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 Ggamma-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 gamma-globin gene expression. We conclude that these data indicate that cJun activates the Ggamma-globin promoter via the G-CRE in a manner comparable with CREB1 and propose a model for gamma-globin activation based on DNA-protein interactions in the G-CRE.
Kodeboyina
Sirisha
S
Department of Molecular and Cell Biology, University of Texas at Dallas, PO Box 830688, FO 3.1, Richardson, TX 75083, USA.
Balamurugan
Parimaladevi
P
Liu
Li
L
Pace
Betty S
BS
D020121:5' Untranslated Regions / Q000235:genetics; D051698:Activating Transcription Factor 2 / Q000378:metabolism; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D056572:Histone Deacetylase Inhibitors / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism / Q000502:physiology*; D020218:Response Elements / Q000235:genetics; D015533:Transcriptional Activation; D055545:gamma-Globins / Q000235:genetics / Q000378:metabolism*
D020121:5' Untranslated Regions; C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D056572:Histone Deacetylase Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D055545:gamma-Globins; D000242:Cyclic AMP