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P38 MAPK-mediated gamma globin induction involves altered DNA-protein interactions in an upstream promoter element


, : P38 MAPK-mediated gamma globin induction involves altered DNA-protein interactions in an upstream promoter element. Blood 102(11): 515a, November 16

Histone deacetylase inhibitors (HDAIs) are validated fetal hemoglobin inducing agents. We have recently demonstrated that induction of gamma globin gene expression by sodium butyrate (NaB) and trichostatin (TSA) is synchronized with p38 MAPK pathway activation. TSA and NaB induce a higher increase in Ggamma compared to Agamma globin mRNA in K562 cells indicating Ggamma is the preferredp38 MAPK target. To identify transcription factors involved in this phenomenon we performed a computer assisted search and identified a cyclic adenosine monophosphate (cAMP) response element (CRE) located at position -1224 in the Ggamma promoter (G-CRE). Interestingly, the G-CRE is a cognate consensus sequence for the ATF-2 and CRE binding proteins (CREB) and both transcription factors are activated by p38 MAPK. We therefore tested the hypothesis that HDAI and p38 MAPK-mediated gamma globin gene induction involves ATF-2 activation and enhances its interaction in the Ggamma promoter. Indeed, we found that NaB and TSA at concentrations of 2 mM and 0.3 muM respectively activated ATF-2 in K562 cells as judged by the appearance of phosphorylated ATF-2 species by immunoblotting. Moreover, we observed increased protein binding to a G-CRE DNA probe with nuclear extracts from HDAI-treated K562 cells. The involvement of the p38 pathway in increased nuclear protein binding to the G-CRE was supported by experiments with the p38 MAPK inhibitor SB203580 (SB). Moreover, a supershifted DNA-protein complex was observed with anti-ATF2/CREB monoclonal antibodies confirming that ATF-2, CREB or both bind to the G-CRE. No DNA-protein complex was observed for the homologous region in the Agamma globin promoter. These findings indicate p38 MAPK-mediated activation and increased binding of ATF-2/CREB to the Ggamma promoter. To assess the functional relevance of the altered DNA-protein interactions, we performed transient transfection studies using luciferase reporter constructs truncated at -1500, -1350 and -1180 in the upstream Ggamma promoter. NaB and TSA increased luciferase activities in the -1500GgammaLuc and -1350GgammaLuc constructs transfected into K562 cells. A fourth construct -1224GgammaLuc, carrying a 280-bp Ggamma fragment from the interval -1500 to -1224 was induced 35-fold and 22-fold by TSA and NaB respectively. Increased reporter activities were specifically inhibited by SB. Significantly, SB had no effect on the activity of the -1180GgammaLuc reporter, which lacks the -1224 G-CRE. Collectively these functional data are consistent with the presence of an HDAI response element in the interval between -1350 and -1224 of the Ggamma promoter. Further insight into the role of ATF-2/CREB in p38 MAPK-mediated gamma globin induction will be gained by analyzing the effect of over-expressing each transcription factor on gamma promoter activity.

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