The histone deacetylase inhibitors (HDACIs) butyrate and trichostatin A activate ␥-globin expression via a p38 mitogenactivating protein kinase (MAPK)-dependent mechanism. We hypothesized that downstream effectors of p38 MAPK, namely activating transcription factor-2 (ATF-2) and cyclic AMP response element (CRE) binding protein (CREB), are intimately involved in fetal hemoglobin induction by these agents. In this study, we observed increased ATF-2 and CREB1 phosphorylation mediated by the HDACIs in K562 cells, in conjunction with histone H4 hyperacetylation. Moreover, enhanced DNAprotein interactions occurred in the CRE in the G ␥-globin promoter (G-CRE) in vitro after drug treatments; subsequent chromatin immunoprecipitation assay confirmed ATF-2 and CREB1 binding to the G-CRE in vivo. Enforced expression of ATF-2 and CREB produced G ␥-promoter IntroductionThe growth factor erythropoietin (Epo) exerts its effects on commitment, proliferation, and differentiation of erythroid progenitors and globin chain synthesis through Janus kinase 2/Stat5 signaling and crosstalk with mitogen-activated protein kinase (MAPK) pathways. 1-3 p38 MAPK signaling is required for Epo mRNA stability and hemoglobin synthesis. 4,5 The reversible inhibition of p38 MAPK using SB203580 blocked Epo-dependent accumulation of mouse globin chains, 6 and studies in p38␣ Ϫ/Ϫ knockout mice showed a failure of definitive ␤ maj -globin gene expression. These studies confirm an Epo-p38 MAPK-dependent mechanism for hemoglobin synthesis. 7 The HDACI sodium butyrate (NaB) induces differentiation in erythroleukemia cells via Stat5 8,9 and p38 MAPK signaling. 10,11 Butyrate is a clinically useful fetal hemoglobin (HbF) inducer which has been used to treat individuals with sickle cell disease 12 and thalassemia 13 ; however, the molecular mechanism for NaB-mediated HbF induction is poorly understood. Recent data from Weinberg et al 14 showed that HbF induction by arginine butyrate is due in part to posttranslational mechanisms and increased ␥-globin mRNAtranslation.Several HDACIs, including trichostatin A (TSA), 10, and scriptaid,15,16 induce ␥-globin expression via p38 MAPK signaling. These studies suggest that different pharmacologic agents converge on the p38 MAPK pathway to activate ␥-globin expression. Four major MAPK pathways have been characterized: ERK1/2, ERK5/BMK1, cJun amino-terminal signal kinases (JNK), and p38. [17][18][19][20] Studies using erythroid progenitors, 21,22 knockout mice, 7 and K562 stable lines 10 suggest that p38␣ is the primary mediator of globin gene regulation.The downstream effector molecules of p38 MAPK signaling include MAPK-activated protein kinases 1 and 2, 23,24 PRAK, 25 ATF-1-4, CREB1, CREB2, and CREM. 26,27 Commonly, p38 phosphorylates ATF-2 and CREB to augment gene transcription. We recently demonstrated a p38 MAPK-dependent mechanism for NaB and TSA-induced ␥-globin expression. 10 Mechanistically, both agents bind a central zinc atom in HDAC to produce hyperacetylation of histone H3 (H3) and H4 28,29 to activate...

Erythroid differentiation recapitulates the temporal switch from γ to β-globin gene expression that occurs during fetal development. We and others have demonstrated a p38 mitogen activated protein kinase (MAPK)-dependent mechanism for γ-globin activation by fetal hemoglobin inducers in the setting of permissive chromatin structure. Furthermore, by electrophoretic mobility shift assay we confirmed that CREB and ATF-2 bind the CRE at nucleotide −1222 in the Gγ-promoter (G-CRE). A naturally occurring C→G mutation in the CRE occurs is associated with the Benin haplotype, producing lower fetal hemoglobin levels in sickle cell patients. To understand the functional relevance of the G-CRE we used a luciferase reporter system established with promoter mutants at nucleotides −1500, −1350 and −1180 to produce the respective reporters -1500Gγluc, −1350Gγluc and −1180Gγluc. Transient transfections were performed in K562 cells by electroporation. In the −1180Gγluc reporter there was a loss of the 15 to 20-fold activation induced by 2mM butyrate and 0.5μM trichostatin in the −1500Gγluc reporter . The loss of trans-activation when the G-CRE was deleted in the −1180 mutant confirms functional relevance for this element. Trans-activation studies were also completed with a wtCREB (pCMV-CREB) and dominant negative, pKCREB expression vector. The latter forms an inactive heterodimer with CREB by blocking the DNA binding domain. To determine the direct trans-activation potential of wtCREB, co-transfection studies were completed with 10 to 40μg of either pCMV-CREB or pKCREB alone. We observed a dose-dependent 1.5 to 11-fold increase in luciferase activity with wtCREB compare to a robust 18 to 100-fold Gγ-promoter induction by pkCREB. This ability of both vectors to activate was reversed when the G-CRE was deleted. Interestingly, when an equal concentration of both vectors was co-transfected, a 40-fold increase in promoter activity was observed. Additional studies with pkCREB and butyrate or trichostatin inductions produced a similar synergistic effect. Collectively, the findings suggest that other transcription factors with greater trans-activation potential bind the G-CRE when CREB is inactivated by its dominant negative protein. We will test the two most likely candidate transcription factors, cJun and ATF-2 which bind the G-CRE and are downstream effectors of p38 MAPK signaling. Transfection experiments with cJun confirmed a dose-dependent 80-fold activation of −1500Gγluc. Interestingly, when cJun was combined with wtCREB, γ-promoter trans-activation dropped 60-fold supporting two possible interactions between cJun and CREB: 1) competitive binding to the G-CRE or 2) the formation of an inactive or low activity heterodimer between the two proteins. Additional studies with site-directed mutants of the G-CRE will be performed to ferret out the DNA-protein interactions in this region, thereby providing gene-based approaches for fetal hemoglobin induction.