The Role of p38 MAPK/CREB1 Signaling In γ-Globin Gene Regulation

Abstract 1012

The clinical symptoms of sickle cell disease can be ameliorated by increased fetal hemoglobin (HbF) levels. Previous work from our laboratory demonstrated that Trichostatin A (TSA) and sodium butyrate (NaB) activate γ-globin expression via p38 MAPK signaling. In addition, cAMP response element binding protein 1 (CREB1) was shown to trans-activate the -1222 Gγ-globin cAMP response element (G-CRE) in a transient assay system. To study the role of p38 MAPK signaling in γ-globin regulation, loss of function siRNA studies were performed in K562 cells. siRNA-mediated knockdown of p38 MAPK resulted in 72% loss of γ-globin transcription. Furthermore, enforced stable expression of MKK3/6 increased the phosphorylated form of p38 MAPK by 70%, which in turn produced a 2- to 3-fold increase in γ-globin mRNA and HbF levels. Likewise, siCREB1 treatment reduced CREB1 levels by 62% and down regulated γ-globin expression 59%. In the same vein, stable expression of recombinant CREB1 activated HbF by 2-fold. These findings were subsequently confirmed in human primary erythroid cells grown in a two-phase liquid culture system. On day 11, we observed 50–70% γ-globin silencing after CREB1 and p38 MAPK siRNA knockdown with 60% target gene silencing. CREB1 enforced expression trans-activated γ-globin 4.5-fold which was accompanied by a 90% increase in HbF-FITC positive cells and HbF levels. Collectively, these data demonstrate that p38 MAPK and CREB1 are required for steady-state γ-globin gene transcription. To determine the role of the G-CRE in γ-globin regulation, the Gγ-globin promoter (-1500 to +36) was cloned into pGL4.17 Luc2/neo to produce pGγLuc2 (wild type) and mutant plasmids: -1225 G/A (m1), -1227 AC/TG (m2) and a scrambled G-CRE (m3s). Five K562 stable lines including KLuc2 (promoterless), KGγLuc2, KGγLuc2(m1), KGγLuc2(m2) and KGγLuc2(m3s) were established. Luciferase activity in KGγLuc2 was 1000-fold higher than in the control Kluc2 line; all mutations produced >90% loss of luciferase activity and a loss of γ-globin trans-activation by TSA and NaB. Next, siRNA studies were completed to determine if the G-CRE is required for γ-globin activation. A dose-dependent loss of promoter activity was observed after p38 MAPK and CREB1 siRNA knockdown of the KGγLuc2 cell line; however, promoter silencing was not observed in the mutant lines supporting a role for the G-CRE in p38 MAPK/CREB1 mediated γ-globin regulation. To study in vivo binding, chromatin immunoprecipitation (ChIP) assays were performed with CREB1 antibody in the KGγLuc2 stable line. We observed comparable 2- to 3-fold chromatin enrichment with CREB1 compared to the control IgG in the G-CRE regions of the pGγLuc2 plasmid and endogenous Gγ-globin promoter. To determine if an enhanceosome complex is bound to the G-CRE, we performed affinity column pull-down/mass spectrometry analysis. K562 nuclear extract was purified on a Heparin Sepharose column, following which fractions eluting at 0.6M NaCl showing peak gel shift binding activities with the G-CRE oligo were loaded into a size selecting Suprose 6 gel exclusion column. G-CRE eluting fractions were then identified by protein microsequencing (MS/MS). We identified CREB1, ATF2, c-Jun, BRG-1, hnRNPC1/C2, and the TCP-1 complex as major components. To determine protein co-localization, promoter pull-down assays were performed using biotinylated wild type and mutant (AC/TG) G-CRE probes and K562 nuclear extracts. We observed simultaneous CREB1, ATF-2 and cJun binding to the G-CRE which was abolished in the mutant probe. However, Brg1 was bound after NaB (2mM) induction. Subsequent co-IP studies showed interactions between ATF-2 and Brg1, CREB1, cJun, and hnRNPC1C2, which was further confirmed by co-elution profile of these molecules observed by sucrose gradient centrifugation, thus implying association as one complex. These data support complex protein-protein interactions in the G-CRE, which modulate γ-globin gene expression. Additional studies will be performed in primary erythroid cells using siRNA-based gene silencing and ChIP assays to determine novel mechanisms of γ-globin regulation and to define in vivo binding of proteins identified in the G-CRE enhanceosome complex.