Role of Upstream cAMP Response Element (CRE) in γ-Globin Gene Regulation.

Abstract 4076

Poster Board III-1011

The reactivation of fetal hemoglobin has been shown to ameliorate symptoms of sickle cell disease. Previous work from our laboratory demonstrated that histone deacetylase inhibitors such as Trichostatin A and sodium butyrate activate γ-globin gene expression via p38 MAPK signaling. Using a transient luciferase genetic reporter system, the trans-factors cAMP response element binding protein 1 (CREB1) and activating transcription factor-2 (ATF-2) were shown to trans-activate the Gγ-globin CRE, located upstream at nucleotide -1222. To study this regulatory element in a native chromatin conformation, we cloned the Gγ-globin promoter (-1500 to +36) into pGL4.17 Luc2/neo to produce the pGγLuc2 reporter construct. In addition, three mutant plasmids were created by site directed mutagenesis including the -1225 G/A mutation (pGγLuc2m1), -1227 AC/TG mutations (pGγLuc2m2) and a scrambled G-CRE, 5'-TCTATGTA-3' (pGγLuc2m3S). Each reporter along with the empty vector control (pGL4.17 Luc2/neo) was transfected into K562 cells and five stable lines including KLuc2, KGγLuc2, KGγLuc2(m1), KGγLuc2(m2) and KGγLuc2(m3s) were established using the G418 selectable marker. Luciferase activity in KGγLuc2 was 700-fold higher than the promoterless Kluc2 stable line. When the G-CRE element was mutated, we observed a 70 to 80 % decrease (p<0.05) in luciferase activity in the three mutant lines. Furthermore, a loss of Gγ-promoter inducibility was observed in the mutant lines. Treatment with butyrate (2mM) or Trichostatin A (0.5μM) resulted in a 3-fold increase of luciferase activity for the wild-type KGγLuc2 line while a 5-fold loss of inducibility was observed in the mutant stable lines. These studies support a functional role for the G-CRE in γ-globin transcription. To expand our understanding of p38 MAPK signaling in G-CRE function, siRNA studies were completed. In wild-type K562 cells, p38 siRNA molecules produced a 95% decrease in target p38 MAPK mRNA levels and a concomitant 65% loss of γ-globin expression was observed. This demonstrated that p38 MAPK is required for steady-state γ-globin gene transcription. When p38 MAPK siRNA knockdown was performed in the KGγLuc2 stable line we observed 30% reduction in luciferase activity, while this effect was lost in the mutant stable lines. Additional siRNA studies will be completed in the stable lines to determine if the target trans-factors CREB1 and ATF-2 mediate the effect of p38 MAPK in the context of chromatin. Chromatin immunoprecipitation assay will also be performed to determine in vivo binding to the G-CRE. Elucidating regulatory elements involved in γ-globin gene regulation will provide novel approaches for fetal hemoglobin induction as a treatment for sickle cell disease.