Butyrate-inducible elements in the human gamma-globin promoter

Objective. Several agents including hydroxyurea, erythropoietin and butyric acid have been shown to reactivate gamma gene expression during adult stag e development by unknown molecular mechanisms. In addition to inhibiting th e enzyme histone deacetylase, butyrate may modulate transcription factor bi nding to specific DNA sequences defined as butyrate response elements (BREs ). The purpose of this study was to identify promoter sequences involved in A gamma gene activation by butyrate using truncation mutants in stable cel l lines. Materials and Methods. A detailed analysis of A gamma gene activation in th e presence of alpha-aminobutyric acid and sodium butyrate was completed in stable mouse erythroleukemia (MEL) cell pools established with seven A gamm a promoter truncation mutants. Functional studies were performed in a trans ient assay system followed by gel mobility shift assays to define protein b inding patterns and to demonstrate transcription factor interactions in the gamma promoter BRE. Results. A gamma promoter analysis in stable MEL cell pools revealed BREs b etween nucleotide-141 and -201, and nucleotide-822 and -893 (gamma BRE), Th e gamma BRE required the minimal A gamma promoter (-201 to +36) to stimulat e gene expression. We observed a 6.1-fold (p < 0.05) increase in CAT activi ty for the minimal A gamma promoter alone compared with an 11.5-fold (p < 0 .05) increase when the gamma promoter was combined with the -822 to -893 fr agment. Protein binding studies demonstrated altered protein-DNA interactio ns in the gamma BRE after butyrate induction. The pattern for binding obser ved suggest both negative- and positive-acting transcription factors may in teract in this region. Conclusion. The data supports the -822 to -893 region as a DNA regulatory e lement that contributes to Ay gene inducibility by butyrate. (C) 2000 Inter national Society for Experimental Hematology. Published by Elsevier Science Inc.