Butyrate-inducible elements in the human gamma-globin promoter

Several agents including hydroxyurea, erythropoietin and butyric acid have been shown to reactivate gamma gene expression during adult stage development by unknown molecular mechanisms. In addition to inhibiting the enzyme histone deacetylase, butyrate may modulate transcription factor binding to specific DNA sequences defined as butyrate response elements (BREs). The purpose of this study was to identify promoter sequences involved in gamma gene activation by butyrate using truncation mutants in stable cell lines.

A detailed analysis of Agamma gene activation in the presence of alpha-aminobutyric acid and sodium butyrate was completed in stable mouse erythroleukemia (MEL) cell pools established with seven Agamma promoter truncation mutants. Functional studies were performed in a transient assay system followed by gel mobility shift assays to define protein binding patterns and to demonstrate transcription factor interactions in the gamma promoter BRE.

Agamma promoter analysis in stable MEL cell pools revealed BREs between nucleotide-141 and -201, and nucleotide-822 and -893 (gammaBRE). The gammaBRE required the minimal Agamma promoter (-201 to +36) to stimulate gene expression. We observed a 6.1-fold (p < 0.05) increase in CAT activity for the minimal Agamma promoter alone compared with an 11.5-fold (p < 0.05) increase when the gamma promoter was combined with the -822 to -893 fragment. Protein binding studies demonstrated altered protein-DNA interactions in the gammaBRE after butyrate induction. The pattern for binding observed suggest both negative- and positive-acting transcription factors may interact in this region.

The data supports the -822 to -893 region as a DNA regulatory element that contributes to Agamma gene inducibility by butyrate.