Identification of functional elements of the chicken ε-globin promoter involved in stage-specific interaction with the β/ε enhancer

Abstract

Expression of the chicken globin genes is regulated in part by competition between the β(A)-globin and ε-globin promoters for the enhancer found between the genes. To understand the determinants of the enhancer- promoter interaction in stage-specific regulation, the functional elements of the embryonic chicken ε-globin promoter were characterized. In vitro assays demonstrated that: (a) the TATA motif at -30 bound GATA-1, (b) Sp1 bound to an element centered at -54, and (c) both Sp1 and another factor, designated CACCC (which appears related to erythroid Kruppel-like factor, EKLF) bound in the -120 to -128 region. The functions of these motifs were tested using transient expression in embryonic erythroid cells. In the absence of the enhancer, promoter point mutants showed that the TATA, Sp1, and CCAAT motifs (but not the CACCC motif) contributed to promoter activity. In contrast, in the presence of the enhancer, all four motifs (including the CACCC motif) contributed to transcription. Developmental regulation of the enhancer activity was observed, with enhancement decreasing sharply from 185-fold at 4 days (cells expressing ε-glohin) to 16-fold at 10 days (when ε-globin is no longer expressed). Taken together, the data suggest that multiple transcription factors contribute to promoter-enhancer interaction and the developmental regulation of ε-globin expression, with EKLF-like factors having an especially important role. Regulation of stage specificity occurs at the level of enhancer/ε-promoter interaction, even in the absence of competition, and is not simply a property of the enhancer or promoter in isolation.