MULTIPLE PROTEINS BINDING TO A GATA-E BOX-GATA MOTIF REGULATE THE ERYTHROID KRUPPEL-LIKE FACTOR (EKLF) GENE

Erythroid Kruppel-like factor (EKLF) is a zinc finger transcription fa ctor required for beta-globin gene expression and is implicated as one of the key factors necessary for the fetal to adult switch in globin gene expression, In an effort to identify factors involved in the expr ession of this important erythroid-specific regulatory protein, we hav e isolated the mouse EKLF gene and systematically analyzed the promote r region. Initially, a reporter construct with 1150 base pairs of the EKLF 5'-region was introduced into transgenic mice and shown to direct erythroid-specific expression. We continued the expression studies in erythroid cells and have identified a sequence element consisting of two GATA sites flanking an E box motif, The three sites act in concert to elevate the transcriptional activity of the EKLF promoter. Each si te is essential for EKLF expression indicating that the three binding sites do not work additively, but rather function as a unit. We furthe r show that GATA-1 binds to the two GATA sites and present evidence fo r binding of another factor from erythroid cell nuclear extracts to th e E box motif. These results are consistent with the formation of a qu aternary complex composed of an E box dimer and two GATA-1 proteins bi nding at a combined GATA-E box-GATA activator element in the distal EK LF promoter.