A (GATA)(7) motif located in the 5 ' boundary area of the human beta-globin locus control region exhibits silencer activity in erythroid cells

A 40-bp DNA, consisting of seven tandem GATA repeats, is located near the H S5 site in the 5' boundary area of the locus control region (LCR) of human beta-globin gene. This (GATA)(7) motif, named 5a, exhibits silencer activit y in erythroid cells, In transfected, recombinant plasmids containing the c hloramphenicol acetyltransferase (CAT) reporter gene, 5a repressed the acti vity of the cis-linked housekeeping phosphoglycerate kinase (pgk) promoter; 5a also repressed the activity of the cis-linked HS2 enhancer regardless o f whether the CAT gene was driven by the pgk or the epsilon-globin promoter . Repression by 5a was most severe when 5a was spliced upstream of HS2 at a distance of less than 200 bases from the HS2 enhancer core. The silencer a ctivity of 5a was independent of whether the component GATA motifs were in head to tail orientation as in the wild type 5a or in head to head or tail to tail orientation as in a mutant 5a, Band shift experiments show that the GATA-1 protein binds to both 5a and the mutant 5a and forms a large protei n complex. Together, the results suggest that GATA-1 bound at 5a is a stron g, proximal repressor of HS2 enhancer activity. (C) 2000 Wiley-Liss, Inc.