FACTOR-BINDING TO THE HUMAN GAMMA-GLOBIN GENE DISTAL CCAAT SITE - CANDIDATES FOR REPRESSION OF THE NORMAL GENE OR ACTIVATION OF HPFH MUTANTS

We have examined factor binding to the distal human gamma-globin CCAAT site and three naturally occurring hereditary persistence of fetal ha emoglobin (HPFH) mutations of this site. Factor binding was examined u sing nuclear extracts from the erythroleukaemic cell lines K562 and ME L, and from A4 cells, a non-transformed mouse bone marrow stem cell li ne, using the electrophoretic mobility shift assay. Under standard bin ding conditions, in addition to the previously reported binding by a C CAAT factor (CPI) and GATA-1, the wild-type (wt) sequence bound high m obility factors which appeared to be GATA-2 isoforms. However, when th e non-specific competitor conditions were varied, the binding profile with K562, but not MEL nuclear extract, was substantially altered, CP1 and GATA-1 were absent, and two new factors were detected, one of whi ch bound preferentially to the Greek and Japanese non-deletion HPFH mu tants, However, binding by the GATA-2 isoforms to the wt sequence was maintained with both cell types, as it was using the A4 cell line. Wit h modified binding conditions, in A4 cells the two non-deletion and th e Black deletion HPFH mutants each had a different protein binding pro file which was lost on erythroid induction of the cells, We discuss th e possibility that the GATA-2 isoforms bound to the wt sequence may fu nction to suppress wt gamma gene expression in the bone marrow Additio nally, those factors which bind preferentially either to the deletion or non-deletion HPFH mutants may play positive roles in establishing a n active chromatin structure.