Sites I and II upstream of the A gamma globin gene bind nuclear factors and affect gene expression.

ABSTRACT

Five broad regions in and near the A gamma globin gene specifically bind proteins in K562 nuclear extracts. Two are located 5' of the gene, one between -1349 and -1094, and the other between -384 and +52. Each of the two introns has a binding region, +184 to +284 and +930 to +1209. The fifth binding region is within the A gamma globin gene enhancer, a 750 bp Hind III fragment located 3' to the gene. The nuclear factors which bind to any or all of these regions may be important for control of regulation of the A gamma globin gene. Since HPFH point mutations occur at -175, -196 and -202, we investigated K562 nuclear factor binding to this region more closely. Footprints were obtained for two binding sites, one from -168 to -185 (site II) and the other from -267 to -293 (site I). Site II contains an octamer sequence (ATGCAAAT) important for protein binding and expression of the histone H2b and immunoglobulin genes. The HPFH mutation at -175 changes the T in the octamer sequence to a C. Site I is upstream of the bases affected by HPFH mutations. A A gamma globin gene promoter - CAT reporter fusion gene was constructed with a clustered - base substitution of site I or a T----C point mutation at -175. The mutation in site I decreases CAT expression 20X compared to wild-type in erythroid and non-erythroid cells. Site I probably binds a positive regulator. The T----C change at -175 increases expression 2-3X over wild-type in erythroid cells, but not in non-erythroid cells. This increase correlates with the effect of the naturally occurring HPFH, and may result from decreased binding of a negative effector, and/or increased positive factor binding.