METHYLATION-ENHANCED BINDING OF SP1 TO THE STAGE SELECTOR ELEMENT OF THE HUMAN GAMMA-GLOBIN GENE PROMOTER MAY REGULATE DEVELOPMENTAL SPECIFICITY OF EXPRESSION

The human gamma-globin gene promoter contains a stage selector element (SSE) responsible for preferential interaction of the promoter with a powerful erythroid-specific enhancer in the fetal developmental stage (S. M. Jane, P. A. Ney, E. F. Vanin, D. L. Gumucio, and A. W. Nienhui s. EMBO J. 11:2691-2699, 1992). The element binds two proteins, the ub iquitous activator Sp1 and a protein previously known as -50gamma and now named the stage selector protein (SSP). Binding of the second prot ein correlates with SSE activity in transient-transfection assays. We now report that a de novo binding site for the SSP is created by the - 202(C-->G) mutation that causes hereditary persistence of fetal hemogl obin (HPFH). This site functions in an analogous manner to the SSE in hybrid beta-promoter/reporter gene constructs transfected into K562 ce lls. In contrast, the wild-type -202 sequence, which fails to bind the SSP, is incapable of activating the beta-gene promoter. Both the -50 and -202 HPFH sites for SSP binding overlap a consensus sequence for t he transcriptional regulator Spl. In addition, both sites contain CpG dinucleotides that are contact bases for SSP. Since the gamma promoter is known to be hypomethylated in fetal cells but fully methylated at CpG residues in adult erythroid cells, we examined the effects of this DNA modification on protein binding to the two regions. Gel mobility shift assays with nuclear extract from K562 cells (which contain both Sp1 and SSP) demonstrate preferential binding of SSP to the SSE and HP FH sites under conditions in which probe was limiting. Methylation of the CpG residues reverses this preference only in the SSE site, with a marked increase in the binding of Sp1 at the expense of the SSP. Puri fied Spl binds with 10-fold higher affinity to the methylated than to the nonmethylated -50 probe but with the same affinity to the -202 HPF H probe. The methylation-induced preferential binding of Sp1 to the SS E at the expense of SSP may be part of the mechanism by which the gamm a genes are repressed in normal adult erythroid cells. In cells contai ning the -202 HPFH mutation, the inability of Sp1 to displace SSP in t he methylated state may explain the persistence of gamma-promoter acti vity and gamma-gene expression observed in adults with this mutation.