Globin genes exhibit tissue, developmental and maturational specificity. It is our purpose to understand the molecular basis of globin gene regulation. Our efforts have focused on the beta-globin gene cluster that contains the epsilon, gamma and beta genes expressed during the embryonic, fetal and adult developmental periods, respectively. These genes are encompassed within a 60 kb segment of DNA on human chromosome 11. Within this cluster are several cis-acting regulatory elements that interact with trans-acting factors (proteins) to modulate globin gene expression. The locus control region (LCR) located upstream from the cluster contains four regulatory elements (5'HS1-4). Other regulatory elements include the individual gene promoters and downstream enhancers. Our work during the past year has focused on the purification and characterization of proteins that interact with these regulatory elements. We have purified and molecularly cloned one component (p45) of human, NF-E2, the basic leucine zipper protein that is responsible for the inducible activity of the HS2 enhancer in erythroid cells. The second component, p18, has been purified to homogeneity and its sequence determined in collaboration with N. Andrews and S. Orkin. The two proteins form a heterodimeric complex having the DNA binding activity and specificity of NF-E2. Stage selector protein (SSP) binds the stage selector element (SSE) in the gamma globin gene promoter and fosters its interaction with HS2 enhancer. SSP has been highly purified and shown to have a molecular weight of 40Kd. Sp1 competes with SSP for binding to the SSE. Methylation of SSE as observed in adult erythroid cells enhances binding of Sp1 at the expense of SSP. We have previously characterized another protein that binds to a regulatory element downstream from the Agamma globin gene. Sequencing of five peptides from the purified protein established its identity to a recently cloned protein (SATB-1) that binds to DNA sequences associated with the nuclear matrix (MAR). The downstream Agamma globin regulatory element binds to the nuclear matrix.
