The developmental stage specific regulation of the human beta globin locus is a complex process. The 5' and 3' flanking regions of these genes play an important role in modulating their expression. This occurs by the binding of trans - acting nuclear proteins to well defined sequences in these regions. Intragenic sequences also play an important role in the regulation of human globin gene expression. One of the goals of this study is to map sites in these regions of DNA that bind these regulatory proteins. This will be accomplished by DNAse I footprinting and gel shift assays. Any unique nuclear factor discovered will be further characterized as to its binding properties, purified by column chromatographic techniques, and eventually cloned from a cDNA library. The functional significance of sites in intervening sequences of the beta and gamma globin genes will be assessed by performing in vitro mutagenesis of nucleotides in these introns to abolish the binding of a given factor. Subsequently, this mutated intron will replace its normal counterpart in a globin gene construct, transfected into the appropriate erythroid cell line and analyzed for expression. A second goal of this study is to gain further insight into the mechanism of regulation of globin synthesis by the only erythroid specific DNA binding protein (GF-1) characterized to date. The human protein cDNA will be cloned and expressed in bacteria. The relative binding affinity of recombinant GF-1 for various sites in the gamma promoter, the 3' beta enhancer and intragenic sequences in these genes will be estimated by gel shift assay. Finally, the role of GF-1 in induction of gamma globin synthesis in K562 cells will be characterized by examining changes in the level of binding activity of the protein and its mRNA transcript during the course of hemin induction.
|Flamm, M; Bornschlegel, K; Wu, S et al. (1996) Expression of a cosmid containing the LCR, (A)gamma, delta, and beta globin genes in mouse erythroleukemia cells. Am J Hematol 53:181-7|