The CACCC element in the human gamma-globin gene promoter is essential for gamma- to beta-globin switching, a developmental process with clinical relevance. Two complementary approaches are described to identify the factor(s) that binds to the gamma-globin CACCC element and regulates the gene.
The first aim i s a functional candidate gene approach. The erythroid Kruppel-like factor (EKLF) regulates the beta-globin gene through its CACCC element. The gamma-globin CACCC-binding regulator is likely to be similar to EKLF. There are 21 C2H2 zinc finger proteins that form a subgroup with EKLF in phylogenetic analyses. Several of the mRNAs for these proteins are expressed in primary erythroid cells from the chick, and in human erythroid K562 and HEL cells. Knockout mice are available for KLF2, 4, 5 and 9. These mice will be bred with mice with the human beta-globin locus, and the effects of these KLFs on globin gene expression will be determined. RNA interference (RNAi) will be used in K562 and HEL cells to knockdown KLF3 and 8, which are expressed in erythroid cells and for which antibodies are available. For those proteins that affect gamma-globin gene expression in these assays, it will be determined whether they bind to the gamma- and/or beta-globin CACCC elements in vitro in gel mobility shift assays and in vivo in chromatin immunoprecipitation (CHIP) assays. In the second aim, gamma-globin CACCC binding factors will be identified and characterized in a non-biased approach. Nuclear extracts will be prepared from purified chicken red blood cells from adults and 9-dpc embryos. The chicken system is ideal because erythroid cells are nucleated and readily available, but if necessary an alternative approach with mouse fetal livers will be used. The nuclear extracts will be affinity-purified to enrich for gamma-globin CACCC binding proteins. Proteins that bind to an affinity column with a wild-type oligonucleotide, but not to a similar oligonucleotide with a CACCC box mutation, will be distinguished by 2-D gel electrophoresis. These proteins will be excised and subjected to MALDI-TOF and/or electrospray ionization tandem mass spectrometry. The human homolog for the chicken (or mouse) protein will be identified using protein sequence comparisons to databases. To determine the importance of the identified protein(s) in gamma-globin gene regulation, RNAi knock down, DNA binding, and ChIP assays will be performed. ? ?
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