Globin gene transcription depends on powerful, upstream regulatory sequences encompassing the locus control region (LCR). Within LCR subregions, binding sites for the erythroid DNA-binding complex NF-E2 mediate enhancer activity. Previously, the investigators characterized and purified the NF-E2 DNA-binding activity of erythroid cells, microsequenced polypeptides and cloned cDNAs encoding its p45 and p18 (maf K) subunits, and disrupted each gene in mice. This combined biochemical-genetic analysis has revealed that p45 NF-E2 is essential for megakaryocyte development but largely dispensable for erythropoiesis, and p18 NF-E2 is entirely dispensable. Studies in cell culture, however, demonstrate that p45 NF-E2 and the p45/p18 heterodimer are both required for polypeptides heterodimerize with p18-like maf- related polypeptides, such as mafG, to substitute for """"""""NF-E2"""""""" function in vivo in erythroid cells. According to this model, small mafs (p18/ mafK and mafG together) are predicted to be essential for normal erythroid development. To test this model, the investigators propose to generate mice in which inactivation of both p18 (mafK) and mafG occurs only in erythroid cells using the cre-loxP system. To accomplish this, they will generate mice with a conditional knockout of the mafG gene and interbreed these with their pre-existing p18 knockout mice. In parallel, they will generate a new mouse strain in which cre recombinase is expressed specifically within the erythroid compartment and validate its usefulness for cre-mediated excisions of condition alleles by making and testing a universal lacZ """"""""reporter"""""""" mouse strain. Ultimately, they will examine erythropoiesis and globin gene expression in the erythroid cells of p18-/-/mafG conditional mice interbred with erythroid cre-expressing mice. Erythroid development and globin gene expression will be analyzed. These studies should permit definitive assessment of the in vivo role of small maf proteins in erythroid gene expression and also establish a general method for the analysis of erythroid-specific gene knockouts in mice.
