The human epsilon and gamma globin genes are expressed at specific times during early development and are silenced in the adult. The experiments outlined in this proposal are designed to test the hypothesis that the stage-specific suppression of epsilon and gamma globin gene activity is accomplished by the binding of regressors to cis elements upstream from these genes. A candidate repressor protein, CSBP-1 (Conserved Sequence Binding Protein-1) binds to nine evolutionarily conserved sequence elements upstream from the gamma and epsilon genes. Three CSBP-1 binding sites were found within a region of the epsilon promoter which is known to have silencing activity. Initial experiments indicated that this ubiquitously distributed protein was a probable zinc finger protein of approximately 50 kd. In transient transfection assays, it exhibited repressor activity. Alignment of the six highest affinity binding sites revealed the binding consensus, CTNNATTTT. Further analysis shows that CSBP-1 is identical to a previously cloned zinc finger protein variously called YY1, NF-E1, delta or UCRBP. The YY1 protein has been shown to act as a switch factor, mediating repression or activation depending upon the cellular milieu. The NF-E1 protein is involved in the stage-specific silencing of another tissue-specific gene, Igk. In this application, the possible role of this interesting protein in the control of gamma and/ or epsilon gene regulation will be explored. Human epsilon or gamma genes containing mutated CSBP-1 binding sites will be tested in transgenic mice to determine whether stage- specific silencing is compromised. Transient transfection assays will be used to further define the binding sites important for CSBP-1 function and to test whether protein-protein interactions influence CSBP-1 function. In vivo footprinting will be employed to determine the degree of occupation of the CSBP-1 binding sites during erythroid development. To dissect the domains of CSBP-1 which are necessary for the function of this protein, a complementation assay will be developed in Drosophila SL-2 cells and in vitro transcription systems will be established. Triplex strategies will also be used to directly interfere with the binding of CSBP-1 to its cognate site in the nucleus. If this results in reactivation of epsilon and gamma gene activity, then it represents a potentially useful therapeutic strategy in the individuals with defective adult beta genes. Finally, the cellular co-factors which interact with CSBP-1 will be identified. Elucidation of such co-factors would not only further our understanding of the biology of hemoglobin switching, but also serve as a second possible point of control in the therapeutic manipulation of epsilon and/ or gamma gene activity.
