The goals of this proposal are to define the roles of putative cis-acting regulatory sequences on the stage specific activation and silencing of the human gamma and beta globin genes. Previous studies demonstrate that the native spatial and sequence organization of the human beta globin locus must be preserved to accurately recapitulate the pattern of gene expression observed during human ontogeny. This group and others have generated beta globin YAC transgenic mouse models in which the pattern of gene expression observed, in transgenic lines carrying the wild type locus, is stage-specific and highly reproducible. However, the purification, injection and mapping of YACs is time consuming and technically difficult. In this proposal we will develop a complementary strategy by making mutations in the human beta globin locus in embryonic stem (ES) cell lines derived from transgenic mice carrying a single copy of the human beta globin YAC. This approach will allow us to directly compare the effects of each mutated human beta globin transgene in a fixed chromosomal position of integration. We will focus initially on elements proximal to the gamma globin genes, the stage selector element (SSE), and putative 5' negative regulator of gamma globin genes. Our studies will provide a more complete understanding of the cis elements involved in globin gene regulation during development, and may ultimately aid in the development of novel genetic and pharmacolgic therapies for the treatment of thalassemias and hemoglobinopathies.
