Developmental control over the vertebrae beta-type globin genes is predominantly transcriptional. The differential transcriptional activity of each of the genes appears to be the direct consequence of a complex set of temporally determined interactions between the promoters of each gene and the locus control region (LCR). Most, but not all, investigators in this field subscribe to the contention that this promoter:enhancer interaction is physically direct, and that differential gene activation is a consequence of """"""""promoter competition"""""""" [i.e. that activation of the various genes within the locus is determined by the stage-specific activation of transcription factors which bind to the promoters and the LCR (a powerful, multifaceted enhancer), causing the enhancer to activate the thermodynamically most stable promoter interaction by DNA looping]. Most current experiments in the field are interpreted in terms of this hypothesis, but it has never actually been proven in the murine or human beta-globin loci. We continue to explore the molecular bases for developmental activation of the human (beta-globin genes. During the past grant period, we developed and then exploited the use of recombinant YACs containing the whole human beta-globin locus to examine the consequences of mutation of various cis-elements in the locus (presumptive enhancers, promoters, silencers) by incorporating site-specific mutations into a wild-type human (beta-globin YAC using homologous recombination in yeast. While providing compelling evidence in its support, these data did not actually prove or disprove the competition hypothesis in the human (beta-globin locus. In the immediate future, we propose to more fully examine the molecular basis for two of the discoveries we made during the recently completed funding period (i.e. synergy in LCR structure and directionality of the LCR). Secondly, we propose to thoroughly examine the contributions of the KLF family of transcription factors to globin gene switching. Finally, we propose experiments which, if successful, will provide conclusive, direct evidence for competition among beta-globin locus genes for the LCR.
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