Gene Activation by Remote Transcriptional Enhancers

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DESCRIPTION (Verbatim from the applicant's abstract): Enhancers that activate promoters several kilobases away are vital for the appropriate expression of several genes that are required for metazoan development. We discovered the Drosophila Nipped-B protein in a genetic screen for factors that support activation by a distant enhancer in the cut gene. Nipped-B also aids activation of the Ultrabithorax (Ubx) gene by remote enhancers, and participates in sister chromatid cohesion in dividing larval neuroblasts. The goal is to understand how Nipped-B participates in gene activation. Nipped-B is homologous to a human protein of unknown function and to the yeast Scc2 (S. cerevisiae) and Mis4 (S. pombe) proteins required for sister chromatid cohesion. Scc2 is needed for the Cohesin protein complex that holds sister chromatids together to bind to chromosomes. The data indicate that Scc2 and Mis4 bind to chromosomes at all stages of the cell cycle and help recruit Cohesin during S phase. The investigator postulates that Nipped-B acts similarly to aid gene activation by recruiting protein complexes that act on chromosome structure to hold enhancers and promoters close together. Cohesin contains two SMC proteins, which may directly affect DNA and chromosome structure. In the proposed work, mutations in genes encoding SMC proteins or other components of the Cohesin complex will be used to determine if these proteins also participate in activation of cut or Ubx. As a more general approach, proteins that interact with Nipped-B will be isolated biochemically, and then mutations in the genes encoding them will be used to explore their in vivo roles in gene expression and chromatid cohesion. Mutations in Nipped-B and constructed deletion mutants will be used to see if the gene expression and chromatid cohesion functions of the Nipped-B protein are separable. Immunostaining and chromatin immunoprecipitation experiments will be used to determine if Nipped-B protein acts directly at cut and Ubx, or at sites distant from these target genes. These experiments will help define the role of Nipped-B in gene activation and increase understanding of how remote enhancers regulate transcription. This will help illuminate the basic mechanisms underlying gene expression defects that occur in some human diseases.