Mastermind enhances the activation of a Notch pathway target in cell culture. B. Yedvobnick , A. Kumar. Dept Biol, Emory Univ, Atlanta, GA.

   Previous studies of the Notch pathway component Mastermind (Mam) have suggested roles both upstream and downstream of Notch activity. For example, overexpression of Delta or Notch can rescue wing margin defects associated with loss of Mam function, suggesting an upstream role. However, in other contexts, loss of Mam function can block effects of pathway activation, more consistent with a downstream role. Since the interpretation of such data is complicated by feedback loops that influence the Notch pathway, we are examining the role of Mam through cell culture transfection assays. Previous cell culture assays established that Notch processing liberates an intracellular (Nintra) fragment that acts as a transcriptional coactivator. This fragment complexes with the DNA-binding protein Su(H), leading to up regulation of E(spl) loci. We have tested for the effects of Mam expression on the activation of the E(spl) m gamma promoter. When coexpressed with Nintra and Su(H), full length Mam augments the levels of m gamma promoter activity. We are also testing the hypothesis that Mam is a transcriptional regulator of the Delta gene. Promoter regions from Delta were cloned into a reporter vector and activated in cell culture through cotransfection with the proneural proteins Achaete and Daughterless. However, we observe that addition of full length Mam does not appear to modulate Delta promoter activity in this assay. Thus, either Mam is not involved in Delta activation, or our current cell culture assay cannot properly model the system. The potential effects of putative Mam partner proteins will be also be examined in this system.