[
International Worm Meeting,
2009]
Cell fate, or how a cell decides to differentiate into a certain cell type, depends largely on which genes are expressed. Transcription factors are proteins responsible for such regulation, although the biochemical characterization of such systems is incomplete. For example, very few gene targets have been identified. In addition, transcription factors often heterodimerize with other factors in order to regulate their activity. The winged-helix transcription factor LIN-31 is involved in cell fate decisions during development of the vulva, an egg laying structure, in the model organism Caenorhabditis elegans. This protein contains a DNA-binding domain and several other regions of unknown function. The current model for LIN-31 function is that it plays two roles in vulval development: 1) it heterodimerizes with another transcription factor, LIN-1, to promote non-vulval cell fates in some cells and 2) when the dimer is disrupted due to cell-cell signaling events, it promotes a vulval cell fate in other cells (Miller et al, 1993 and Tan et al, 1998). In C. elegans, the LIN-31 protein is expressed in very few cells, making direct biochemical analysis difficult. Attempts to express and purify this protein in bacterial and insect cells have been impeded by low expression levels and insolubility. To learn more about the role of this protein without purifying it, we are using a yeast-based system for two-hybrid (Y2H) analysis. Prior to conducting a full-scale screen, we are directly testing interactions between LIN-31 and its interaction partner, LIN-1. First, we used the Y2H system to confirm LIN-31''s interaction with LIN-1. Previous Y2H experiments in other labs had failed to see this interaction, but removal of LIN-31''s DNA binding domain allowed interaction with LIN-1 in our Y2H system. Second, we showed that LIN-31''s small acidic domain is required for this interaction. Elimination of this domain by non-conservative amino acid substitutions resulted in disruption of the LIN-31/LIN-1 interaction. This is consistent with previous site-directed mutagenesis experiments indicating that this acidic region is required for proper LIN-31 function in non-vulval cells (Morris et al, in prep).