The C. elegans pharynx is distinct neuromuscular organ consisting of several very different cell types, including muscles, neurons and epithelia cells. Our long term goal is to understand the development of the pharynx as a simple model for cell-type specification and organogenesis. The primary focus of this application is on the mechanisms regulating expression and function of the T-box transcription factor TBX-2 during pharyngeal muscle development. TBX-2 is essential for development of the pharyngeal muscles derived from the embryonic ABa blastomere, and it physically interacts with enzymes catalyzing protein sumoylation and with the Gro/TLE family co-repressor UNC-37. We hypothesize TBX-2 is a transcriptional repressor, and this function depends on post-translational sumoylation and complex formation with UNC-37. We further hypothesize sumoylation and interaction with Gro/TLE co-repressors is a conserved feature of T-box transcriptional repressors in all organisms. T-box factors related to TBX-2 have been implicated in human congenital disease and cancer, but a role for sumoylation and Gro/TLE interaction has not been examined. An understanding of C. elegans TBX-2 will provide crucial information that may direct development of treatments for diseases resulting from T-box factor defects. This proposal includes 4 Specific Aims.
The first aim i s to characterize the position of tbx-2 in the pathway leading to ABa-derived pharyngeal muscle. Genetic and molecular approaches will be used to identify upstream regulators of tbx-2 expression, while microarray analyses will be used to identify downstream genes regulated by TBX-2.
The second aim i s to determine the role of sumoylation in TBX-2 function, and the third aim is to examine the role of TBX-2 interaction with UNC-37/Groucho. Mutant forms of TBX-2 defective in sumoylation or UNC-37 interaction will be expressed in transgenic C. elegans and in cell culture assays, and the function and localization pattern of these proteins will be examined.
The fourth aim i s to determine whether sumoylation and Gro/TLE co-repressor interaction are common features of T-box factor function in C. elegans and mammals. Predicted sumoylation sites and Gro/TLE interaction motifs will be mutated in the C. elegans MAB-9 and MLS-1 T-box factors, and function of these factors will be assayed in transgenic animals. Similarly, a sumoylation site and a Gro/TLE interaction motif will be mutated in the mouse repressor Tbx2, and activity of these mutants will be examined using established co-transfection assays in mammalian cultured cells. T-box genes encode a phylogenetically conserved family of transcription factors that play crucial roles in the development of all multicellular organisms. Interest in this important transcription factor family has recently increased, because defects in the function or regulation of T-box factors are associated with a number of human congenital diseases and cancers. This proposal aims to characterize mechanisms controlling the expression and function of T- box factors in the simple model organism C. elegans, and the results obtained will have a direct bearing on our understanding of these factors in all organisms. ? ? ?
