Discipline: Biological Sciences
Bethanie-Michelle Statler - University of Illinois at Chicago
Co-Author(s): Peter Okkema, University of Illinois at Chicago, IL
T-box transcription factors play a key role in the development of many multicellular organisms. However, misregulation of T-box factors TBX2 and TBX3 in humans can cause congenital diseases and a variety of cancers (1). We are studying the closely related C. elegans ortholog of these factors TBX-2 to understand the molecular mechanism by which these conserved factors function. Our genetic results show that C. elegans TBX-2 is a transcriptional repressor whose activity depends on post-translational modification with the SUMO peptide (2, 4). Previous data also shows that TBX-2 represses its own promoter in a nega-tive feedback loop (3). We can use a Ptbx-2::mCherry reporter as a molecular readout to analyze the expression of TBX-2 in TBX-2 mutants defective in SUMOylation. We hypothesize that mutating the SUMOylation site on TBX-2 will result in ectopic expression of Ptbx-2::mCherry. We use transgenically expressed wild-type TBX-2 as a control that effectively represses Ptbx2::mCherry so that it is only expressed in the head neurons. In contrast, a conservative mutation of a key lysine residue that is conjugated to SUMO to engineer mutant TBX-2K400R reduces its repressor activity. In the mutant strain, Ptbx-2::mCherry is ectopically expressed in the hypodermic seam cells and posterior gut at a significantly higher rate than strains expressing wild-type TBX-2. Our findings support our hypothesis that TBX-2 repressor activity depends on SUMOylation. Our results will help elucidate the molecular mechanism for TBX-2 repressor activity in C. elegans. Future research involves investigating other protein interactions that may also be required for TBX-2 activity and identifying target genes of TBX-2. If our project is successful, we can then translate this research into mammalian cell culture assays. As SUMO-conjugation sites are also found in human TBX2 and TBX3, we expect the mechanism of regulation to be conserved (1). These results may help guide the identification of targets in possible therapies for diseases involving these factors.
References (1) Crum, T.L., Okkema, P.G., 2007. SUMOylation-dependent function of a T-box transcription-al repressor in Caenorhabditis elegans. Biochemical Society Transactions. 35(6), 1424-1426.
(2) Huber, P., Crum, T., Clary, L.M., Ronan, T., Packard, A.V., Okkema, P.G., 2013. Function of the C. ele-gans T-box factor TBX-2 depends on SUMOylation. Cell. Mol. Life Sci. 70, 4157-4168.
(3) Milton, A.C., Okkema, P.G., 2015. Caenorhabditis elegans TBX-2 directly regulates its own expression in a negative autoregulatory loop. G3 5, 1177-1186.
(4) Chowdhuri, S.R., Crum, T., Woollard, A., Aslam, S., Okkema, P.G., 2006. The T-box factor TBX-2 and the SUMO conjugating enzyme UBC-9 are required for ABa-derived pharyngeal muscle in C. elegans. Dev. Biol. 295, 664-677.
Funder Acknowledgement(s): Funding was provided by the NSF Bridge to the Doctorate fellowship to B-M. Statler.
Faculty Advisor: Peter Okkema, firstname.lastname@example.org
Role: I used optical microscopy to analyze and score multiple TBX-2 mutants for expressing Ptbx-2::mCherry. I created additional strains that will be used in future research involving the interactions of other proteins with TBX-2.