Gene expression is controlled by transcription factors, proteins that recognize and bind to specific sequences in the genome and facilitate the expression of target genes. This project focuses on how related transcription factors that share a common DNA binding domain and thus recognize similar DNA sequences regulate the expression of their targets when they are found in the same cells. One such example is the T-box transcription factors, T and Tbx6, which activate target gene expression during mesoderm formation in the mouse embryo. To understand how these related proteins coordinate gene expression to regulate cell fate decisions, transgenic approaches will be used to mis-express each of the proteins during mouse embryogenesis. Molecular and histological techniques will then be used to analyze the resulting embryos. While these proteins can recognize similar DNA sequences, whether they regulate similar target genes in the embryo is unknown. To identify T and Tbx6 target genes in embryonic tissues, biochemical assays will be used. Once targets are identified, transgenic embryos will be examined to determine how changes in the relative levels of T or Tbx6 affect target gene expression, thus linking target gene expression to embryonic consequences. Understanding how this T-box family coordinates target gene selection when they are found in the same cells will serve as a paradigm for other transcription factor families. These experiments will provide the PI, one graduate student and several undergraduates an opportunity to engage in research at a university that actively integrates research and teaching. Undergraduates have multiple opportunities to present their research, including science fairs and undergraduate research symposia. Examples from this research will be used for local K-12 outreach programs, including a middle school science club led by the PI.
T-box transcription factors are essential for normal development in organisms as widely diverse as nematodes and humans. Family members are expressed in a variety of tissues and functional studies have demonstrated the important roles played by these genes during development, including cell type specification and morphogenesis. Maintaining the appropriate levels of these factors is critical since humans heterozygous for T-box genes display phenotypes such as cleft lip and palate, and heart and limb defects. T-box proteins all share a similar DNA binding domain and thus have the potential to regulate similar target genes. We have used complementary in vivo and in vitro approaches to understand how two of these related proteins (T and Tbx6) regulate target gene expression and how this regulation leads to the formation of different mesoderm populations during mouse embryonic development. Genetic data from our lab and others suggests that T is critical for the formation and maintenance of the primitive streak (the source of mesoderm) and Tbx6 is essential for the formation of one specific type of mesoderm – the paraxial mesoderm. We have shown that while both T and Tbx6 can bind to the same regulatory regions of target genes, they have different activities at these targets, with Tbx6 activating expression ten-fold higher than T. We further showed that T and Tbx6 have different affinities for the same recognition sites within these enhancers. Differences in activity and affinities of these transcription factors can thus lead to differential target gene regulation. We hypothesize that these differences create a situation where T and Tbx6 can compete for target genes when they are found in the same cells, specifically in the primitive streak. Thus modulating the amount of these proteins, particularly in mutant embryos, can lead to phenotypes in heterozygotes, similar to the haploinsufficiency seen in humans. To test this hypothesis, we have attempted to identify T and Tbx6 targets and test whether the expression of these genes changes in various mutant backgrounds. In addition we have generated genetic models to increase the levels of T and Tbx6 in the developing embryo to test whether T and Tbx6 can compete in vivo as we have shown in vitro in tissue culture cells. Altogether our results suggest that T-box factors can compete when co-expressed, however this may be through blocking the ability of a related factor from regulating its normal target genes, and not by up-regulation of their own target genes. Future studies will concentrate on altering the levels of these proteins in their normal domains to more directly test how this affects normal target gene expression and behavior of the cells in the context of a whole embryo. The benefits to society of this research include a better understanding of how related transcription factors interact. In addition this research has provided numerous training opportunities not only during the funding period but has also set up future training opportunities as we continue to analyze our target gene data and compare this with other publically available data on T-box targets. During the funding period, the PI and lab members have been involved with a variety of outreach activities. We have trained 1 female post-doctoral fellow, 2 rotating graduate students, and 9 undergraduates (of these 9 undergraduates, 1 was an African American male and 8 were female) and one high school student. Training in the lab ranged from 14 months for the post-doctoral fellow, 1-3 academic years for the undergraduates, to 10 weeks in the summer for the high school student. These trainees have been involved in various aspects of the project from molecular biology, tissue culture, cloning and transcriptional assays to establishing and maintaining breeding colonies and embryo phenotypic characterization. Trainees have presented their data in local development meetings and as an undergraduate poster. The PI and post-doctoral fellow have further been involved in numerous outreach activities, including K-12 presentations and high school and teacher workshops; altogether making developmental biology and research on mammalian model systems more accessible and exciting to students and teachers alike.
