Cell fate decisions lead to highly reproducible patterns in metazoan development. However, these decisions hinge upon the system overcoming stochastic transcriptional events at the molecular level. The goal of the proposed study is to establish the molecular mechanisms underlying transcription in living fly embryos and to understand the dynamic and stochastic nature of these mechanisms that eventually lead to precise patterns. To gain insights into the regulatory processes that span such vastly differing temporal and spatial scales-molecular transcription events vs. formation of macroscopic patterns-we need to establish a connection between DNA regulatory elements and cell fate decisions in terms of stable transcriptional output, resolved in both time and space. Therefore we will determine a relationship between regulatory DNA elements and transcription activity using quantitative measurements to define a set of parameters characterizing transcription dynamics, such as, e.g., polymerase loading and elongation rates. Such a relationship will give us a link between sequence and fate, and will allow us to formulate quantitative models that lead to predictions that are directly testable in vivo. We propose three approaches that will contribute to the establishment of these structure- function relationships at different and complementary levels: 1) we will dissect the structure of small regulatory enhancers and measure the transcription activity and output in reporter fly constructs. 2) Using genome editing we will measure the endogenous transcription activity of enhancers and combinations of enhancers for specific genes. 3) We will scale up the approach to large numbers of genes in fixed tissue to identify classes of genes according to their dynamic transcription properties. Establishing a quantitative structure-function relationship will ultimately lead us to regulate and re-engineer the transcription programs underlying development and disease processes.
Embryonic development is an extraordinarily reliable process. It is robust against genetic and environmental perturbations, and it is highly reproducible from one embryo to the next. On the other hand, the smallest error, down at the molecular level, can have very severe consequences, such as developmental or birth defects, and errors in developmental signaling pathways can lead to the formation of cancer. Answers to these problems are of molecular nature, and here we propose a quantitative approach to uncover the rules governing one of the most fundamental aspects of development-the link between genomic sequence and cell fate decision. The goal is to use these rules to regulate and re-engineer the transcription programs underlying development and disease processes.
| Zoller, Benjamin; Little, Shawn C; Gregor, Thomas (2018) Diverse Spatial Expression Patterns Emerge from Unified Kinetics of Transcriptional Bursting. Cell 175:835-847.e25 |
| Garcia, Hernan G; Gregor, Thomas (2018) Live Imaging of mRNA Synthesis in Drosophila. Methods Mol Biol 1649:349-357 |
| Chen, Hongtao; Levo, Michal; Barinov, Lev et al. (2018) Dynamic interplay between enhancer-promoter topology and gene activity. Nat Genet 50:1296-1303 |
| Little, Shawn C; Gregor, Thomas (2018) Single mRNA Molecule Detection in Drosophila. Methods Mol Biol 1649:127-142 |
| Gregor, Thomas (2017) Beyond D'Arcy Thompson: Future challenges for quantitative biology. Mech Dev 145:10-12 |
| Bothma, Jacques P; Garcia, Hernan G; Ng, Samuel et al. (2015) Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo. Elife 4: |
| Tka?ik, Gašper; Dubuis, Julien O; Petkova, Mariela D et al. (2015) Positional information, positional error, and readout precision in morphogenesis: a mathematical framework. Genetics 199:39-59 |
| Tikhonov, Mikhail; Little, Shawn C; Gregor, Thomas (2015) Only accessible information is useful: insights from gradient-mediated patterning. R Soc Open Sci 2:150486 |
| Bothma, Jacques P; Garcia, Hernan G; Esposito, Emilia et al. (2014) Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos. Proc Natl Acad Sci U S A 111:10598-603 |
| Petkova, Mariela D; Little, Shawn C; Liu, Feng et al. (2014) Maternal origins of developmental reproducibility. Curr Biol 24:1283-8 |
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