During the past 25-30 years the two grants under consideration for merger in the MIRA program (GM46638 and GM34431) have provided numerous insights into the mechanisms underlying the control of gene expression during animal development. Specific highlights include evidence that Hox proteins function as sequence-specific transcription factors, the characterization of the complex eve stripe 2 enhancer, and the elucidation of the affinity threshold model for the differential regulation of gene expression by te Dorsal (NF-kB) gradient. These studies have established the early Drosophila embryo as a premiere system for the study of gene activity in animal development. It is the goal of the proposed MIRA grant to exploit the advent of new imaging technologies to uncover novel mechanisms of gene control. During the past year we have begun to use live imaging methods to visualize the dynamic regulation of gene expression during Drosophila embryogenesis. These studies have led to a number of striking observations that we wish to follow-up during the upcoming funding period, including transcriptional bursts of eve stripe 2 expressions, the non-additive activities of shadow enhancers, transcriptional memory, and allelic communication. We will determine whether transcriptional bursting is due to unstable enhancer-promoter looping interactions, and examine the possibility that the two alleles at a given genetic locus somehow communicate to ensure balanced levels of expression during development. The research plan includes four specific aims: 1) investigate the dynamics by which a single enhancer activates two different target promoters; 2) explore the possibility that temporal precision depends on enhancer switching, whereby enhancers with overlapping activities work in a sequential manner during development; 3) determine whether transcriptional memory depends on specific histone modifications such as methylation; and 4) substantiate our preliminary evidence for allele communication by examining a variety of sensitized transgenes in living embryos.

Public Health Relevance

The development of new technologies enables discovery in the life sciences. The spotlight has shined brightly on the advent of new DNA sequencing methods and genome assays. Imaging technologies have been equally transformative during the past decade. It is now possible to 'cheat' the limits of wavelength resolution and observe living processes at scales under 100 nm. We have begun to use these methods for the analysis of gene activity in living Drosophila embryos. These studies provide evidence for transcriptional memory and the occurrence of transcriptional bursts. We propose to push the envelope on these methods to visualize dynamic enhancer-promoter interactions during the development of living embryos. The insights that will be gained will help illuminate similar processes in more complex systems, including humans. In particular, there is emerging evidence that the human genomes contains 400,000-1 million enhancers, and represent major genetic targets for population diversity and predilection to disease. The proposed study will provide new insights into the function of developmental enhancers.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZGM1)
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Sledjeski, Darren D
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Princeton University
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Lim, Bomyi; Heist, Tyler; Levine, Michael et al. (2018) Visualization of Transvection in Living Drosophila Embryos. Mol Cell 70:287-296.e6
Furlong, Eileen E M; Levine, Michael (2018) Developmental enhancers and chromosome topology. Science 361:1341-1345
Lim, Bomyi; Fukaya, Takashi; Heist, Tyler et al. (2018) Temporal dynamics of pair-rule stripes in living Drosophila embryos. Proc Natl Acad Sci U S A 115:8376-8381
Fukaya, Takashi; Lim, Bomyi; Levine, Michael (2017) Rapid Rates of Pol II Elongation in the Drosophila Embryo. Curr Biol 27:1387-1391
Lim, Bomyi; Levine, Michael; Yamazaki, Yuji (2017) Transcriptional Pre-patterning of Drosophila Gastrulation. Curr Biol 27:286-290
Rogers, William A; Goyal, Yogesh; Yamaya, Kei et al. (2017) Uncoupling neurogenic gene networks in the Drosophila embryo. Genes Dev 31:634-638
Fukaya, Takashi; Levine, Michael (2017) Transvection. Curr Biol 27:R1047-R1049