The early Drosophila embryo represents one of the premier systems for visualizing differential gene activity in animal development. During a one-hour interval, from 2 to 3 hours after fertilization, hundreds of genes are activated during the mid-blastula transition. These genes exhibit localized stripes and bands of expression that establish the different segments and tissues of larvae and adults. During this time the embryo is composed of a simple grid of synchronized nuclei located near the surface of the egg. It is therefore possible to visualize basic transcriptional processes, such as the regulation of enhancer-promoter interactions. We will use this system to investigate the physiological properties of transcriptional processes that are mainly studied in vitro or in cultured cells. Most of our proposed studies focus on mechanisms of transcriptional precision. For example, Snail, a key regulator of epithelial-mesenchyme transitions, is expressed in 800 mesoderm progenitor cells slated for invagination during gastrulation. Snail exhibits remarkably invariant expression in these cells (~180 mRNAs/cell), and the sharp lateral limits of its expression pattern delineate the boundary separating the future mesoderm and ectoderm. These features, homogenous expression and sharp lateral limits, are highly reproducible among the different embryos of a population. We have obtained evidence that this precision depends on paused RNA polymerase (paused Pol II), which appears to foster synchronous transcription in the different cells of a tissue. Preliminary studies suggest that replacing the paused Snail promoter with a nonpaused promoter results in stochastic activation of Snail expression and a curious bistable phenotype. Approximately 20% of the resulting embryos exhibit an essentially normal Snail pattern, including homogenous expression and sharp lateral limits, whereas 80% of sibling embryos lack Snail expression and display the same loss of mesoderm invagination seen in sna- mutants. These results suggest that transcriptional synchrony is important for the coordination of gene activity underlying the invagination of the mesoderm. We will attempt to determine the basis for this gastrulation bistability and explore the mechanisms responsible for transcriptional synchrony. In particular, we will test the idea that paused Pol II displaces inhibitory promoter-positioned nucleosomes (PPNs). We will also investigate the role of the promoter in developmental timing. Moreover, just as paused Pol II might serve to """"""""prime"""""""" the core promoter for timely activation of gene expression, we will explore the role of histone modifications and general transcription factors for priming distal enhancers. And finally, we will use quantitative imaging methods to attempt to visualize long-range enhancer-promoter interactions in the Drosophila embryo.
Switching genes on and off underlies many developmental and disease processes. We propose to use the unique advantages of the fruit fly embryo to study the mechanisms responsible for the reliable activation of gene expression in large populations of cells and tissues. Many of the proposed experiments focus on emerging evidence that genes are primed or prepared for activation in order to respond quickly and synchronously to developmental cues.
|Esposito, Emilia; Lim, Bomyi; Guessous, Ghita et al. (2016) Mitosis-associated repression in development. Genes Dev 30:1503-8|
|Fukaya, Takashi; Lim, Bomyi; Levine, Michael (2016) Enhancer Control of Transcriptional Bursting. Cell 166:358-68|
|Farley, Emma K; Olson, Katrina M; Zhang, Wei et al. (2015) Suboptimization of developmental enhancers. Science 350:325-8|
|Farley, Emma K; Olson, Katrina M; Levine, Michael S (2015) Regulatory Principles Governing Tissue Specificity of Developmental Enhancers. Cold Spring Harb Symp Quant Biol 80:27-32|
|Levine, Michael (2014) The contraction of time and space in remote chromosomal interactions. Cell 158:243-4|
|Levine, Michael; Cattoglio, Claudia; Tjian, Robert (2014) Looping back to leap forward: transcription enters a new era. Cell 157:13-25|
|Boettiger, Alistair Nicol; Levine, Michael (2013) Rapid transcription fosters coordinate snail expression in the Drosophila embryo. Cell Rep 3:8-15|
|Lagha, Mounia; Bothma, Jacques P; Esposito, Emilia et al. (2013) Paused Pol II coordinates tissue morphogenesis in the Drosophila embryo. Cell 153:976-87|
|Lagha, Mounia; Bothma, Jacques P; Levine, Michael (2012) Mechanisms of transcriptional precision in animal development. Trends Genet 28:409-16|
|Chopra, Vivek S; Kong, Nikki; Levine, Michael (2012) Transcriptional repression via antilooping in the Drosophila embryo. Proc Natl Acad Sci U S A 109:9460-4|
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