The precellular Drosophila embryo provides a unique opportunity to study mechanisms of transcription regulation in the context of development. Between 2 and 3 hours after fertilization, hundreds of genes are activated within restricted spatial limits, e.g., segmentation stripes, that control the patterning of the embryo and establish the blueprint of the adult fly. Due to the remarkable rapidity of development, the transcription of key patterning genes approaches the kinetic limits of eukaryotic gene expression. During the past few years it has become clear that most patterning genes contain paused Pol II prior to activation, leading to a sea change in our thinking about gene regulation in development. Pol II elongation, not recruitment, appears to be the key rate-limiting step in the activation of gene expression during embryognesis. The proposed study will explore the regulation and function of paused Pol II in the anterior-posterior patterning of the Drosophila embryo. Whole-genome microccocal nuclease assays will be used to determine whether paused Pol II fosters reliable and rapid activation of gene expression by excluding the assembly of inhibitory nucleosomes at the core promoter. We will also employ ChIP-Seq assays to examine interactions between paused Pol II and Pol II elongation factors. Particular efforts will focus on two elongation complexes, SEC and PAF1C. The proposed study will also investigate the basis for Polycomb-mediated silencing of gene expression. Polycomb repressors have been implicated in a variety of developmental and disease processes and appear to be essential for stable gene silencing in differentiated tissues. We recently obtained evidence that many developmentally regulated genes that are transiently expressed in the early embryo lose paused Pol II as they are silenced in larval tissues at the conclusion of embryogenesis. However, Pol II is retained in the promoter regions of these genes in Polycomb mutants. We will investigate the possibility that Polycomb repressors, particularly the PRC2 repression complex, remove paused Pol II from silent genes by fostering the assembly of inhibitory nucleosomes at the core promoter. A final goal of the proposed study is to investigate the basis for the unusually long 3'UTRs seen at hundreds of genes expressed in the Drosophila CNS. We recently obtained evidence that the synthesis of these UTRs depends on the suppression of proximal poly(A) signals by the RNA-binding protein ELAV, a key neuronal determinant. We will determine whether paused Pol II is important for the selective recruitment of ELAV to target genes containing ultra-long 3'UTRs.

Public Health Relevance

There has been a sea change in our view of how genes are switched on and off in animal development, since recent studies have shown that a significant fraction of all genes (~30%) contain preloaded RNA Polymerase II (paused Pol II) prior to their activation. This is seen in both model organisms such as the fruitfly Drosophila and in human tissues, including pluripotent stem cells. The purpose of this study is to exploit the well-defined Drosophila embryo to understand the regulation and function of paused Pol II in development.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Sledjeski, Darren D
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University of California Berkeley
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Fukaya, Takashi; Lim, Bomyi; Levine, Michael (2017) Rapid Rates of Pol II Elongation in the Drosophila Embryo. Curr Biol 27:1387-1391
Ferraro, Teresa; Esposito, Emilia; Mancini, Laure et al. (2016) Transcriptional Memory in the Drosophila Embryo. Curr Biol 26:212-218
El-Sherif, Ezzat; Levine, Michael (2016) Shadow Enhancers Mediate Dynamic Shifts of Gap Gene Expression in the Drosophila Embryo. Curr Biol 26:1164-9
Fukaya, Takashi; Lim, Bomyi; Levine, Michael (2016) Enhancer Control of Transcriptional Bursting. Cell 166:358-368
Oktaba, Katarzyna; Zhang, Wei; Lotz, Thea Sabrina et al. (2015) ELAV links paused Pol II to alternative polyadenylation in the Drosophila nervous system. Mol Cell 57:341-8
Hilgers, Valérie (2015) Alternative polyadenylation coupled to transcription initiation: Insights from ELAV-mediated 3' UTR extension. RNA Biol 12:918-21
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:
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
Levine, Michael; Cattoglio, Claudia; Tjian, Robert (2014) Looping back to leap forward: transcription enters a new era. Cell 157:13-25
Levine, Michael (2014) The contraction of time and space in remote chromosomal interactions. Cell 158:243-244

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