The proposed study is a continuation of current efforts to analyze the regulation of the Drosophila segmentation gene, even-skipped (eve). It encodes a homeobox protein that is transcribed in a series of 7 stripes in the early embryo. These stripes foreshadow the subdivision of the embryo into segments, and it is hoped that a detailed understanding of how eve stripes are formed will provide general insights into the segmentation process. It is known that individual eve stripes are controlled by separate enhancers in the eve promoter. For example, the regulation of stripe #2 depends on a 500 bp enhancer that is located between -1.5 Kb and -1 Kb upstream of the transcription start site. The stripe #2 enhancer is activated by broadly distributed activators, including the bicoid morphogen; the stripe borders are formed by selective repression. Activation and repression is mediated by tightly linked binding sites within the stripe #2 enhancer. The overall goal of the proposed study is to determine how the eve stripe enhancers function as integrating pattern elements that direct sharp on/off stripes of gene expression in response to crude gradients of transcriptional activators and repressors. This information should provide general insights into how eukaryotic enhancers work. For example, the kappa light chain enhancer probably directs B-cell specific expression by discerning relatively small differences in the levels of activators and repressors that are common to many different hematopoietic lineages. The research plan includes 5 specific aims. first, in vitro binding assays will be done to determine whether stripe #2 activators bind DNA cooperatively, and whether repressors compete with activators for common sites. Second, in vivo assays will investigate the detailed regulation of eve stripe #2, with a particular emphasis on the mechanisms underlying repression and multiplicative activation. Third, the stripe #3 enhancer will be characterized to determine whether there are general rules governing stripe formation. Fourth, the organization of the eve promoter will be examined by altering the spacing between individual stripe enhancers. And finally, the origins of segment polarity will be investigated by performing genetic complementation assays with defective eve promoters.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM034431-10
Application #
3285364
Study Section
Molecular Biology Study Section (MBY)
Project Start
1984-12-01
Project End
1997-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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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