Complex animals use hundreds of transcription factors (TFs) to accurately control cell-specific gene expression during the differentiation of specialized cell types within each organ. While genomic approaches have shown that many TFs bind thousands of overlapping regions, deciphering which DNA binding events and TF interactions are biologically meaningful remains a major challenge. The long-term goal of this application is to obtain a high-resolution understanding of the TFs, transcriptional mechanisms, and cis-regulatory logic used to ensure robust cell-specific EGF signaling during Drosophila development. Our experimental system is the transcriptional activation of the rhomboid (rho) protease that triggers EGF secretion from specific abdominal sensory organ precursor cells (SOPs) to induce metabolic cells (oenocytes) needed for animal growth and viability. Since only a subset of abdominal but not thoracic SOPs activate rho and the transcriptional levels of rho dictate the number of oenocytes specified, the regulation of rho serves as a great model to understand how regional- and tissue-restricted transcription factors are integrated to control robust cell-specific gene expression and phenotypic outcomes. Our findings during the first funding cycle of this grant revealed that: A) rho contains multiple cis-regulatory modules (CRMs) that activate abdominal SOP gene expression;B) A rho CRM contains numerous overlapping TF binding sites that directly integrate five TFs including an Abdominal-A (Abd-A) Hox complex containing the Extradenticle and Homothorax Hox cofactors and two neuronal transcription factors (Senseless and Pax2);C) AbdA-Senseless antagonism is a novel conserved Hox transcriptional mechanism that controls both EGF signaling in flies and blood cell proliferation and leukemia progression in mice. Building on these findings, this application has three aims: 1) Determine how the regional Abd-A Hox factor is integrated with the neural-restricted Pax2 factor to activate rho and assess which other Hox factors use Pax2 as a cofactor. 2) Define the role of additional neuronal transcriptional inputs that regulate rho in a specific subset of SOPs. 3) Use the underlying cis-regulatory logic to develop a bioinformatics algorithm to predict additional rho CRMs that ensure robust expression levels and phenotypes. Our approach combines the advantages of Drosophila genetics, non-biased mutagenesis reporter assays, and BAC genomic rescue assays with the speed of cell culture, biochemistry and bioinformatics. The successful completion of these aims has a high potential to uncover novel TF interactions that will open up new avenues of research. In addition, by coupling high-resolution mutagenesis studies with genomic rescue assays that provide a biologically meaningful readout, we will obtain new insights into how CRMs and transcription factors control robust cell-specific gene expression within a complex animal. Since the TFs and biological processes studied are highly conserved between flies and mammals, we are optimistic our mechanistic studies will continue to uncover new gene regulatory mechanisms relevant to human health and development.

Public Health Relevance

The precise control of gene expression by transcription factors is essential for the proper formation of specialized cell types within each organ system. Unfortunately, we lack an understanding of how transcription factors are integrated to yield cell-specific gene expression within a complex organism. The goal of this research proposal is to determine how region-specific Hox transcription factors interact with tissue-specific transcription factors to regulate cell-specific gene expression during development. Our proposed studies utilize powerful genetic and biochemical approaches in the fruit- fly Drosophila melanogaster, and we have already used this approach to uncover novel gene interactions relevant to human development and leukemia. Thus, since both the processes and the transcription factors studied are highly conserved from flies to mammals, our findings will reveal fundamental new insights relevant to vertebrate development and human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079428-07
Application #
8716770
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Hoodbhoy, Tanya
Project Start
2008-04-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
7
Fiscal Year
2014
Total Cost
$290,700
Indirect Cost
$100,700
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
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Fujioka, Miki; Gebelein, Brian; Cofer, Zenobia C et al. (2012) Engrailed cooperates directly with Extradenticle and Homothorax on a distinct class of homeodomain binding sites to repress sloppy paired. Dev Biol 366:382-92
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Uhl, Juli D; Cook, Tiffany A; Gebelein, Brian (2010) Comparing anterior and posterior Hox complex formation reveals guidelines for predicting cis-regulatory elements. Dev Biol 343:154-66
Witt, Lorraine M; Gutzwiller, Lisa M; Gresser, Amy L et al. (2010) Atonal, Senseless, and Abdominal-A regulate rhomboid enhancer activity in abdominal sensory organ precursors. Dev Biol 344:1060-70
Lomberk, Gwen A; Imoto, Issei; Gebelein, Brian et al. (2010) Conservation of the TGFbeta/Labial homeobox signaling loop in endoderm-derived cells between Drosophila and mammals. Pancreatology 10:74-84
Li-Kroeger, David; Witt, Lorraine M; Grimes, H Leighton et al. (2008) Hox and senseless antagonism functions as a molecular switch to regulate EGF secretion in the Drosophila PNS. Dev Cell 15:298-308
Gebelein, Brian (2008) The control of EGF signaling and cell fate in the Drosophila abdomen. Fly (Austin) 2:257-8

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