A key question in developmental biology is how to identify the functional DNA sequences in enhancer cis--?regulatory modules (CRMs), those regions of the genome which are critical to the activation of gene expression in all eukaryotes. The regulatory output from an enhancer is known to be controlled by the combination of transcription factor proteins that bind within the enhancer sequence. However, these transcription factors often bind many sites within the genome with varying affinity and their binding sites frequently undergo extensive turnover through the process of evolution. Despite these properties, in many cases the functional activity of the enhancers across different species remains robust. The precise requirements for enhancer functional activity are thus still unclear. The goal of this project is to investigate the regulatory grammar that controls the activity of transcription factor binding at enhancers and subsequent gene regulation. The Hox genes of the Drosophila bithorax complex are critical genes that determine the animal body plan during embryonic development. The spatial and temporal expression of Hox genes in the developing embryo is controlled by CRMs. Our preliminary studies show that sequences at the CRMs of the Drosophila bithorax complex are rapidly evolving within the subset of Drosophila species that we studied. This is in sharp contrast to the protein--?coding Hox genes themselves, which are highly conserved across all bilaterian animals. The recent genomic sequencing of additional Drosophila species, for a total of twelve species, as well as the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera, provides an enticing opportunity to expand the evolutionary scope of our bioinformatic and functional analysis of Hox gene CRMs into emerging model organisms. The wide range of insect genomes, representing over 300 million years of evolutionary separation enables a more accurate measure of transcription factor binding site turnover in CRMs and the functional significance of such changes at the sequence level. The ultimate goal of this proposal is to elucidate the molecular mechanisms that control functional activity of the embryonic enhancer CRMs at the sequence level and to extend this investigation to emerging model species. The long term scientific goal of the PIs is to fully investigate the mechanisms by which the genomic regulatory regions achieve coordinated control of gene expression across the entire bithorax complex. The major focus of this proposal will be on decoding the regulatory logic of transcription factor binding site architecture within CRMs from the Hox complexes in different insect species. The tools developed from the preliminary studies will be used to identify clusters of transcription factor binding sites within known CRM orthologs, to discover novel CRMs across a wide range of Anopheles, Apis and Drosophila species and to address the functional constraints on enhancer evolution. These studies will offer insight into the molecular mechanisms that control the functional plasticity and robustness of the CRMs that regulate embryonic development in divergent insect species. Undergraduate students will be an integral part of the success of this project. Indeed, the contribution from students will be essential to the completion of the proposed research and as a result they will receive extensive guidance and share authorship on publications. Clark University has a strong tradition of excellence in undergraduate research. During the last eight years, Dr. Drewell has personally trained 75 undergraduate students in the laboratory and published 15 papers with 44 student co--?authors, representing 20 different students. Each student in the Drewell laboratory is fully encouraged to take ownership of his or her own research project. Almost all students who have graduated from the Drewell laboratory were accepted into prestigious PhD programs including those at UCSF, Harvard, Caltech, Duke, Columbia and MIT. Special attention will continue to be given to recruiting female, minority and first--?generation college student researchers, who are currently highly represented in the Drewell laboratory. The newly established Dresch laboratory will work in close collaboration on the proposed project. During Dr. Dresch's one year as a postdoctoral fellow at Harvey Mudd College and two years as an Assistant Professor at Amherst College she has worked directly with 20 undergraduate students on collaborative research projects with the Drewell lab in areas including bioinformatics, thermodynamic--?based modeling and evolutionary algorithms for parameter estimation. The projects from these interdisciplinary interactions has already resulted in four publications, with a further three papers currently submitted for publication. The work in this proposal will also generate exciting material for incorporation into courses taught by the PIs at Clark University, while the results of the work will be presented by students at international conferences. The efforts from this project will therefore concurrently advance discovery and promote teaching, training and learning.

Public Health Relevance

The issue of gene regulation is at the heart of our understanding of development and disease. Carefully timed and spatially controlled expression of key developmental genes, many of which are transcription factors that regulate the expression of downstream genes, serves as the critical basis for normal development. The early patterns of Hox gene expression in the embryo initiate a cascade of regulatory events at the molecular level that has profound consequences for later development. Currently our understanding of the mechanisms of cis--?regulation for the Hox genes is still very superficial. Our preliminary studies have led us to investigate the function of cis--?regulatory modules and how transcription factors act at the regulatory sequence level to control the expression of Hox genes at the bithorax complex of Drosophila. In conjunction with the recent publication of genomic sequences for twelve Drosophila species, the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera, we will extend our bioinformatic and functional analysis of CRM function to non--?traditional Drosophila, Anopheles, and Apis species. Our proposed studies promise to elucidate the regulatory grammar, function and molecular evolution of CRMs in embryonic development.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
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Special Emphasis Panel (ZRG1)
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Sledjeski, Darren D
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Clark University (Worcester, MA)
Schools of Arts and Sciences
United States
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Elmas, Abdulkadir; Wang, Xiaodong; Dresch, Jacqueline M (2017) The folded k-spectrum kernel: A machine learning approach to detecting transcription factor binding sites with gapped nucleotide dependencies. PLoS One 12:e0185570
Pettie, Kade P; Dresch, Jacqueline M; Drewell, Robert A (2016) Spatial distribution of predicted transcription factor binding sites in Drosophila ChIP peaks. Mech Dev 141:51-61
Dresch, Jacqueline M; Zellers, Rowan G; Bork, Daniel K et al. (2016) Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome. Gene Regul Syst Bio 10:21-33