Genome sequencing and the identification of epigenetic marks by projects such as ENCODE and the Epigenomics Roadmap Project have transformed biomedical research. Technologies for targeted manipulation of these epigenetic properties are necessary to transform the knowledge gained from these projects into tangible scientific advances and benefits for human health, such as gene therapies that modify the epigenetic code at targeted regions of the genome and the engineering of epigenome-specific drug screening platforms. To address this technology gap, we are developing a suite of well-characterized tools for custom locus- and cell type-specific modification of any epigenomic property with precise spatiotemporal control. These tools consist of fusion proteins of programmable DNA-binding proteins and enzymes that control genome structure and function. These epigenetic modifiers (EGEMs) can be specifically targeted to nearly any site in the genome. Optimized EGEM designs will be tested on both proximal and distal regulatory elements that represent diverse chromatin states, including active, repressive, bivalent, and imprinted marks. The generality of EGEMs will be shown on additional high-value targets that have broad relevance to disease. Importantly, all of these tools function independent of cell- and species-type, and therefore are useful to all fields of biologic research. Comprehensive characterization of EGEM activity in human cells will be provided by targeted and genome-wide analysis of DNA-binding, chromatin structure, and gene regulation. A validated optogenetic approach for controlling protein localization with blue light will be used to achieve precise spatiotemporal control of EGEM activity. The utility of the tool set of epigenetic modifiers will b demonstrated by impacting gene regulation in a manner that is robust, specific, and heritable. We will test the working hypothesis that different genes will require a customized set of epigenetic modification(s) to achieve efficient changes in gene expression. The specificity and stability of epigenetic modifications will be of broad utility to the fields of genomics, epigenomis, imprinting, gene therapy, developmental biology, regenerative medicine, and drug development.

Public Health Relevance

In addition to the sequence of the human genome, it has become clear that genome structure plays a critical role in health and disease. However, there are currently no tools to manipulate this genome structure in cells in a precise manner. As a result, it is not possible to reverse diseases or disorders related to genome structure or to perform experiments that increase our understanding of the role of genome structure in biological systems. In this proposal, we will develop tools for precisely modifying genome structure that will catalyze innovative advances in gene and cell therapy, drug development, and basic science.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA036865-03
Application #
8866379
Study Section
Special Emphasis Panel (ZRG1-BST-N (50))
Program Officer
Satterlee, John S
Project Start
2013-09-15
Project End
2018-05-31
Budget Start
2015-06-01
Budget End
2016-05-31
Support Year
3
Fiscal Year
2015
Total Cost
$486,983
Indirect Cost
$173,426
Name
Duke University
Department
Biomedical Engineering
Type
Schools of Engineering
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
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Thakore, Pratiksha I; Kwon, Jennifer B; Nelson, Christopher E et al. (2018) RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors. Nat Commun 9:1674
McDowell, Ian C; Barrera, Alejandro; D'Ippolito, Anthony M et al. (2018) Glucocorticoid receptor recruits to enhancers and drives activation by motif-directed binding. Genome Res 28:1272-1284
Black, Joshua B; Gersbach, Charles A (2018) Synthetic transcription factors for cell fate reprogramming. Curr Opin Genet Dev 52:13-21
D'Ippolito, Anthony M; McDowell, Ian C; Barrera, Alejandro et al. (2018) Pre-established Chromatin Interactions Mediate the Genomic Response to Glucocorticoids. Cell Syst 7:146-160.e7
Gersbach, Charles A; Barrangou, Rodolphe (2018) Pulling the genome in opposite directions to dissect gene networks. Genome Biol 19:42
Klann, Tyler S; Black, Joshua B; Gersbach, Charles A (2018) CRISPR-based methods for high-throughput annotation of regulatory DNA. Curr Opin Biotechnol 52:32-41
Black, Joshua B; Perez-Pinera, Pablo; Gersbach, Charles A (2017) Mammalian Synthetic Biology: Engineering Biological Systems. Annu Rev Biomed Eng 19:249-277
Adkar, Shaunak S; Brunger, Jonathan M; Willard, Vincent P et al. (2017) Genome Engineering for Personalized Arthritis Therapeutics. Trends Mol Med 23:917-931

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