Overview: Manipulating Epigenetic Mechanisms to Enhance Non-Viral Transgene Expression In the same way that words are neatly organized into the pages of books in a library, human DNA is condensed by histone proteins to form chromosomes in the nucleus. While this process allows human cells to maintain a large library of genetic information (~6 billion bases), it also prevents the expression of genes that are hidden deep within compacted chromosomes. Consequently, cells control gene expression with a family of epigenetic enzymes that make site-specific histone modifications that either release DNA to activate genes (i.e. enhancers) or tightly bind DNA to silence genes (i.e. repressors). Histones have also been shown to condense and silence foreign DNA (e.g. transgenes in episomes or plasmids), thereby limiting the efficacy of clinical gene therapy treatments and reducing recombinant protein yields in the biotechnology industry. The overall goal of this project is to use novel synthetic biology approaches that modify host epigenetic mechanisms to enhance non-viral transgene expression in mammalian cells. To achieve this goal, we will: 1. Use inhibitors to prevent transgene condensation and silencing by epigenetic repressors 2. Design and construct novel plasmids with binding sites that recruit epigenetic enhancers to activate transgenes 3. Use synthetic fusion proteins to artificially induce epigenetic activation of transgenes To the best of our knowledge, the effects of epigenetic enzymes on non-viral transgene expression have not been investigated. Our proposed research will fill this critical gap in the field. Intellectual Merit Epigenetic regulation of gene expression via site-specific histone modifications (i.e. the histone code) has recently emerged as a crucial factor in controlling cell differentiation, signaling pathways, etc. Based on our preliminary results, we hypothesize that specific histone modifications also inhibit the expression of foreign DNA, thereby limiting the efficacy of non-viral transgene expression. We will test this hypothesis by screening inhibitors of epigenetic enzymes (Objective 1) to reveal host regulatory mechanisms that repress transgene expression. Histones bound to plasmid DNA will also be isolated and characterized to determine key histone modifications that correlate with plasmid DNA sequences. We also hypothesize that host epigenetic mechanisms can be manipulated to activate and enhance transgene expression. To test this hypothesis, we will develop new synthetic biology strategies to enhance transgene expression. These include the development of new expression plasmids with binding sites that recruit transcription factors and other enzymes that modify histones to activate transgene expression (Objective 2), and the design of novel fusion proteins that bind plasmid DNA sequences and/or specific histone modifications to activate transgenes (Objective 3). Overall, this work will enhance our understanding of epigenetic regulatory mechanisms involved in responses to foreign DNA and provide several new synergistic strategies to enhance non-viral transgene expression. Objective 1: Identifying Epigenetic Inhibitors of Transgene Expression. A library of small molecule inhibitors (SMIs) of epigenetic repressor enzymes will be screened to identify epigenetic repressors that inhibit transgene (luciferase) expression. The effects of lead inhibitors on cell viability and intracellular transport will also be investigated. Objective 2: Designing Plasmids that Recruit Epigenetic Enhancers. The effects of commonly used plasmid sequences (e.g. viral or human promoters) on histone modifications will be determined. Novel expression plasmids containing DNA binding sites for epigenetic enhancer enzymes will also be screened to optimize transgene expression in mammalian cells. Objective 3: Development of Synthetic Fusion Proteins to Activate Transgenes. A DNA-binding motif from yeast (Gal4) will be fused to several activation-associated proteins. These proteins will then be delivered with plasmids containing a Gal4 binding site to enhance their expression. Broader Impacts Our work will advance the fields of gene therapy and recombinant protein production by providing at least three novel strategies to enhance the expression of therapeutic or commercially valuable transgenes in mammalian cells. These strategies may also be combined to synergistically enhance transgene expression even further. We will also conduct outreach activities aimed at exposing high school and college students to biotechnology and synthetic biology. We will also prepare genetic engineering experiments for high school students to perform at several different schools in Arizona and Pennsylvania through the Quanta Program.
|Effective start/end date||8/1/14 → 7/31/19|
- National Science Foundation (NSF): $426,386.00