Small molecule dependent molecular switches that control gene expression are important tool in understanding biological cellular processes and for regulating gene therapy. Nuclear receptors are ligand activated transcription factors that have been engineered to selectively respond to synthetic ligands and used as regulators of gene expression. In this work the retinoid X receptor (RXR), has been used to develop an inducible molecular switch with a near drug like compound LG335. Three RXR variants (Q275C; I310M; F313I), (I268A; I310A; F313A; L436F), (I268V; A272V; I310M; F313S; L436M) were created via site-directed mutagenesis and a structure based approach, such that they preferentially bind to the synthetic ligand LG335 and not its natural ligand, 9-cis retinoic acid. These variants show reverse ligand specificity as designed and have an EC50 for LG335 of 80 nM, 30 nM, 180 nM, respectively. The ligand binding domains of the RXR variants were fused to a yeast transcription factor Gal4 DNA binding domain. This modified chimeric fusion protein showed reverse response element specificity as designed and recognized the Gal4 response element instead of the RXR response element. The modified RXR protein did not heterodimerize with wild type RXR or with other nuclear receptor such as retinoic acid receptor. These RXR-based molecular switches were tested in retroviral vectors using firefly luciferase and green fluorescence protein and they maintain their inducible behavior with LG335. These experiments demonstrate the orthogonality of RXR variants and their possible use in regulating gene therapy.

Epigenetics is the study of heritable biological variation not related to changes in DNA sequence. Epigenetic processes are responsible for establishing and maintaining transcriptional programs that define cell identity. Defects to epigenetic processes have been linked to a host of disorders, including mental retardation, aging, cancer and neurodegenerative diseases. The ability to control and engineer epigenetic systems would be valuable both for the basic study of these critical cellular processes as well as for synthetic biology. Indeed, while synthetic biology has made progress using bottom-up approaches to engineer transcriptional and signaling circuitry, epigenetic systems have remained largely underutilized. The predictive engineering of epigenetic systems could enable new functions to be implemented in synthetic organisms, including programmed phenotypic diversity, memory, reversibility, inheritance, and hysteresis. This thesis broadly focuses on the development of foundational tools and intellectual frameworks for applying synthetic biology to epigenetic regulation in the model eukaryote, Saccharomyces cerevisiae. Epigenetic regulation is mediated by diverse molecular mechanisms: e.g. self-sustaining feedback loops, protein structural templating, modifications to chromatin, and RNA silencing. Here we develop synthetic tools and circuits for controlling epigenetic states through (1) modifications to chromatin and (2) self-templating protein conformations. On the former, the synthetic tools we develop make it possible to study and direct how chromatin regulators operate to produce distinct gene expression programs. On the latter, we focus our studies on yeast prions, which are self-templating protein conformations that act as elements of inheritance, developing synthetic tools for detecting and controlling prion states in yeast cells. This thesis explores the application of synthetic biology to these epigenetic systems through four aims: Aim 1. Development of inducible expression systems for precise temporal expression of epigenetic regulators Aim 2. Construction of a library of chromatin regulators to study and program chromatin-based epigenetic regulation. Aim 3. Development of a genetic tool for quantifying protein aggregation and prion states in high-throughput Aim 4. Dynamics and control of prion switching Our tools and studies enable a deeper functional understanding of epigenetic regulation in cells, and the repurposing of these systems for synthetic biology toward addressing industrial and medical applications. / 2019-10-08T00:00:00Z

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus has long been considered a master circadian pacemaker that drives rhythms in physiology and behavior in mammals. The recent discovery of self-sustained and cell-autonomous circadian oscillators in peripheral tissues has challenged this position. This dissertation tested the general hypothesis that the SCN has properties that distinguish it from other oscillators, thereby positioning it atop a circadian hierarchy. The general approach was to compare the consequences of altering the molecular circadian clock on tissue-autonomous rhythmicity in mice. In the first experiments, the role of the SCN as a master clock was tested by manipulating the expression of a circadian gene in the brain. Specifically, the expression of the short period tau mutation of casein kinase-1-epsilon (CK1ε) was controlled in an anatomically- and a temporally-specific manner via a tetracycline transactivator regulatory system. This inducible expression of CK1εtau affected the period of activity rhythms when expressed in the SCN, but did not affect the tissue-autonomous rhythmic properties in the peripheral tissues. Second, real-time bioluminescence imaging of tissues from PER2::LUCIFERASE mice revealed that period and phase of different circadian oscillators were tissue specific. Various circadian gene mutations (Cry1-/-, Cry2-/-, Cry1-/-;Cry2-/-, Clock∆19/∆19) produced little difference in rhythmic properties between the SCN and peripheral oscillators, although Cry1-/- SCN had more robust and persistent rhythms compared with the periphery. Third, the loss of Bmal1, which produces behavioral arrhythmicity, eliminated rhythms in the peripheral tissues, but not in the SCN. Bmal1-/- SCN rhythms were highly variable in period and amplitude, fitting a stochastic, but not a deterministic model of rhythm generation. Unlike mutations in other circadian genes, rhythmicity was completely abolished in single SCN neurons in Bmal1-/- mice, indicating that rhythms in Bmal1-/- SCN tissue are a property of the tissue organization rather than an averaging of single-cell autonomous rhythms. The SCN, therefore, has a unique anatomical organization that contributes to long-term stability and temporal organization of the circadian hierarchy.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus has long been considered a master circadian pacemaker that drives rhythms in physiology and behavior in mammals. The recent discovery of self-sustained and cell-autonomous circadian oscillators in peripheral tissues has challenged this position. This dissertation tested the general hypothesis that the SCN has properties that distinguish it from other oscillators, thereby positioning it atop a circadian hierarchy. The general approach was to compare the consequences of altering the molecular circadian clock on tissue-autonomous rhythmicity in mice. In the first experiments, the role of the SCN as a master clock was tested by manipulating the expression of a circadian gene in the brain. Specifically, the expression of the short period tau mutation of casein kinase-1-epsilon (CK1ε) was controlled in an anatomically- and a temporally-specific manner via a tetracycline transactivator regulatory system. This inducible expression of CK1εtau affected the period of activity rhythms when expressed in the SCN, but did not affect the tissue-autonomous rhythmic properties in the peripheral tissues. Second, real-time bioluminescence imaging of tissues from PER2::LUCIFERASE mice revealed that period and phase of different circadian oscillators were tissue specific. Various circadian gene mutations (Cry1-/-, Cry2-/-, Cry1-/-;Cry2-/-, Clock∆19/∆19) produced little difference in rhythmic properties between the SCN and peripheral oscillators, although Cry1-/- SCN had more robust and persistent rhythms compared with the periphery. Third, the loss of Bmal1, which produces behavioral arrhythmicity, eliminated rhythms in the peripheral tissues, but not in the SCN. Bmal1-/- SCN rhythms were highly variable in period and amplitude, fitting a stochastic, but not a deterministic model of rhythm generation. Unlike mutations in other circadian genes, rhythmicity was completely abolished in single SCN neurons in Bmal1-/- mice, indicating that rhythms in Bmal1-/- SCN tissue are a property of the tissue organization rather than an averaging of single-cell autonomous rhythms. The SCN, therefore, has a unique anatomical organization that contributes to long-term stability and temporal organization of the circadian hierarchy.

Uncontrolled spreading of genetically modified (GM) plants is one of the main concerns about their cultivation. Inducible RNA interference against an essential gene could be a tool for control of GM plants. After spraying with a chemical inducer, the essential gene will be silenced so the treated GM plant will die. For testing this strategy we chose two key enzymes of nitrogen metabolism, glutamate synthase (GOGAT) and glutamine synthetase (GS). GS processes ammonium ions into glutamine, then GOGAT transfers the amide group from glutamine to 2-oxoglutarate to form two glutamates. GS/GOGAT cycle is the main pathway for assimilation of ammonium ions, which could be toxic to plants in a higher concentration. Disruption of ammonium assimilation during photorespiration causes a strong inhibition of photosynthesis. The aim of this work was to describe the effects of silencing GOGAT and GS genes in Arabidopsis thaliana. To induce silencing, RNAi hairpin constructs under a control of constitutive or estradiol-inducible promoter were prepared. In selected independent transformants with the inducible hairpin against GOGAT, chlorosis and reduced growth were observed after the estradiol treatment in in vitro conditions. However, the spraying with estradiol was tricky, at the whole plant level, the induction of...