Thesis (Ph. D.)--Ohio State University, 2004. / Document formatted into pages; contains xvi, 162 p.; also includes grpahics (some col.). Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 May 4.

Multicellular organisms are composed of a number of different specialised cells that all carry the same genetic material but are highly divergent in their functions and characteristics. This diversity is only allowed because sets of specific genes are expressed in one type of cells and silent in others. A precise control mechanism is required to fine-tune gene regulation and relies on chromatin structure and regulatory proteins. One of the largest families of DNA-binding factors that influence this in human and mouse is the KRAB zinc finger protein (KZFP) family. KZFPs are thought to have rapidly evolved alongside transposable elements and be mediators of transcriptional repression. The few KZFPs that have been characterised so far have been shown to be involved in a wide range of regulatory and biological processes; hence it is hard to make functional generalisations. During my PhD, I studied one member of the KZFP family in mouse, ZFP263, with the aim of understanding its mechanism of action in mouse embryonic stem cells (mESCs) and its role in mice. My work has shown that ZFP263 is an ancient protein highly conserved in mammals and under purifying selection. One of its two functional domains however is divergent from the consensus sequence found in most KZFPs and suggests that ZFP263 might have lost the ability to recruit repressive chromatin states. My research identified the targets of ZFP263 binding in mESCs and showed that it does not bind and silence transposable elements. Indeed it targets unique regions of the genome, mostly within transcribed regions of genes. These genes show a wide range of expression levels and are involved in several key biological processes. Surprisingly, binding sites are not associated with the canonical KZFP co-factor but mostly co-localize with active histone marks. My findings lead me to hypothesise that ZFP263 has evolved to target active epigenetic states to unique regions that are positive regulators of transcription, in contrast to the more canonical model of KZFP function. To test this hypothesis, I have generated targeted mutations at Zfp263 in mice using CRISPR-Cas9 and my preliminary results suggest that Zfp263 mutants have growth defects indicating a role for this protein in mouse development. My findings indicate that ZFP263 is a unique KZFP with non-canonical properties and provide novel insights into the evolution and functions of KZFPs in mammals.

The Krüppel-associated box (KRAB) zinc finger protein (ZFP) genes comprise one of the largest gene families in the mammalian genome, encoding transcription factors with an N-terminal KRAB domain and C-terminal zinc fingers. The KRAB domain interacts with a co-repressor protein, KAP-1, which can recruit various factors causing transcriptional repression of genes to which KRAB ZFPs bind. Little is currently known about the gene targets of the ~400 human and mouse KRAB ZFPs. Many KRAB ZFPs interact with factors other than KAP-1. To identify proteins that may interact with one particular KRAB ZFP, Zfp647, I previously carried out a yeast two-hybrid screen using the full-length Zfp647 sequence and a mouse embryonic cDNA library. I have now tested the interactions from this screen for their specificity for Zfp647. I show that Zfp647 can interact with itself and at least 20 other KRAB ZFPs through their zinc finger domains, and have confirmed the Zfp647 self-interaction by in vitro co-immunoprecipitation. In my yeast two-hybrid screen, Zfp647 bound to KAP-1 as well as another related protein, ARD1/Trim23. Zfp647 also interacts with proteins that function in ubiquitylation. I have found evidence to suggest that Zfp647 may also interact with proteins encoding jumonji domains both by yeast two-hybrid assay and by co-immunoprecipitation from NIH/3T3 cell extracts. We have previously found that Zfp647 localises to non-heterochromatic nuclear foci in differentiated ES cells, which also contain KAP-1 and HP1, and which lie adjacent to PML nuclear bodies in a high proportion of cells. I have found that these foci are also visible in pMEFs, but not NIH/3T3 tissue culture cells. Immunofluorescence studies with antibodies against proteins from the yeast twohybrid screen have not shown any significant co-localisation with Zfp647. KAP-1 is sumoylated ex vivo, as are two human KRAB ZFPs. Because Zfp647 lies adjacent to PML nuclear bodies and can associate with proteins involved in posttranslational modification, I tested whether Zfp647 is also modified. I characterised a sheep _-Zfp647 antibody previously created in the lab and have shown that it detects Zfp647 by western blot, but not by immunofluorescence. I show that treatment of NIH/3T3 cells with NEM, which prevents the removal of protein modifications, leads to the appearance of higher molecular weight forms of Zfp647. Modification of Zfp647 is not dependent on KAP-1, which is known to function as a SUMO E3 ligase. Attempts to classify the modification as either ubiquitin, SUMO or NEDD8 have suggested that Zfp647 may be mono-ubquitylated. The larger modified forms of Zfp647 are present in both NIH/3T3 and ES cells. Interestingly, I found that the modification profile of the protein changes over the course of ES cell differentiation, during which time Zfp647 relocalises to punctate nuclear foci; thus Zfp647 modification may be involved in this process.

Potassium ion channels play a key role in the generation and propagation of action potentials. The S4-S5 linker peptide (L45) is believed to be responsible for the anesthetic/alcohol response of voltage-gated K+ channels. We investigated this region to define the structural basis of 1-alkanol binding site in dShaw2 K+ channel. L45 peptides derived from dShaw2 and hKv3.4 K+ channel, which, if part of the complete channel, demonstrate different sensitivity to 1-alcohols. Specifically, dShaw2 is alcohol sensitive and hKv3.4 is alcohol resistant. Structural analysis of L45 with NMR and CD suggested a direct correlation between alpha-helicity and the inhibition of dShaw2 channel by 1-butanol. We used CD and NMR to determine the structure of L45 peptides in micelles and vesicles. We measured spin-lattice relaxation time (T1) and determined the location and surface accessibility of L45 in micelles. These experiments confirm that L45 of dShaw2 adopts an α-helical conformation, partially buried in the membrane and parallel to the surface. The binding and accumulation of rev proteins to an internal loop of RRE (rev responsive element) of unspliced mRNA precursors is a key step of propagation of human immunodeficiency (HIV) virus. Molecules that interfere with this process can be expected to show anti-HIV activity. Our work is based on an assumption that zinc fingers could compete with rev proteins, therefore impeding the life cycle of HIV and stopping its infection. We studied the influence of different cations, anions, and the concentration of salts and osmolytes on the binding affinity with Polyacrylamide Gel Electrophoresis (PAGE) and Isothermal Titration Calorimetry (ITC). We conclude that the types of anions and/or cations and their concentrations affect the enthalpy and entropy of the binding interacitons. Using a gel assay, we confirm that there are three products in RNA-Protein reaction, and both EDTA and salts (and their concentrations) in the gel or samples interfere with RNA-protein complex mobility.

The Hepatitis B virus (HBV) chronically infects 350 million individuals worldwide, leading to mortality by end-stage liver disease, liver cirrhosis, and hepatocellular carcinoma. The vaccine to prevent HBV infection is highly effective but is not extensively available in endemic areas, resulting in high infection rates. Nucleoside analogue treatment of HBV has allowed for higher rates of viral clearance in infected individuals, but most patients must remain on therapy long term and viral resistance to the drugs is growing. The HBV viral genome is an episome in the nucleus of infected hepatocytes. It is called covalently closed circular (ccc) DNA and is highly stable, has a long half-life, and is the template for all viral transcription and progeny production. Nucleoside analogues do not directly target cccDNA, therefore many patients experience rebound when antiviral therapy is stopped. I have designed novel DNA binding proteins called zinc finger proteins (ZFPs) to specifically bind to the cccDNA in infected cells and inhibit viral transcription. Seven ZFPs targeting the model duck HBV (DHBV) and ten ZFPs targeting HBV were developed. Kinetic analyses of the purified ZFPs were performed, characterizing their specificity and binding properties. Using the DHBV tissue culture model system, I have demonstrated that the DHBV-specific ZFPs can specifically inhibit transcription from the viral template, resulting in reduced viral RNA, protein products and progeny virions. The DHBV-specific ZFPs were tested in primary duck hepatocytes (PDH) and in vivo in the Pekin duck model. ZFPs failed to express in PDH transduced by baculovirus vectors when DHBV was present in the cells. In vivo gene delivery of the ZFPs was carried out by portal vein injection of chitosan-based nanospheres. Unfortunately, non-specific reductions in viral levels masked any direct effect by the ZFPs. Testing of the HBV-specific ZFPs in tissue culture was hindered by a lack of transfectable cell culture model. A number of different transfection methods were tested to express the HBV-specific ZFPs, all without success. Further work is being carried out using baculovirus vectors to deliver the HBV-specific ZFPs to HBV-harbouring cell lines and HBV-infected scid-Alb/uPA chimeric mice with human liver cells. / Virology

Potassium ion channels play a key role in the generation and propagation of action potentials. The S4-S5 linker peptide (L45) is believed to be responsible for the anesthetic/alcohol response of voltage-gated K+ channels. We investigated this region to define the structural basis of 1-alkanol binding site in dShaw2 K+ channel. L45 peptides derived from dShaw2 and hKv3.4 K+ channel, which, if part of the complete channel, demonstrate different sensitivity to 1-alcohols. Specifically, dShaw2 is alcohol sensitive and hKv3.4 is alcohol resistant. Structural analysis of L45 with NMR and CD suggested a direct correlation between alpha-helicity and the inhibition of dShaw2 channel by 1-butanol. We used CD and NMR to determine the structure of L45 peptides in micelles and vesicles. We measured spin-lattice relaxation time (T1) and determined the location and surface accessibility of L45 in micelles. These experiments confirm that L45 of dShaw2 adopts an α-helical conformation, partially buried in the membrane and parallel to the surface. The binding and accumulation of rev proteins to an internal loop of RRE (rev responsive element) of unspliced mRNA precursors is a key step of propagation of human immunodeficiency (HIV) virus. Molecules that interfere with this process can be expected to show anti-HIV activity. Our work is based on an assumption that zinc fingers could compete with rev proteins, therefore impeding the life cycle of HIV and stopping its infection. We studied the influence of different cations, anions, and the concentration of salts and osmolytes on the binding affinity with Polyacrylamide Gel Electrophoresis (PAGE) and Isothermal Titration Calorimetry (ITC). We conclude that the types of anions and/or cations and their concentrations affect the enthalpy and entropy of the binding interacitons. Using a gel assay, we confirm that there are three products in RNA-Protein reaction, and both EDTA and salts (and their concentrations) in the gel or samples interfere with RNA-protein complex mobility.

No description available.

ZNF292 is a gene that encodes for a large multifunctional zinc finger protein. ZNF292 has a role in Growth Hormone transcription, developmental disorders on the autism spectrum, and in the initiation of tumorigenesis. Cancer cells have revealed ZNF292 as a gene with unique features: it is present in both linear and circular RNA (circRNA) forms. Circular ZNF292 RNAs vary in size depending on the number of exons that are back-spliced together forming a nested set of babushkas or “Russian dolls” – larger forms add an exon to a smaller circle. To determine whether anti-cancer treatments change the expression of circRNA forms as well as the linear form of ZNF292, we performed quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) analysis. Primers used were designed to amplify only the specified form of ZNF292, either the linear form or one of four targeted circular forms. Control and flavone (3,5 dihydroxy-7-methoxyflavone)-treated cell lines were grown, harvested, and total RNA extracted. Then, samples were analyzed by qRT-PCR with specific ZNF292 primer sets for each product using a standard curve for comparisons. All results were normalized to actin levels in each sample prior to statistical analysis. When compared to untreated controls, two linear ZNF292 RNAs were each reduced to 52% of control levels (p Funded by the Bill Gatton College of Pharmacy.

<p>The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.</p><p>Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible.</p><p>The recent emergence of the field of optogenetics--the ability to control gene expression using light--has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.</p><p>Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific three base-pair DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or eGFP transgene enabled expression of the transgene in response to blue-light. Gene activation was both reversible and tunable based on duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be spatially patterned by illuminating the cell culture through photomasks containing various patterns.</p><p>Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a light-activated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the N-terminal fragment of CIB1 to the N-, C-, or N- and C- terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask. </p><p>Finally, we engineered a light-activated genetic "on" switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase-mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system,1 could be used to express MyoD or the synthetic transcription factor VP64-MyoD2 in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated upregulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T½ cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased microvessel density compared to mice that were not illuminated. Analysis of human VEGF and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.</p><p>In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.</p> / Dissertation

Meiotic recombination is a fundamental biological process in sexually reproducing organisms, enabling offspring to inherit novel combinations of mutations, and ensuring even segregation of chromosomes into gametes. Recombination is initiated by programmed Double Strand Breaks (DSBs), the genomic locations of which are determined in most mammals by PRDM9, a rapidly evolving DNA-binding protein. In crosses between different mouse subspecies, certain Prdm9 alleles cause infertility in hybrid males, implying a critical role in fertility and speciation. Upon binding to DNA, PRDM9 deposits a histone modification (H3K4me3) typically found in the promoters of expressed genes, suggesting that binding might alter the expression of nearby genes. Many other questions have remained about how PRDM9 initiates recombination, how it causes speciation, and why it evolves so rapidly. This body of work investigates these questions using complementary experimental and analytical methodologies. By generating a map of human PRDM9 binding sites and applying novel sequence analysis methods, I uncovered new DNA-binding modalities of PRDM9 and identified sequence-independent factors that predict binding and recombination outcomes. I also confirmed that PRDM9 can affect gene expression by binding to promoters, identifying candidate regulatory targets in meiosis. Furthermore, I showed that PRDM9’s DNA-binding domain also mediates strong protein-protein interactions that produce PRDM9 multimers, which may play an important functional role. Finally, by generating high-resolution maps of PRDM9 binding in hybrid mice, I provide evidence for a mechanism to explain PRDM9-mediated speciation as a consequence of the joint evolution of PRDM9 and its binding targets. This work reveals that PRDM9 binding on one chromosome strongly impacts DSB formation and/or repair on the homologue, suggesting a novel role for PRDM9 in promoting efficient homology search and DSB repair, both critical for meiotic progression and fertility. One consequence is that PRDM9 may play a wider role in mammalian speciation.