<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

Bioinorganic chemistry strives to understand the roles of metals in biological systems, whether in the form of naturally occurring or addition of non-essential metals to natural systems. Metal ions play vital roles in many cellular functions such as gene expression/regulation and DNA transcription and repair. The study of metal-protein-DNA/RNA interactions has been relatively unexplored. It is important to understand the role of metalloprotein interactions with DNA/RNA as this enhanced knowledge may lead to better understanding of diseases and therefore more effective treatments. A major milestone in the development of this field was the discovery of the cytotoxic properties of cisplatin in 1965 and its FDA approval in 1978. Since then, two other chemotherapeutic drugs containing platinum, carboplatin and oxaliplatin, have been used in the clinic. These three compounds are all bifunctional with the ligands surrounding platinum In the cis conformation and rearrangement of the ligands to the trans orientation results in a loss of cytotoxic properties due to rapid deactivation through binding to S-containing proteins. This enhanced reactivity yields new opportunities to study the reactions between proteins and DNA. One of the first crosslinking experiments used transplatin to crosslink NCp7 to viral RNA in order to understand how/where the protein bound to RNA. We have studied the interaction between cis and trans dinuclear platinum complexes and the C-terminal zinc finger (ZF). The trans complex reacts at a faster rate than the cis isomer and causes N- terminal specific cleavage of the ZF. The dinuclear structure plays a critical role in the peptide cleavage as studies with transplatin (the mononuclear derivative) does not result in cleavage. Monofunctional trans platinum-nucleobase complexes (MPNs) serve as a model for the binding of transplatin to DNA. This provides an interesting opportunity to study their reactions with S-containing proteins, such as HIV1 NCp7. MPNs have been shown to bind to the C-terminal ZF of HIV1 NCp7, resulting in zinc ejection. This occurs through a two-step process where the nucleobase π-stacks with Trp37 on the ZF, followed by covalent binding at the labile Cl site to Cys. MPNs have also shown antiviral activity in vitro. The labile Cl on MPNs reduces specificity of these compounds, as it leaves an available coordination site on the platinum center for binding to other S-proteins or DNA. Therefore, we have moved to an inert PtN4 coordination sphere, [Pt(dien)L]2+ (dien= diethylenetri- amine). Due to the strong bond between platinum and nitrogen, covalent reactions are highly unlikely to occur at rapid rates. The strength of the pi-stacking interaction between nucleobases (free and platinated) and the aromatic amino acid, tryptophan (Trp), showed an enhanced binding constant for platinated nucleobases. This was confirmed by density functional theory (DFT) calculations as the difference in energy between the HOMO of Trp and the LUMO of the nucleobase was smaller for the platinum complex. The studies were extended to the Trp-containing C-terminal ZF of HIV1 NCp7 and an increase in association constant was seen compared to free Trp. Reaction of PtN4 nucleobases compounds with a short amino acid sequence con- taining either Ala (no pi-stacking capabilities) or Trp (pi-stacking interactions) revealed an enhanced rate of reactivity for the Trp-containing peptide. This result supports the theory of a two-step reaction mechanism where the platinum-nucleobase complex recognizes the pep- tide through a pi-stacking interaction with Trp followed by covalent binding to the platinum center. The [Pt(dien)L]2+ motif allows for systematic modification of the structural elements surrounding platinum in a search for the most effective compound. Methylation of the dien ligand should, in theory, increase lipophilicity of the compounds, however, due to 2+ charge of the compounds, this simple association does not hold true. Analysis of the cellular accumulation profiles showed little change in the uptake with the addition of methyl groups to the dien ligand, in agreement with the non-linear change in lipophilicity. Modification of L using different nucleobases allows for the tuning of the strength of the π-stacking interaction between Trp and the platinum complex. The addition of inosine (which lacks a H-bonding donor/acceptor at the C2 position) resulted in a lower association constant with both N-AcTrp and the C-terminal zinc finger of HIV1 NCp7. Interestingly, the addition of xanthosine resulted in an ehanced pi-stacking interaction with the C-terminal zinc finger of HIV1 NCp7; likely as a results of the addition of a H-bonding donor (double-bonded O) at the C2 position. The ability of PtN4 nucleobase complexes to inhibit formation of the NCp7 complexation with viral RNA was studied by mass spectrometry and gel electrophoresis. Dissociation of the NCp7-RNA complex was seen upon addition of PtN4 compounds. These compounds were also able to retard formation of the NCp7-RNA complex when pre-incubated with the protein. These results have important implications as inhibition of complex formation between NCp7 and viral RNA has negative implications for viral replication. Despite the success of platinum-nucleobase compounds, it is important to evaluate all potential pi-stacking ligands. A series of pyridine- and thiazole-based compounds were evaluated for the strength of the pi-stacking interaction with N-AcTrp and the C-terminal ZF of HIV1 NCp7. There was notable increase in association constant for the platinum- DMAP (4-dimethylaminopyridine) complex compared to other ligands studied. This result highlights the importance of exploring multiple avenues for the design of specifically targeted inhibitors and further confirms the viability of the medicinal chemistry dual approach of target recognition (non-covalent) followed by target fixation (covalent).

As reações luminosas da fotossíntese em plantas envolvem quatro complexos proteicos multi-unidades na membrana dos tilacóides incluindo o fotossistema II (PSII), o complexo citocromo b6f, o fotossistema I (PSI) e o complexo ATP sintase. Uma atividade apropriada desse processo exige um mecanismo de controle de qualidade mediado por chaperonas, DnaJs e proteases, como o complexo FtsH. Esse conjunto de proteínas garantem um dobramento correto de proteínas, as montagens devidas dos complexos e a degradação de algumas subunidades danificadas quando necessário. Neste trabalho nós mostramos o envolvimento de FIP, uma proteína com um domínio dedo-de-zinco localizada nos tilacóides de cloroplastos de A. thaliana, no mecanismo de resposta à estresses abióticos. Plantas mutantes fip foram, fenotipicamente, mais tolerantes à estresses abióticos de alta luminosidade, elevado potencial osmótico e excesso de sal. Também mostramos que a expressão de FIP é diminuída em resposta às diferentes condições de estresse, assim como o acúmulo de transcritos de genes relacionados à estresse foi menor nas plantas mutantes fip. Análises por immunoblot mostraram que os mutantes fip acumulam menos proteínas PsaA e PsaB do fotossistema I e plastocianina (PC) do que as plantas selvagens, no entanto não são afetados quanto ao acúmulo de proteínas do fotossistema II e do Complexo do Citocromo b6f sob condições controle. Esses mutantes também acumulam menos FtsH5 nos tilacóides, sem afetar a eficiência dos fotossistemas I e II. Foi testado também o potencial redutase do domínio dedo-de-zinco da proteína recombinante FIP (6xHis-FIP) em ensaios in vitro de redução de insulina. Vimos que FIP apresenta atividade redutase, significantemente, maior que o controle negativo nas condições testadas. Considerando todos os resultados obtidos até o momento, acreditamos que FIP possa estar agindo como uma redutase na membrana dos tilacóides, tendo como alvos não somente FtsH5, mas também outras proteínas com resíduos de cisteína nas suas estruturas, e que sua atividade tem influência no acúmulo de proteínas dependentes de redução para a maturação como PsaA, PsaB e PC. Uma investigação mais aprofundada da atividade de FIP nos cloroplastos ainda é necessária para o completo entendimento da sua função. / The light-driven photosynthetic reactions in plants take place within four multi- subunit protein complexes in the thylakoid membranes, including photosystem II (PSII), the cytochrome b6f complex, photosystem I (PSI) and the ATP synthase complex. Regulation of all these molecular machineries requires a fine-tuning control mechanism mediated by specific proteins, including chaperones, DnaJs, and proteases, such as the FtsH complex. These set of proteins guarantee the proper folding, assembly and degradation of the photosynthetic complexes\' subunits. In this work we showed the involvement of FIP, a zinc-finger protein localized in the thylakoid membranes in A. thaliana, in the abiotic stress response mechanism. Mutants fip knockdown plants were phenotypically more tolerant to abiotic stresses like high light, increased osmotic potential and salt excess. We also showed that FIP is down-regulated by different abiotic stresses, with lower levels of stress-related gene transcripts accumulation in mutant fip plants. Analysis of accumulation of photosynthetic proteins by immunoblot under control conditions showed that mutants fip displayed lower levels of PsaA, PsaB (PSI) and Plastocyanin (PC) proteins than wild-type plants, however are not affected for PSII and Cyt b6f proteins accumulation under the same growth conditions. In addition, the mutants accumulated slightly less FtsH5 proteins in thylakoid membranes, without affecting PSII and PSI efficiency. We tested the putative reductase activity probably mediated by FIP zinc-finger domain, using the recombinant form of the protein 6xHis-FIP in in vitro insulin reduction assays. FIP presented a reductase activity higher than the negative control under the same assay conditions. Taking all together, these results suggest that FIP may be acting as a reductase in the thylakoid membranes, having as targets not only FtsH5 but other targets with available cysteine residues, depending on the reduction step for proper accumulation such as PsaA, PsaB and PC. Further investigations regarding the role of FIP in chloroplasts are still necessary to completely understand its function.

Transcriptional regulation of genes is vital to cell success making it an important aspect of research. Transcriptional regulation can occur in many ways; transcription factors bind to the promoter region and block transcription, disrupt an activator protein, or interact with histones to lead to higher order chromatin. Plant HomeoDomain can recognize and bind to different methylation states of histone tails. PHD proteins use other functional regions to carry out functions. Two associated domains having DNA-binding capacity were characterized in this study; the ARID domains of JARID1A and JARID1C and the DDT domains of BAZ1A, BAZ1B and BAZ2A. These genes are important because of their roles in various diseases such as cancer. The consensus sequences for BAZ1A-DDT is GGACGGRnnGG, GnGAGRGCRnnGGnG, RAGGGGGRnG and CRYCGGT. Consensus sequences for BAZ1B-DDT were CGnCCAnCTTnTGGG and YGCCCCTCCCCnR. Consensus sequences for BAZ2A-DDT were TACnnAGCnY and CnnCCRGCnRTGnYY. Consensus sequence for JARID1A-ARID was GnYnGCGYRCYnCnG. Consensus sequences for JARID1C-ARID was RGGRGCCRGGY. / by Emmanuel MacDonald. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Transcriptional regulation by the family of SNAG (Snail/Gfi-1) zinc fingers has been shown to play a role in various developmental states and diseases. These transcriptional repressors have function in both DNA- and protein-binding, allowing for multiple interactions by a single family member. This work aims to characterize the SNAG members Slug, Smuc, Snail, Scratch, Gfi-1, Gfi-1B, and IA-1 in terms of both DNA-protein and protein-protein interactions. The specific DNA sequences to which the zinc finger regions bind were determined for each member, and a general consensus of TGCACCTGTCCGA, was developed for four of the members. Via these studies, we also reveal thebinding affinities of E-box (CANNTG) sequences to the members, since this core is found for multiple members' binding sites. Additionally, protein-protein interactions of SNAG members to other biological molecules were investigated. The Slug domain and Scratch domain have unknown function, yet through yeast two-hybrid screening, we were able to determine protein interaction partners for them as well as for other full length SNAG members. These protein-interacting partners have suggested function as corepressors during transcriptional repression. The comprehensive information determined from these studies allow for a better understanding of the functional relationship between SNAG-ZFPs and other genes. The collected data not only creates a new profile for each member investigated, but it also allows for further studies to be initiated from the results. / by Cindy Chiang. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Transcriptional regulation is an important area of research due to the fact that it leads to gene expression. Transcription factors associated with the regulation can either be activators or repressors of target genes, acting directly or with the aid of other factors. A majority of transcriptional repressors are zinc finger proteins (ZFPs) which bind to specific DNA sequences. The Snail/Gfi (SNAG) domain family, with members such as Slug, Smuc, Snail, and Scratch, are transcriptional repressors shown to play a role in various diseases such as cancer. The SNAG transcription factors contain a conserved SNAG repression domain and DNA binding domain zinc fingers. The specific DNA sequences to which each SNAG-ZFP binds, as well as a general consensus -TGCACCTGTCCGA, have been determined. Also, putative protein-protein interactions in which the Slug domain participates has been identified via binding assays. All these results contribute to better understanding of SNAG-ZFP functions. / by Cindy Chung-Yue Chiang. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Zhang, Zhiyi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves ). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

There are approximately 20,000 genes in the human genome. Around 2% of these genes code for transcriptional repressors known as KRAB-ZFPs. It is already known that Zinc-Finger Proteins contain two main functional domains at either end of the polypeptide. In today's database, you will find a KRAB (Kruppell-associated Box) domain at one end and a tandem array of Zinc-finger repeats at the other end. The carboxyl terminal tandem Zinc-finger repeats function as sequence-specific DNA-binding domains. The amino terminal KRAB domain serves as a repressor domain, which will recruit a co-repressor termed KAP-1 (KRAB Associated Protein-1). Located in between these two domains is a region of uncharacterized DNA referred to as the "Linker Region". This thesis will explore the DNA-binding domains of 6 known KRAB-ZFPs, as well as utilize the linker regions to derive an evolutionary history for this superfamily. / by Alain Chamoun. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Hepatitis B virus chronically infects 350-400 million people worldwide. It often leads to hepatocellular carcinoma, which causes >1 million deaths yearly. Current therapies prevent new viral genome formation but do not target pre-existing viral genomic DNA, thus curing only ~1/2 of patients. We targeted hepatitis B virus DNA for cleavage using zinc finger nucleases, which cleave as dimers. Co-transfection of our zinc finger nuclease pair with a target plasmid containing the hepatitis B virus genome resulted in specific cleavage. After three days in culture, 26% of the target remained linear, while ~10% was cleaved and mis-joined tail-to-tail. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the zinc finger nuclease sites. Targeted cleavage and misrepair destroys the XbaI site. After three days in culture, ~6% of plasmids were XbaI resistant. 13 of 16 clones sequenced contained frameshift mutations that would lead to dramatic truncations of the viral core protein. These results demonstrate for the first time the feasibility of targeting episomal viral DNA genomes in cells using zinc finger nucleases. This strategy is broadly applicable toward inactivating other DNA viruses within cells. A major concern for the therapeutic use of zinc finger nucleases is off-target cleavage. To measure specificity, we employed in vitro assays and developed a bioinformatics method to find off-target cleavage sites in cultured cells. These sites can then be PCR amplified and tested using a mutation detection assay that we developed.

Doctor of Philosophy (PhD) / Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.