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An introduction to Chord Algorithm.

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GWAS have identified several chromosomal loci associated with ovarian cancer risk. However, the mechanism underlying these associations remains elusive. We identify candidate functional Single Nucleotide Polymorphisms (SNPs) at the 9p22.2 ovarian cancer susceptibility locus, several of which map to transcriptional regulatory elements active in ovarian cells identified by FAIRE-seq (Formaldehyde assisted isolation of regulatory elements followed by sequencing) and ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) in relevant cell types. Reporter and electrophoretic mobility shift assays (EMSA) determined the extent to which candidate SNPs had allele specific effects. Chromosome conformation capture (3C) reveals a physical association between Basonuclin 2 (BNC2) and SNPs with functional properties. This establishes BNC2 as a major target of four candidate functional SNPs in at least two distinct elements. BNC2 codes for a putative transcription regulator containing three pairs of zinc finger (ZF) domains. Furthermore, bnc2 mutation in zebrafish leads to developmental defects including dysmorphic ovaries and sterility, clearly implicating this protein in cellular processes associated with ovarian development. We show that BNC2 is a transcriptional regulator with a specific DNA recognition sequence of targets enriched in genes involved in cell communication through DNA binding assays, ChIP-seq, and expression analysis. This study reveals a comprehensive regulatory landscape at the 9p22.2 locus and indicates that a likely mechanism of susceptibility to ovarian cancer may include multiple allele-specific changes in DNA regulatory elements some of which alter BNC2 expression. This study begins to identify the underlying mechanisms of the 9p22.2 locus association with ovarian cancer and aims to provide data to support advances in care based on one’s genetic composition.

The United States Navy incorporates many different occupations to ensure it achieves its overall mission. These occupations are extremely diversified and present a wide spectrum of occupational exposures. Many of these exposures have been well studied and documented. However, shipboard pneumatic tool hand-arm vibration, (HAV) and how it relates to different body postures is an area of occupational exposure that has received little attention. The chief objective of this study was to assess whether there is a difference in hand-arm vibration levels, while working on one of two surface orientations (e.g., horizontal and vertical) among distinctly different pneumatic tools while cleaning or not cleaning. The design of the study evaluated three pneumatic tools cleaning both horizontal and vertical surfaces and the fourth tool only cleaning a horizontal surface. HAV levels were measured to identify the effect horizontal and vertical surface orientations had on the tool. Five subjects were used in the evaluation of the four tools by a random sequencing order. Each subject was required to hold the tool in an idle condition, an activated without cleaning condition, and an activated cleaning condition, (surface contact) for 20 seconds each. These conditions were evaluated in two different surface orientations; horizontal and vertical (except for the 4th tool). Each subject repeated each of the cleaning/not cleaning conditions three times for a total of 7 measurements per surface. The idle condition was only conducted one time for each tool and surface. The measurements were collected from a Quest, HAVPro instrument using an accelerometer on the pneumatic tool following ISO 5349-1:2001 and ISO 5349-2:2001 methods.A three-way ANOVA (subjects by tool, by condition, (cleaning vs. not cleaning) and tool vs. condition) with replicates (not including idle conditions) was conducted on the data. The analysis included the main effects and the interaction of tool and surface orientation. The subjects were treated as a blocking variable. All the main effects and the interaction were significant at p<0.0001, except for surface, p<0.6396. Surface orientation does not affect HAV levels in pneumatic tools.

United States (U.S.) Navy sailors are exposed to a very large number of hazards, both chemical and physical. Occupational vibration from pneumatic air tools is one of the potential exposure hazards. There are very limited data as to the exposures to one type of tool, a needle gun or needle scaler, used by the sailors.The purpose of this study was to characterize the vibration levels generated by a needle gun used in the U.S. Navy. The design of the study evaluated the difference pressure had on the acceleration levels generated from the needle scaler. Five subjects were used in the evaluation of the tool. Each subject was required to hold the tool for twenty seconds activated without contact and activated on a surface and at two different pressures, 60 and 80 pound per square inch (psi). Each subject repeated each of the conditions three times for a total of 12 measurements. Each subject was also required to hold the tool in hand without the tool activated. The measurements were collected from an accelerometer on the needle gun following ISO 5349-1:2001 and ISO 5349-2:2001 methods. Significant differences were observed individually in pressure (p<0.0001), contact (p<0.0001)), and subjects (p<0.001). In addition, there was a significant interaction between contact and pressure (p<0.001). It was concluded that U.S. Navy sailors are not likely at significant risk to Hand-Arm Vibration Syndrome for lifetime exposures to hand transmitted vibration.

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. Aberrant DNA methylation in general occurred more often within H3K27me3 stem cell domains. We found a striking association between enrichment of H3K9me3 stem cell domains and toxicant-induced agglomerative DNA methylation. Global gene expression profiling of the arsenite-transformed prostate epithelial cells showed that gene expression changes and DNA methylation changes were negatively correlated, but less than 10% of the hypermethylated genes were down-regulated. These studies confirm that a majority of the DNA hypermethylation events occur at transcriptionally repressed, H3K27me3 marked genes. In contrast to aberrant DNA methylation targeting H3K27me3 pre-marked silent genes, we found that actively expressed ZNF genes marked with H3K9me3 on their 3' ends, are preferred targets of DNA methylation linked gene silencing. H3K9me3 mediated gene silencing of ZNF genes was widespread, occurring at individual ZNF genes on multiple chromosomes and across ZNF gene family clusters. At ZNF gene promoters, H3K9me3 and DNA hypermethylation replaced H3K4me3, resulting in a widespread down-regulation of ZNF gene expression which accounted for 8% of all the down-regulated genes in the arsenical-transformed cells. In summary, these studies associate arsenical exposure with agglomerative DNA methylation of gene family clusters and widespread silencing of ZNF genes by DNA hypermethylation-linked H3K9me3 spreading, further implicating epigenetic dysfunction as a driver of arsenical-induced carcinogenesis.

Zinc finger nucleases (ZFNs) induce double-strand DNA breaks at specific recognition sites. ZFNs can dramatically increase the efficiency of incorporating desired insertions, deletions, or substitutions in living cells. These tools have revolutionized the field of genome engineering in several model organisms and cell types including zebrafish, rats, and human pluripotent stem cells. There have been numerous advances in ZFN engineering and characterization strategies, some of which are detailed in this work. The central theme of this dissertation is improving the activity and specificity of engineered zinc finger nucleases with the ultimate goal of increasing the safety and efficacy of these tools for human therapy. As a first step, I undertook a large-scale effort to demonstrate that the modular assembly method of ZFN synthesis has a significantly higher failure rate than previously reported in the literature. This strongly suggested that engineering of ZFNs should better account for context-dependent effects among zinc fingers. The second advance reported in this dissertation is a method for biasing repair of zinc finger protein-induced DNA breaks toward homology-driven rather than error-prone repair in the presence of a donor template. Catalytically inactivating one monomer of a ZFN dimer results in a zinc finger nickase (ZFNickase) whose cleavage preference is directed at only one DNA strand. In human cell reporter assays, these ZFNickases exhibit a higher likelihood of repair by homology-driven processes, albeit with reduced absolute rates of correction. With further optimization, zinc finger nickases could provide a safer alternative to ZFNs in the context of gene correction therapies. Third, realizing there was no robust method for determining off-target cleavage sites of ZFNs in a genome-wide manner, I validated a collaborator’s novel in vitro selection system in human cells by identifying eight new potential off-target cleavage sites for a ZFN pair currently being used in clinical trials. Although it is unlikely these low-frequency mutations would be deleterious to patients, these results demonstrated that ZFNs induced more off-target effects than had been appreciated by previous work in the field. Collectively, the findings of this dissertation have contributed to more robust strategies for designing and evaluating ZFNs.

The field of synthetic biology seeks to revolutionize the scope and scale of what is currently feasible by genetic engineering. By focusing on engineering general signal processing platforms using modular genetic parts and devices rather than `one-off' systems, synthetic biologists aim to enable plug-and-play genetic circuits readily adaptable to different contexts. For mammalian systems, the goal of synthetic biology is to create sophisticated research tools and gene therapies. While several isolated parts and devices exist for mammalian systems there are few signal processing platforms available. We addressed this need by creating a transcriptional regulatory framework using programmable zinc finger (ZF) and TALE transcription factors and a conceptual framework for logical T-cell receptor signaling. We first engineered a large set of ZF activator and repressor transcription factors and response promoters. ZFs are scalable elements as they can be engineered to bind to given DNA sequences. We demonstrated that we could ‘tune’ the activity of the ZF transcription factors by fusing them to protein homo-dimerization domains and by modifying their response promoters. We also created OR and NOR logic gates using hybrid promoters and AND and NAND logic gates by reconstituting split zinc finger activators and repressors with split inteins. Next, using a computational algorithm we designed a series of TALE transcriptional activators and repressors predicted to be orthogonal to all 2kb human promoter regions and thus minimally interfere with endogenous gene expression. TALEs can be designed to bind to even longer DNA sequences than ZFs, however off-target binding is predicted to occur. We tested our computationally designed TALEs in human cells demonstrating that they activated their intended target genes, but not their likely endogenous off-target genes, nor synthetic promoters with binding site mismatches. Finally, we created a conceptual framework for logical T-cell-mediated killing of target cells expressing combinations of surface antigens. The systems consist of conventional and novel chimeric antigen receptors (CARs) containing inhibitory or co-stimulatory cytoplasmic signaling domains. In co-incubation assays of engineered T-cells with target cells we demonstrated a functioning OR-Gate system and progress toward development of a functional NOT-Gate system using the CD300a and CD45 inhibitory receptor domains.

Engineered DNA-binding proteins enable targeted manipulation of the genome. Zinc fingers are the most well characterized DNA-binding domain and for many years research has focused on understanding and manipulating the sequence-specificities of these proteins. Recently, major advances in the ability to engineer zinc finger proteins, as well as the discovery of a new class of DNA-binding domains - transcription activator-like effectors (TALEs), have made it possible to rapidly and reliably engineer proteins targeted to any sequence of interest. With this capability, focus has shifted to exploring the applications of this powerful technology. In this dissertation I explore three important applications of engineered DNA-binding proteins.