<p>Krüppel-related zinc finger proteins constitute the largest single class of transcription factors within the human genome. Members of this protein family have the ability to either activate or repress transcription depending on the presence of specific activator or repressor domains within the protein. Approximately one third of the Krüppel-related zinc finger proteins contain an evolutionarily well-conserved repressor domain termed the KRAB domain. This domain acts as a potent repressor of transcription by interacting with the co-repressor protein, TIF1β. TIF1β then, in turn, recruits HP1 proteins, HDACs and probably other proteins involved in gene silencing. In order to identify novel KRAB-containing zinc finger proteins, one mouse monocytic cDNA library and two testis cDNA libraries were screened for novel members of this multigene family. Six novel KRAB-ZNF cDNAs, four mouse and two human, were isolated. The corresponding proteins were all shown to contain N-terminally located KRAB domains as well as varying numbers of C-terminally located zinc finger motifs. An extensive comparative sequence analysis of the KRAB domains of these proteins together with KRAB domains from a large number of previously identified KRAB-ZNF proteins resulted in a clear subdivision into three different subfamilies, A+B, A+b and A. Later, we also isolated a fourth KRAB box, which is present downstream of the KRAB A box in a few proteins of the KRAB A family. This module was named KRAB C. Potential functional differences between these different subfamilies were investigated. In line with previous observations, the KRAB A box was shown to repress transcription, an activity which was enhanced by the presence of the KRAB B box. However, addition of neither the KRAB b box nor the KRAB C box had any effect on repression. Moreover, all KRAB A motifs had the ability to bind TIF1β, and this binding was increased both by the presence of the KRAB B box and by the KRAB C box. The KRAB b box, however, did not seem to contribute to TIF1β-binding. One of the novel human cDNAs, HKr19, was found to be a member of the large ZNF91 family of KRAB zinc finger genes. Interestingly, the expression of HKr19 and a number of other closely related genes were restricted to lymphoid cells, indicating that these genes may be involved in regulating lineage commitment. The effect of HKr19 on cell viability was investigated by transfection into human embryonic kidney cells (HEK 293). The results indicated that HKr19, or its zinc finger domain in isolation, were toxic to these cells when expressed at high levels. The MZF6D protein, on the other hand, showed a testis-specific expression. <i>In situ</i> hybridization analysis located this expression to meiotic germ cells, suggesting a role for this protein in spermatogenesis. Further, the evolutionary perspectives of this large gene family were addressed, and its enormous expansion throughout evolution probably includes numerous duplication events. The results from two extensive sequence analyses give clues to how the repetitive nature of the ZNF motif has given rise to both internal duplications of single motifs as well as duplications of entire genes resulting in gene clusters.</p>

<p>All living organisms consist of cells and the identity of a cell is defined by the genes it expresses. To assure proper function, a cell receives continuous information on which genes to turn on and off. This information is, to a large extent, provided by transcription factors. Krüppel-related zinc finger proteins probably constitute the largest family of transcription factors in mammals and many of these proteins carry a potent repressor domain called Krüppel-associated box (KRAB). The human genome alone encodes more than 200 KRAB zinc finger proteins but still very little is known about their biological functions. </p><p>The Krüppel-related zinc finger genes appear to have been involved in a massive expansion throughout evolution. To unravel some of the secrets underlying this evolutionary success, we studied the molecular evolution of KRAB zinc finger genes. We show that the frequently occurring duplications of these genes are accompanied by a low sequence constraint in their zinc finger region. In addition, we show that the number of zinc finger motifs carried within these proteins is far from fixed. New zinc finger motifs are frequently added while others are inactivated or even discarded from the coding region. The structurally independent Krüppel zinc finger motif has, through these mechanisms, served as a highly adaptive building block for the generation of new transcriptional regulators. </p><p>The mouse, rat and human genomes carry four different variants of the KRAB domain – KRAB(AB), KRAB(Ab), KRAB(AC) and KRAB(A). This thesis presents the identification of a novel KRAB domain, KRAB C, as well as a functional analysis of the different KRAB domains. We conclude that all different KRAB domains share a common co-repressor, TIFβ, and effectively repress transcription. These functions are mainly mediated by the KRAB A box but are clearly influenced by the presence of a KRAB B, b or C box. Furthermore, we show that all KRAB zinc finger gene subfamilies originate from the KRAB(AB) zinc finger genes.</p><p>In addition, this thesis includes a structural and functional analysis of four novel mouse and human KRAB zinc finger genes; <i>MZF6D</i>, <i>HKr18</i>, <i>HKr19</i> and <i>HZF12</i>. Whereas <i>HKr18</i> and <i>HZF12</i> seem to be ubiquitously expressed, <i>MZF6D</i> and <i>HKr19</i> show a more restricted expression pattern. Northern blot and <i>in situ</i> hybridisation analyses of <i>MZF6D</i> showed that the expression of this gene is restricted to meiotic germ cells. <i>MZF6D</i> might thus be involved in the formation of male gametes. The expression of <i>HKr19</i>, on the other hand, seems to be restricted to lymphoid cells, indicating a possible role for this KRAB zinc finger gene in the regulation of lineage commitment.</p>

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 138-153).

Krüppel-related zinc finger proteins constitute the largest single class of transcription factors within the human genome. Members of this protein family have the ability to either activate or repress transcription depending on the presence of specific activator or repressor domains within the protein. Approximately one third of the Krüppel-related zinc finger proteins contain an evolutionarily well-conserved repressor domain termed the KRAB domain. This domain acts as a potent repressor of transcription by interacting with the co-repressor protein, TIF1β. TIF1β then, in turn, recruits HP1 proteins, HDACs and probably other proteins involved in gene silencing. In order to identify novel KRAB-containing zinc finger proteins, one mouse monocytic cDNA library and two testis cDNA libraries were screened for novel members of this multigene family. Six novel KRAB-ZNF cDNAs, four mouse and two human, were isolated. The corresponding proteins were all shown to contain N-terminally located KRAB domains as well as varying numbers of C-terminally located zinc finger motifs. An extensive comparative sequence analysis of the KRAB domains of these proteins together with KRAB domains from a large number of previously identified KRAB-ZNF proteins resulted in a clear subdivision into three different subfamilies, A+B, A+b and A. Later, we also isolated a fourth KRAB box, which is present downstream of the KRAB A box in a few proteins of the KRAB A family. This module was named KRAB C. Potential functional differences between these different subfamilies were investigated. In line with previous observations, the KRAB A box was shown to repress transcription, an activity which was enhanced by the presence of the KRAB B box. However, addition of neither the KRAB b box nor the KRAB C box had any effect on repression. Moreover, all KRAB A motifs had the ability to bind TIF1β, and this binding was increased both by the presence of the KRAB B box and by the KRAB C box. The KRAB b box, however, did not seem to contribute to TIF1β-binding. One of the novel human cDNAs, HKr19, was found to be a member of the large ZNF91 family of KRAB zinc finger genes. Interestingly, the expression of HKr19 and a number of other closely related genes were restricted to lymphoid cells, indicating that these genes may be involved in regulating lineage commitment. The effect of HKr19 on cell viability was investigated by transfection into human embryonic kidney cells (HEK 293). The results indicated that HKr19, or its zinc finger domain in isolation, were toxic to these cells when expressed at high levels. The MZF6D protein, on the other hand, showed a testis-specific expression. In situ hybridization analysis located this expression to meiotic germ cells, suggesting a role for this protein in spermatogenesis. Further, the evolutionary perspectives of this large gene family were addressed, and its enormous expansion throughout evolution probably includes numerous duplication events. The results from two extensive sequence analyses give clues to how the repetitive nature of the ZNF motif has given rise to both internal duplications of single motifs as well as duplications of entire genes resulting in gene clusters.

All living organisms consist of cells and the identity of a cell is defined by the genes it expresses. To assure proper function, a cell receives continuous information on which genes to turn on and off. This information is, to a large extent, provided by transcription factors. Krüppel-related zinc finger proteins probably constitute the largest family of transcription factors in mammals and many of these proteins carry a potent repressor domain called Krüppel-associated box (KRAB). The human genome alone encodes more than 200 KRAB zinc finger proteins but still very little is known about their biological functions. The Krüppel-related zinc finger genes appear to have been involved in a massive expansion throughout evolution. To unravel some of the secrets underlying this evolutionary success, we studied the molecular evolution of KRAB zinc finger genes. We show that the frequently occurring duplications of these genes are accompanied by a low sequence constraint in their zinc finger region. In addition, we show that the number of zinc finger motifs carried within these proteins is far from fixed. New zinc finger motifs are frequently added while others are inactivated or even discarded from the coding region. The structurally independent Krüppel zinc finger motif has, through these mechanisms, served as a highly adaptive building block for the generation of new transcriptional regulators. The mouse, rat and human genomes carry four different variants of the KRAB domain – KRAB(AB), KRAB(Ab), KRAB(AC) and KRAB(A). This thesis presents the identification of a novel KRAB domain, KRAB C, as well as a functional analysis of the different KRAB domains. We conclude that all different KRAB domains share a common co-repressor, TIFβ, and effectively repress transcription. These functions are mainly mediated by the KRAB A box but are clearly influenced by the presence of a KRAB B, b or C box. Furthermore, we show that all KRAB zinc finger gene subfamilies originate from the KRAB(AB) zinc finger genes. In addition, this thesis includes a structural and functional analysis of four novel mouse and human KRAB zinc finger genes; MZF6D, HKr18, HKr19 and HZF12. Whereas HKr18 and HZF12 seem to be ubiquitously expressed, MZF6D and HKr19 show a more restricted expression pattern. Northern blot and in situ hybridisation analyses of MZF6D showed that the expression of this gene is restricted to meiotic germ cells. MZF6D might thus be involved in the formation of male gametes. The expression of HKr19, on the other hand, seems to be restricted to lymphoid cells, indicating a possible role for this KRAB zinc finger gene in the regulation of lineage commitment.

The fidelity of an organism’s genome is central to biology. DNA, however, is constantly being damaged and modified by a variety of sources. As a result of these changes, repair enzymes, polymerases, and other interrogating biomolecules must be able to recognize, repair, and adapt to a multitude of different structures and dynamics presented. Manipulation of natural systems via the development and introduction of novel bases and DNA structures only adds to this complexity. In addition, specific RNA sequences are becoming more prevalent therapeutic and diagnostic targets. These include retroviruses and other viruses that maintain their genome with RNA. Unlike DNA, RNA poses a unique challenge as targets due to their highly diverse secondary and tertiary structures. In this manuscript, three different nucleic acid systems were chosen to investigate how intramolecular and intermolecular interactions impact their own structure as well as giving further insight into how nucleic acids are recognized and distorted by interrogating damage specific enzymes as well as structure specific proteins.

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.

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.