abstract: The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic adverse events following receiving of adeno-associated virus (AAV) as one of the clinically relevant delivery agents, and CRISPR off-target activity in the genome, reinforces the necessity for improving the safety of CRISPR and the gene therapy vehicles. Research into designing more advanced CRISPR systems will allow for the increased ability of editing efficiency and safety for human applications. This work 1- develops strategies for decreasing the immunogenicity of CRISPR/Cas9 system components and improving the safety of CRISPR-based gene therapies for human subjects, 2- demonstrates the utility of this system in vivo for transient repression of components of innate and adaptive immunity, and 3- examines an inducible all-in-one CRISPR-based control switch to pave the way for controllable CRISPR-based therapies. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2020

Insertion of transposable elements within an operon has been known not only to abolish expression of the gene interrupted by the insertion, but also to exert a strong polar effect on the expression of downstream genes in the same operon. In this dissertation, I have shown that insertions of the transposable ampicillin-resistance element Tn1, either in the polar or nonpolar orientation, in the 3' untranslated region of the bacteriophage P22 antirepressor (ant) operon reduce the rate of upstream ant gene expression; insertions of Tn1 in the nonpolar orientation reduce the rate of ant gene expression more significantly than those in the polar orientation. This effect appears to be due to reduced stability of ant mRNA. Tn1 deletion mutants of one of the nonpolar Tn1 insertion mutations have been isolated. Two classes of Tn1 deletions are obtained. Class I retains a 68 bp Tn1 sequence that shows a potential 14 bp stem and 37 bp loop conformation, while class II retains 147 bp Tn1 sequence that shows a potential 69 bp stem and 6 bp loop conformation. These two classes of Tn1 deletions do not delete any P22 sequences. Class I but not class II Tn1 deletion mutants restore the rate of ant gene expression and ant mRNA stability. Six different Ant+ revertants of the class II Tn1 deletion mutant simultaneously restore the rate of ant gene expression and ant mRNA stability. They all have deletions that remove all or part of the class II Tn1 sequence. In one case, the Tn1 sequence retained shows a potential 15 bp stem and 8 bp loop conformation, in the other cases, no secondary structure is predicted to form. The results of the Tn1 deletion mutants suggest that the stem-and-loop structures and the length of stems potentially formed by the Tn1 sequences in mRNA may affect its stability.

Chapter II Abstract Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub1Δ mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate. Chapter III Abstract Previously I established that (1) PC4 significantly suppresses oxidative mutagenesis via its single-strand DNA binding activity, (2) a partial suppression of H2O2-induced lethality was observed in a sub1Δ rad2Δ yeast double mutant compared to the sub1Δ mutant, and (3) PC4 interacts with XPG physically and functionally. These results led me to believe that suppression of oxidative mutagenesis and lethality by PC4 is partially due to its function in an XPG/Rad2-dependent pathway and through additional unidentified mechanism(s). In this chapter, I present studies aimed at investigating different DNA repair pathways in which PC4/Sub1 might participate. I address the possible roles of PC4/Sub1 in transcription-coupled repair (TCR) in terms of its binding specificity to oxidative DNA lesions and its ability to allow efficient resumption of transcription after oxidative DNA damaging treatment. To ask if PC4/Sub1 interacts with other DNA repair proteins to protect cells from oxidative DNA damage, I analyzed spontaneous mutation rates among a series of isogenic, haploid yeast mutant strains deficient of SUB1, base excision repair (BER) and/or nucleotide excision repair (NER) functions. I further analyzed genetic interactions between SUB1 and genes critical to various DNA damage avoidance/tolerance mechanisms, such as mismatch repair (MMR), homologous recombination (HR) and translesion synthesis (TLS).

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18095号 / 理博第3973号 / 新制||理||1573(附属図書館) / 30953 / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.

BMI1 est une protéine appartenant à la famille des polycombs impliquée dans la régulation épigénétique de la transcription. Il a été montré que cette protéine est essentielle à la régulation de la prolifération, de la sénescence et du métabolisme ainsi qu’à l’auto-Renouvellement des cellules souches hématopoïétiques et cancéreuses. Ce répresseur transcriptionnel au fort potentiel oncogénique est retrouvé surexprimé dans de nombreux types de cancer ; dans le cas de la Leucémie Myéloïde Chronique (LMC) le niveau d’expression de BMI1 augmente avec l’aggravation de la pathologie. Cependant, les voies de signalisation impliquées dans sa surexpression et le rôle qu’il joue au sein de cette maladie demeurent méconnus. En réprimant l’expression de BMI1 par ARN interférence nous avons pu mettre en évidence que ce polycomb était essentiel à la prolifération cellulaire ainsi qu’au potentiel clonogénique des cellules de LMC. Nous avons également démontré pour la première fois que BMI1 soutenait la croissance tumorale à travers la répression d’un processus autophagique délétère pour la cellule cancéreuse. Une approche transcriptomique nous a permis d’identifier la cible transcriptionnelle impliquée dans ce processus, la Cycline G2. Nous avons, pour finir, trouvé une molécule, via une approche bioinformatique, capable de réinduire l’expression de la Cycline G2 dans les cellules de LMC, l’alexidine dihydrochloride. Cette molécule induit une forte autophagie dans les cellules cancéreuses ainsi que de l’apoptose. Elle s’est également montrée capable de resensibiliser à l’imatinib (un inhibiteur de BCR-ABL) une lignée pourtant résistante. / The polycomb protein Bmi1 is a major epigenetic regulator. It has been shown that this protein is essential for the regulation of cell proliferation, senescence and metabolism but also self-Renewal of hematopoïetic and cancer stem cells. This transcriptional repressor, with a strong oncogenic potential, is overexpressed in many types of cancer. In case of Chronic Myeloid Leukemia (CML) the expression level of BMI1 is associated with worsening prognosis. However, the signaling pathways involved in its overexpression and its role in this disease remains unclear. By using RNAi to repress BMI1 expression we highlighted that this polycomb was essential for proliferation and clonogenicity of CML cells. We also demonstrated, for the first time, that BMI1 supported tumor growth through repression of deleterious cancer cell autophagy. A transcriptomic approach allowed us to identify a transcriptional target involved in this process: the Cyclin G2. Through a bioinformatic approach, we finally found a molecule capable of expression re-Induction of Cyclin G2 in CML cells : alexidine dihydrochloride. This molecule induced a high level of autophagy as well as apopotosis in cancer cells. It had also been able to re-Sensitize to imatinib a resistant cell line. In conclusion, our results revealed a new role for the polycomb BMI1 in supporting the CML pathology. Moreover, our work allowed the identification of two new approaches for therapeutically targeting this oncogene functions.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 179-198). Also issued on the Internet.

The ability of Candida albicans to change morphology plays several roles in its virulence and as a human commensal. The yeast-to-hyphae transition is tightly regulated by several sets of activating and repressing pathways. The DNA-binding proteins Rfg1p, Nrg1p and the global repressor Tup1p are part of the repressors found to regulate this morphogenesis. Knowledge of these repressors is based on extrapolations from homology to S. cerevisiae and from expression studies of mutants in inducing conditions, all of which are indirect means of determining a protein's function. We proposed a genome-wide location study of the Nrg1 and Rfg1 transcription factors to obtain direct data to identify their in vivo targets. Our results suggest different avenues for Nrg1p function and a regulation behaviour diverging from the previously suggested model: Nrg1p acts not only as a repressor but also as a transcription activator. Furthermore it regulates its target genes through binding in their coding regions instead binding to the expected regulatory elements on promoters.