Regulation of gene expression in a spatiotemporal manner specifies cellular identity. Transcription factors (TFs) bind to DNA regulatory elements to remodel chromosome structure, to recruit transcription machinery to initiate gene transcription or to prevent the assembly of such machinery to repress gene transcription, thus they lie at the heart of gene regulation. Given important roles of TFs in gene regulation, numerous attentions have been attracted for engineered transcription factors (eTFs). The recent advance of generating customized DNA-sequence specific binding domains, including transcription activator-like effectors (TALEs) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene Cas9, has greatly accelerated the study and application of eTFs. The eTFs with these new binding domains offer a powerful and precise approach for modulating gene expression. / Oct4 is an important TF and it plays essential roles in the formation of inner cell mass during embryogenesis, and the maintenance of embryonic stem cells in culture as well as the reinstatement of cellular pluripotency from somatic cells. / In this study, we systematically investigated the potential of TALE-TFs and CRISPR/Cas9-TFs in activating Oct4. We designed a number of TALEs and small guide RNAs (sgRNAs) targeting various regions in the mouse and human Oct4 promoters. Using luciferase assays, we found that the most efficient TALE-VP64s bound on the region −120 to −80 bp upstream of transcription start site (TSS), while highly effective sgRNAs targeted −147 to −89 bp upstream of TSS to induce high activity of luciferase reporters. This positional effect can serve as a simple guideline for designing eTFs for activating transcription from a reporter system. Next, we examined the potential of TALE-VP64 and sgRNAs to activate endogenous Oct4 transcription. We found that the positional effect was less obvious as individual eTFs exhibited marginal activity to up-regulate endogenous gene expression. Interestingly, we found that when multiple eTFs were applied simultaneously, Oct4 could be induced significantly and synergistically. This phenomenon was well supported by activation of human SOX2, KLF4, cMYC, CDH1 and NANOG by TALE-VP64s. / Using optimized combinations of TALE-VP64s, we successfully enhanced endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64- and sgRNA/dCas9-VP64-induced transcription of endogenous OCT4. Taken together, this study demonstrated that engineered TALE-TFs and dCas9-TFs are useful tools for modulating gene expression in mammalian cells. / 基因表達調控是決定細胞命運的關鍵。轉錄因子可以結合到DNA調控序列上，以重塑染色體的結構；而且可以募集轉錄機器，以起始轉錄, 或者幹擾轉錄機器的組裝，從而抑制基因轉錄；因此，在基因表達調控過程中轉錄因子處於核心地位。由于轉錄因子在基因調控方面的重要作用，研究者們越來越多的關注人工轉錄因子的研究。DNA 序列特異性結合域的發現與發展很大程度上促進了人工轉錄因子的研究與應用。最近從TALE和CRISPR/Cas9衍生而來的人工轉錄因子給我們提供了一個強大而且精確的調控基因表達的方法。Oct4是一個重要的轉錄因子，對胚胎發育過程中內細胞團的形成，和體外培養的胚胎幹細胞的維持，以及細胞多能性的重塑等多方面都至關重要。 / 在本研究中，我們系統性地探討了TALE和CRISPR/Cas9衍生而來的人工轉錄因子在激活Oct4基因方面的潛能。我們針對小鼠和人的Oct4的啓動子設計了一序列的TALEs和sgRNAs。通過熒光素酶實驗，我們發現結合到轉錄起始位點上遊120‐80bp位置的TALE‐VP64s，或者結合到147‐89bp位置的sgRNAs可以最有效地誘導熒光素酶報告基因的表達。在激活報告基因方面，這種位置效應可以作爲一條設計人工轉錄因子的簡單原則。然後，我們進一步檢測了這些人工轉錄因子在激活內源性Oct4轉錄方面的效果。結果顯示上述觀察到的位置效應並不明顯，因爲每一單個的人工轉錄因子都幾乎不能上調內源性基因的表達。但是，當同時導入多個人工轉錄因子時，我們可以顯著地激活Oct4的表達，而且可以觀察到明顯的疊加效應。利用人工轉錄因子激活SOX2, KLF4, cMYC, CDH1和NANOG,我們進一步證明了這種疊加效應。 / 通過篩查不同的人工轉錄因子組合，我們在小鼠NIH3T3細胞系把Oct4基因的表達提供到了原來水平的30多倍，而在人的HEK293T中，提高了20多倍。更重要的是，我們可以檢測到蛋白質表達水平的提高。通過檢測不同的表觀調控因子，我們發現組蛋白乙酰化轉移酶p300可以進一步提升這些人工轉錄因子誘導的Oct4基因表達。因此，本研究表明這些人工轉錄因子是調節哺乳動物細胞內基因表達的有效工具。 / Hu, Jiabiao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014.y066 / Includes bibliographical references (leaves 132-157). / Abstracts also in Chinese. / Title from PDF title page (viewed on 13, December, 2016). / Hu, Jiabiao. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

The L1 region of the porcine adenovirus (PAdV)-3 genome encodes a protein of 622 amino acids named IIIa. Although it binds a neighboring group of nine (GON) hexons at the capsid level and cement the icosahedral shell that contains the viral DNA, little is known regarding its function with respect to viral life cycle. Moreover, the known location of IIIa protein in the capsid may help to express targeting ligands for altering the tropism of PAdV-3. The objective of this study was to characterize the IIIa protein of porcine adenovirus Type 3 (PAdV-3). <p> In order to characterize the IIIa protein, polyclonal antisera were raised in rabbits against different regions of IIIa. Anti-IIIa sera detected a specific protein of 70 kDa in PAdV-3 infected cells using Western blot assay. Immunofluorescence studies indicated that IIIa is predominantly localized in the nucleus of PAdV-3 infected cells. Analysis of PAdV-3 IIIa using antibodies specific for N- and C- terminal domains of the protein suggested that although the N-terminus and C-terminal domains of IIIa are immunogenic, they are not exposed on the surface of PAdV-3 virions. These results were further confirmed by our inability to isolate a chimeric PAdV-3 virion containing a heterologous protein fused to the N-terminus or C-terminus of IIIa. <p>Functional analysis suggested that IIIa may transactivate the major late promoter and down regulate the early region (E) 1A promoter. In order to locate the domains of IIIa responsible for different functions, in-frame deleted/truncated forms of IIIa were constructed. Analysis of the deleted/truncated forms of IIIa suggested that a) the sequences located between amino acids 273-410 and between amino acids 410-622b) affect the nuclear localization and transactivation function respectively.<p>Since protein- protein interactions are important for the biological functions of the protein, we determined the interaction of PAdV-3 IIIa with other viral proteins. IIIa was found to interact with DNA binding protein (DBP), E3 13.7 kDa protein, hexon, fiber, and pIX. These results suggest that PAdV3 IIIa may do more in the viral life cycle than merely act as cement between the hexons to maintain capsid stability and may actually be involved in regulating early to late gene transcription at appropriate stages during viral infection.

Cells are continuously exposed to a variety of physiological and environmental stresses that can lead to protein aggregation and/or denaturation, and eventually cell death. In order to ensure survival, cells have evolved a stress response that monitors, detects, and responds to changes within the cellular environment. The stress response is characterized by the up-regulation of heat shock protein (hsp) genes whose products can mediate the assembly and/or degradation of misfolded or aggregated proteins within the cell. This stress-induced upregulation of heat shock protein encoding genes is under the regulation of heat shock transcription factor 1 (HSF1) and its associated proteins that together form what is known as the HSF1 heterocomplex. In eukaryotic cells, HSF1 exists as a non-DNA binding monomer in the absence of stress. Upon exposure to stress, HSF1 undergoes trimerization and acquires the ability to bind heat shock elements (HSEs) located upstream of all hsp genes and after further modification, can become converted into a transcriptionally active form. Following prolonged stress or after removal of stress, HSF1 loses its ability to bind DNA and transcription ceases in a process termed attenuation. <p>Several studies have suggested that the DNA-binding and transcriptional activities of HSF1 are regulated by phosphorylation and dephosphorylation events and by chaperone-based folding mechanisms similar to those involved in the regulation of glucocorticoid receptors. Protein phosphatase 5 (PP5) has been identified as a member of the glucocorticoid receptor chaperone complex and its phosphatase activity has been shown to regulate the maturation and activation of the receptor. It has been suggested that PP5 may regulate HSF1 in a manner similar to that of glucocorticoid receptors however it has not yet been determined how PP5 interacts with the HSF1 heterocomplex or if PP5 functions to regulate HSF1-DNA binding and/or HSF1 transactivation.<p>Utilizing the Xenopus model system, I tested the hypothesis that PP5 regulates the DNA binding and transcriptional activities of HSF1 through interactions with the HSF1 heterocomplex. Increasing the activity of PP5, either through the elevation of PP5 protein levels or by activating endogenous PP5, resulted in decreased HSF1-DNA binding as well as accelerated attenuation after the removal of stress. Conversely, inhibiting the phosphatase activity of PP5 using okadaic acid or by immunotargetting, where an antibody recognizing PP5 was microinjected into the nuclei of oocytes, resulted in delayed HSF1 attenuation. Transcription assays performed using activated PP5 also demonstrated that PP5 acts to decrease HSF1-mediated transcription. Immunoprecipitation and gel mobility supershift assays were also used to show that PP5 interacts with the HSF1 heterocomplex and PP5-HSP90 binding mutants illustrated that PP5 may exert its repressive effects independently of binding directly to HSP90.

Cells are continuously exposed to a variety of physiological and environmental stresses that can lead to protein aggregation and/or denaturation, and eventually cell death. In order to ensure survival, cells have evolved a stress response that monitors, detects, and responds to changes within the cellular environment. The stress response is characterized by the up-regulation of heat shock protein (hsp) genes whose products can mediate the assembly and/or degradation of misfolded or aggregated proteins within the cell. This stress-induced upregulation of heat shock protein encoding genes is under the regulation of heat shock transcription factor 1 (HSF1) and its associated proteins that together form what is known as the HSF1 heterocomplex. In eukaryotic cells, HSF1 exists as a non-DNA binding monomer in the absence of stress. Upon exposure to stress, HSF1 undergoes trimerization and acquires the ability to bind heat shock elements (HSEs) located upstream of all hsp genes and after further modification, can become converted into a transcriptionally active form. Following prolonged stress or after removal of stress, HSF1 loses its ability to bind DNA and transcription ceases in a process termed attenuation. <p>Several studies have suggested that the DNA-binding and transcriptional activities of HSF1 are regulated by phosphorylation and dephosphorylation events and by chaperone-based folding mechanisms similar to those involved in the regulation of glucocorticoid receptors. Protein phosphatase 5 (PP5) has been identified as a member of the glucocorticoid receptor chaperone complex and its phosphatase activity has been shown to regulate the maturation and activation of the receptor. It has been suggested that PP5 may regulate HSF1 in a manner similar to that of glucocorticoid receptors however it has not yet been determined how PP5 interacts with the HSF1 heterocomplex or if PP5 functions to regulate HSF1-DNA binding and/or HSF1 transactivation.<p>Utilizing the Xenopus model system, I tested the hypothesis that PP5 regulates the DNA binding and transcriptional activities of HSF1 through interactions with the HSF1 heterocomplex. Increasing the activity of PP5, either through the elevation of PP5 protein levels or by activating endogenous PP5, resulted in decreased HSF1-DNA binding as well as accelerated attenuation after the removal of stress. Conversely, inhibiting the phosphatase activity of PP5 using okadaic acid or by immunotargetting, where an antibody recognizing PP5 was microinjected into the nuclei of oocytes, resulted in delayed HSF1 attenuation. Transcription assays performed using activated PP5 also demonstrated that PP5 acts to decrease HSF1-mediated transcription. Immunoprecipitation and gel mobility supershift assays were also used to show that PP5 interacts with the HSF1 heterocomplex and PP5-HSP90 binding mutants illustrated that PP5 may exert its repressive effects independently of binding directly to HSP90.

The L1 region of the porcine adenovirus (PAdV)-3 genome encodes a protein of 622 amino acids named IIIa. Although it binds a neighboring group of nine (GON) hexons at the capsid level and cement the icosahedral shell that contains the viral DNA, little is known regarding its function with respect to viral life cycle. Moreover, the known location of IIIa protein in the capsid may help to express targeting ligands for altering the tropism of PAdV-3. The objective of this study was to characterize the IIIa protein of porcine adenovirus Type 3 (PAdV-3). <p> In order to characterize the IIIa protein, polyclonal antisera were raised in rabbits against different regions of IIIa. Anti-IIIa sera detected a specific protein of 70 kDa in PAdV-3 infected cells using Western blot assay. Immunofluorescence studies indicated that IIIa is predominantly localized in the nucleus of PAdV-3 infected cells. Analysis of PAdV-3 IIIa using antibodies specific for N- and C- terminal domains of the protein suggested that although the N-terminus and C-terminal domains of IIIa are immunogenic, they are not exposed on the surface of PAdV-3 virions. These results were further confirmed by our inability to isolate a chimeric PAdV-3 virion containing a heterologous protein fused to the N-terminus or C-terminus of IIIa. <p>Functional analysis suggested that IIIa may transactivate the major late promoter and down regulate the early region (E) 1A promoter. In order to locate the domains of IIIa responsible for different functions, in-frame deleted/truncated forms of IIIa were constructed. Analysis of the deleted/truncated forms of IIIa suggested that a) the sequences located between amino acids 273-410 and between amino acids 410-622b) affect the nuclear localization and transactivation function respectively.<p>Since protein- protein interactions are important for the biological functions of the protein, we determined the interaction of PAdV-3 IIIa with other viral proteins. IIIa was found to interact with DNA binding protein (DBP), E3 13.7 kDa protein, hexon, fiber, and pIX. These results suggest that PAdV3 IIIa may do more in the viral life cycle than merely act as cement between the hexons to maintain capsid stability and may actually be involved in regulating early to late gene transcription at appropriate stages during viral infection.

Thesis (Ph.D.). / Written for the Division of Experimental Medicine, Dept. of Medicine. Title from title page of PDF (viewed 2008/03/12). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [79]-97).

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23609号 / 医科博第132号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 森信 暁雄, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/12/10). Includes bibliographical references.