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.

Thesis (Ph.D. in Physiology & Biophysics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 125-138). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

The circadian clock is a molecular mechanism that synchronizes physiological changes with environmental variations. Disruption of the circadian clock has been linked to increased risk in diseases and a number of disorders (e.g. jet lag, insomnia, and cancer). Period 2 (Per2), a circadian protein, is at the center of the clock's function. The loss or deregulation of per2 has been shown to be common in several types of cancer including breast and ovarian [1, 2]. Epidemiological studies established a correlation between circadian disruption and the development of estrogen dependent tumors. The expression of estrogen receptor alpha (ERα) mRNA oscillates in a 24-hour period and, unlike Per2, ERα peaks during the light phase of the day. Because up regulation of ERα relates to tumor development, defining the mechanisms of ERα expression will contribute to our comprehension of cellular proliferation and regulation of normal developmental processes. The overall goal of this project is to investigate the molecular basis for circadian control of ERα transcription. Transcriptional activation of ERα was measured using a reporter system in Chinese hamster ovary (CHO) cell lines. Data show that Per2 influences ERα transcription through a non-canonical mechanism independent of its circadian counterparts. Breast cancer susceptibility protein 1 (BRCA1) was confirmed to be an interactor of Per2 via bacterial two-hybrid assays, in accordance with previous studies [2]. BRCA1 is a transcriptional activator of ERα promoter in the presence of octamer transcription factor-1 (OCT-1) [3]. Our results indicate that the DNA binding domain of OCT-1, POU, to directly interact with Per2 and BRCA1, in vitro. Pull-down assays were used to map direct interaction of various Per2 and BRCA1 recombinant proteins and POU. Chromatin immunoprecipitation assays confirmed the recruitment of PER2 and BRCA1 to the estrogen promoter by OCT-1 and the recruitment of Per2 to the ERα promoter decreases ERα mRNA expression levels in MCF-7 cells. Our work supports a circadian regulation of ERα through the repression of esr1 by Per2 in MCF-7 cells. / Ph. D.

β-Casein is a major milk protein, which is synthesized in mammary alveolar secretory epithelial cells (MECs) upon the stimulation of lactogenic hormones, mainly prolactin and glucocorticoids (HP). Previous studies revealed that the proximal promoter (-258 bp to +7 bp) of the β-casein gene is sufficient for induction of the promoter activity by HP. This proximal region contains the binding sites for the signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), and octamer transcription factors (Oct). STAT5 and GR are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. This study investigated the functions of Oct-1 and Oct-2 in HP induction of β-casein gene expression. By transiently transfection experiment, we showed that individual overexpression of Oct-1 and Oct-2 further enhanced HP-induced β-casein promoter activity, respectively, while Oct-1 and Oct-2 knockdown significantly inhibited the HP-induced β-casein promoter activity, respectively. HP rapidly induced the binding of both Oct-1 and Oct-2 to the β-casein promoter, and this induction was not mediated by either increasing their expression or inducing their translocation to the nucleus. In MECs, Oct-2 was found to physically interact with Oct-1 regardless of HP treatment. However, HP induced physical interactions of Oct-1 or Oct-2 with both STAT5 and GR. Although the interaction between Oct-1 and Oct-2 did not synergistically stimulate HP-induced β-casein gene promoter activity, the synergistic effect was observed for the interactions of Oct-1 or Oct-2 with STAT5 and GR. The interactions of Oct-1 with STAT5 and GR enhanced or stabilized the binding of STAT5 and GR to the promoter. Abolishing the interaction between Oct-1 and STAT5 significantly reduced the hormonal induction of β-casein gene transcription. Thus, our study indicates that HP activate β-casein gene expression by inducing the physical interactions of Oct-1 and Oct-2 with STAT5 and GR in mouse MECs. There is a high and increasing demand for insulin because of the rapid increase in diabetes incidence worldwide. However, the current manufacturing capacities can barely meet the increasing global demand for insulin, and the cost of insulin production keeps rising. The mammary glands of dairy animals have been regarded as ideal bioreactors for mass production of therapeutically important human proteins. We tested the feasibility of producing human proinsulin in the milk of transgenic mice. In this study, four lines of transgenic mice were generated to harbor the human insulin gene driven by the goat β-casein gene promoter. The recombinant human proinsulin was detected in the milk by Western blotting and enzyme-linked immunosorbent assay. The highest expression level of human proinsulin was as high as 8.1 μg/µl in milk of transgenic mice at mid-lactation. The expression of the transgene was only detected in the mammary gland during lactation. The transgene expression profile throughout lactation resembled the milk yield curve, with higher expression level at middle lactation and lower expression level at early and late lactation. The blood glucose and insulin levels and major milk compositions of transgenic mice were not changed. The mature insulin derived from the milk proinsulin retained biological activity. Thus, our study indicates that it is practical to produce high levels of human proinsulin in the milk of dairy animals, such as dairy cattle and goat.

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Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.

Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.