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.

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.