Dissertations / Theses on the topic 'Kidney Transcription Factors Transcriptional Activation'

In recent years an increasingly substantial body of data, supports a role for C-peptide in several biological activities. However, the precise molecular mechanisms of C-peptide action are not fully understood. The aim of this thesis was to study the intracellular signalling pathways and the transcription factors that C-peptide activates in proximal tubular cells using opossum kidney cells (OK) as a model. Using specific inhibitors and phospho-specific antibodies, intracellular signalling pathways activated by C-peptide were examined by kinase assay and Western blotting. The results show that C-peptide is able to activate extracellular signal regulated kinase (ERK), phosphatidylinositol 3-kinase (PI 3-kinase) and PKC-a. ERK activation was attenuated by PKC inhibitor pre-treatment and activation of ERK and PKC-a were abolished in the absence of extracellular Ca2+. Elevations of [Ca2+]i were examined using confocal microscopy. C-peptide induced transient increase in [Ca2+]i but the response of cells was variable. Thymidine incorporation assay was used to assess proliferation. C-peptide was found to be a functional mitogen in this cell type stimulating significantly increased cell proliferation. Proliferator-activated receptor (PPAR) transcriptional activity was measured using a luciferase reporter assay in OK cells. C-peptide induced concentration-dependent stimulation of PPARy activity. C-peptide also substantially augmented ciglitazone-stimulated PPARy activity. GW9662, an irreversible PPARy antagonist, blocked PPARy activation by ciglitazone, but had no effect on C-peptide-stimulated PPARy activity. C-peptide stimulation of PPARy was attenuated by wortmannin pre-treatment, and by expression of a dominant negative PI 3-kinase p85 regulatory subunit (Ap85). C-peptide had no effect on protein expression levels of PPARy. PPARy phosphorylation was examined by [32P]-orthophosphate labelling of OK cells and immunoprecipitation of phospho-PPARy. C-peptide-induced PI 3-kinase dependent phosphorylation of PPARy. C-peptide is able to protect against tumor necrosis factor-alpha- (TNF-a) induced proximal tubular cells toxicity. Stimulation with 300ng/ml TNF-a for 24 hours resulted in significant reduction of cell viability which was reversed by pretreatment with C-peptide. TNF-a induced apoptosis was detected by measuring histone associated DNA fragments and DNA nick end-labelling of OK cells. Incubation of cells with 300ng/ml TNF-a for 24 hours induced apoptosis, but C-peptide pr-etreatment protected against TNF-a induced apoptosis. The protective effects of C-peptide were associated with activation of nuclear factor kB (NFkB) and increased expression of TNF receptor-associated factor 2, the product of an NFkB-dependent survival gene. This was dependent upon activation of PI 3-kinase, but not ERK. All C-peptide effects were abolished by pretreatment with PTX implicating a G-protein coupled receptor (GPCR), to either Goii or GOo, in the transduction of these events. C-peptide increased [35S]-GTPyS binding to Ga* in OK cell membranes. This study has now for the first time demonstrated specifically that Ga* proteins are activated by C-peptide binding to a GPCR. Despite being ignored for many years it is now clear that C-peptide possesses important biological properties and may potentially protect against diabetic complications.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here, we find that transcriptional activation mediated by the GLI family of transcription factors, although dispensable for pancreatic development, is required for KRAS induced pancreatic transformation. Inhibition of GLI using a dominant-negative repressor (Gli3T) inhibits formation of precursor Pancreatic Intraepithelial Neoplasia (PanIN) lesions in mice, and significantly extends survival in a mouse model of PDAC. Further, ectopic activation of the GLI1/2 transcription factors in mouse pancreas accelerates KRAS driven tumor formation and reduces survival, underscoring the importance of GLI transcription factors in pancreatic tumorigenesis. Interestingly, we find that although canonical GLI activity is regulated by the Hedgehog ligands, in the context of PDAC, GLI transcription factors initiate a unique ligand-independent transcriptional program downstream of KRAS, that involves regulation of the RAS, PI3K/AKT, and NF-кB pathways. We identify I-kappa-B kinase epsilon (IKBKE) as a PDAC specific target of GLI, that can also regulate GLI transcriptional activity via positive feedback mechanism involving regulation of GLI subcellular localization. Using human PDAC cells, and an in vivo model of pancreatic neoplasia, we establish IKBKE as a novel regulator pf pancreatic tumorigenesis that acts as an effector of KRAS/GLI, and mediates pancreatic transformation. We show that genetic knockout of Ikbke leads to a dramatic inhibition of initiation and progression of pancreatic intraepithelial viii neoplasia (PanIN) lesions in mice carrying pancreas specific activation of oncogenic Kras. Furthermore, we find that although IKBKE is a known NF-кB activator, it only modestly regulates NF-кB activity in PDAC. Instead, we find that IKBKE strongly promotes AKT phosphorylation in PDAC in vitro and in vivo, and that IKBKE mediates reactivation of AKT post-inhibition of mTOR. We also show that while mTOR inhibition alone does not significantly affect pancreatic tumorigenesis, combined inhibition of IKBKE and mTOR has a synergistic effect leading to significant decrease tumorigenicity of PDAC cells. Together, our findings identify GLI/IKBKE signaling as an important oncogenic effector pathway of KRAS in PDAC that regulates tumorigenicity, cell proliferation, and apoptosis via regulation of AKT and NF-кB signaling. We provide proof of concept for therapeutic targeting of GLI/IKBKE in PDAC, and support the evaluation of IKBKE as a therapeutic target in treatment of pancreatic cancer, and IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here, we find that transcriptional activation mediated by the GLI family of transcription factors, although dispensable for pancreatic development, is required for KRAS induced pancreatic transformation. Inhibition of GLI using a dominant-negative repressor (Gli3T) inhibits formation of precursor Pancreatic Intraepithelial Neoplasia (PanIN) lesions in mice, and significantly extends survival in a mouse model of PDAC. Further, ectopic activation of the GLI1/2 transcription factors in mouse pancreas accelerates KRAS driven tumor formation and reduces survival, underscoring the importance of GLI transcription factors in pancreatic tumorigenesis. Interestingly, we find that although canonical GLI activity is regulated by the Hedgehog ligands, in the context of PDAC, GLI transcription factors initiate a unique ligand-independent transcriptional program downstream of KRAS, that involves regulation of the RAS, PI3K/AKT, and NF-кB pathways. We identify I-kappa-B kinase epsilon (IKBKE) as a PDAC specific target of GLI, that can also regulate GLI transcriptional activity via positive feedback mechanism involving regulation of GLI subcellular localization. Using human PDAC cells, and an in vivo model of pancreatic neoplasia, we establish IKBKE as a novel regulator pf pancreatic tumorigenesis that acts as an effector of KRAS/GLI, and mediates pancreatic transformation. We show that genetic knockout of Ikbke leads to a dramatic inhibition of initiation and progression of pancreatic intraepithelial viii neoplasia (PanIN) lesions in mice carrying pancreas specific activation of oncogenic Kras. Furthermore, we find that although IKBKE is a known NF-кB activator, it only modestly regulates NF-кB activity in PDAC. Instead, we find that IKBKE strongly promotes AKT phosphorylation in PDAC in vitro and in vivo, and that IKBKE mediates reactivation of AKT post-inhibition of mTOR. We also show that while mTOR inhibition alone does not significantly affect pancreatic tumorigenesis, combined inhibition of IKBKE and mTOR has a synergistic effect leading to significant decrease tumorigenicity of PDAC cells. Together, our findings identify GLI/IKBKE signaling as an important oncogenic effector pathway of KRAS in PDAC that regulates tumorigenicity, cell proliferation, and apoptosis via regulation of AKT and NF-кB signaling. We provide proof of concept for therapeutic targeting of GLI/IKBKE in PDAC, and support the evaluation of IKBKE as a therapeutic target in treatment of pancreatic cancer, and IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic.

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.