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Biological function of E2F7 and E2F8 is essential for embryo developmentLi, Jing 02 September 2009 (has links)
No description available.
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Methylation of Geminivirus Genomes: Investigating its role as a host defense and evaluating its efficacy as a model to study chromatin methylation in plantsRaja, Priya 26 August 2010 (has links)
No description available.
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Mechanistic insights into translational modulation of selected RNAs by RNA helicase ARanji, Arnaz K. 21 March 2011 (has links)
No description available.
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Characterization of the BTB/POZ protein ZBTB4 as a transcriptional regulator of cyclin D1Doherty, Patrick W. 10 1900 (has links)
<p>The POZ-ZF transcription factor ZBTB4 was initially identified due to its sequence homology to the dual-specificity DNA-binding transcription factor Kaiso. Subsequent characterization of ZBTB4 revealed that it is also a dual-specificity DNA-binding protein; it recognizes a specific oligonucleotide sequence C<sup>T</sup>/<sub>C</sub>GCCATC, coined the <strong>Z</strong>BTB<strong>4</strong> <strong>B</strong>inding <strong>S</strong>equence (Z4BS) as well as methylated CpG-dinucleotides. Interestingly, ZBTB4 also binds to the highly similar consensus <strong>K</strong>aiso <strong>B</strong>inding <strong>S</strong>ite (KBS) <em>in vitro</em>.</p> <p>ZBTB4 is misexpressed in cancer, and follows a stage-specific pattern of expression in breast carcinoma tissues; low ZBTB4 levels are found in late stages while high ZBTB4 expression is detected in early stages of disease progression. Ongoing studies have begun to elucidate the molecular interactions that mediate ZBTB4’s apparent tumour suppressor role in tumourigenesis, however no study has investigated the nature of ZBTB4’s ability to interact with both the Z4BS and the KBS <em>in vivo</em>, and how this may expand ZBTB4’s repertoire of potential target genes.</p> <p>Recently Kaiso has been characterized as a transcriptional repressor of the cell cycle regulatory gene <em>cyclin D1</em>, and thus we used <em>cyclin D1 </em>as a model to investigate the nature of ZBTB4’s interaction with the KBS <em>in vivo</em>. The <em>cyclin D1</em> minimal promoter contains two partial Z4BS at the same location as the KBS sites and we found that ZBTB4 binds to the +69 Z4BS/KBS site, but not to the -1067 site. Because the +69 Z4BS/KBS is immediately flanked by a CpG dinucleotide, this interaction may be a methylation-dependent interaction. To determine the consequence of this interaction, we conducted minimal promoter luciferase assays, and observed that ZBTB4 mediates an activation of the -1748-CD1 minimal promoter activity.</p> / Master of Science (MSc)
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Developing Generally Applicable Tools to Investigate TetR Family Transcriptional RegulatorsAhn, Sang Kyun 04 1900 (has links)
<p>Bacteria adapt to changes in their environment by regulating gene transcription. TetR family transcriptional regulators (TFRs) constitute one of the largest groups of bacterial transcription factors and thus, characterization of TFRs is anticipated to be crucial for a better understanding of prokaryotic physiology. Of significant importance, the majority of TFRs are predicted to respond to small-molecule signals and an emerging paradigm suggests that identifying ligands of TFRs can provide direct insight into the biochemical functions of the genes they regulate. Regulatory target genes and small-molecule ligands are unknown for all but a few TFRs and therefore, generally applicable tools for identifying these basic elements of TFRs are highly desirable. We first investigated the use of genome context as a predictive tool for identifying regulatory targets of TFRs. We find that the majority of TFRs are divergently oriented from a neighboring gene, and those with a“200 bp rule” should allow us to predict at least one regulatory target for more than half of all TFRs in the public databases. Second, we developed a biosensor mechanism amenable to high-throughput screening for identifying ligands of TFRs of unknown function. Significantly, one of our biosensors has played an integral role in characterizing the ligands of a previously uncharacterized TFR. Thus, the combined use of the tools we have developed will provide considerable benefit in understanding bacterial small-molecules responses mediated by TFRs.</p> / Doctor of Philosophy (PhD)
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TRANSCRIPTIONAL REGULATION OF OSTEOACTIVIN EXPRESSION BY BMP-2 IN OSTEOBLASTSSingh, Maneet January 2011 (has links)
Osteoactivin (OA) is a glycoprotein required for the differentiation of osteoblasts. In osteoblasts, Bone Morphogenetic Protein-2 (BMP-2) activated Smad1 signaling enhances OA expression. However, the transcriptional regulation of OA gene expression by BMP-2 is still unknown. The aim of this study was to characterize BMP-2-induced transcription factors that regulate OA gene expression during osteoblast differentiation. The stimulatory effects of BMP-2 on OA transcription were established by cloning the proximal 0.96kb of rat OA promoter region in a luciferase reporter vector in various osteogenic cell types. Further, by deletion and mutagenesis analyses of the cloned OA promoter, key binding sites for osteogenic transcription factors namely, Runx2, Smad1, Smad4 and homeodomain proteins (Dlx3, Dlx5 and Msx2) were identified and characterized. Utilizing specific siRNAs to knock down Runx2, Smad1, Smad4, Dlx3, Dlx5 or Msx2 proteins in osteoblasts, we found that Runx2, Smad1, Smad4, Dlx3 and Dlx5 proteins up-regulate OA transcription, whereas, Msx2 down-regulated OA gene expression. These specific effects of transcription factors on OA promoter regulation were confirmed by forced expression of transcription factors. Most notably, BMP-2-stimulated cooperative and synergistic interactions between Runx2-Smad1 proteins and Dlx3-Dlx5 proteins that up-regulate OA promoter activity. Electrophoretic mobility shift and supershift assays demonstrated that BMP-2 stimulates interactions between Runx2, Smad1 and Smad4 and homeodomain transcription factors with the OA promoter regions flanking the -585 Runx2 binding site, the -248 Smad binding site and the region between the -852 and the -843 homeodomain binding sites relative to transcription start site. The OA promoter region was occupied by Runx2 and also Dlx3 transcription factors during proliferation stages of osteoblast differentiation. As the osteoblasts progress from proliferation to matrix maturation stages of differentiation, the OA promoter was predominantly occupied by Runx2 and to a lesser extent Dlx5 in response to BMP-2. Finally, during matrix mineralization stages of osteoblast differentiation, BMP-2-induced a robust recruitment of Dlx5, Smad1, Dlx3 and Msx2 proteins with simultaneous dissociation of Runx2 from the OA promoter region. In conclusion, the BMP-2-induced osteogenic transcription factors Runx2, Smad1, Smad4, Dlx3, Dlx5 and Msx2 provide key molecular switches that regulate OA transcription during osteoblast differentiation. / Cell Biology
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Characterization of cyclin D1 as a Putative Kaiso Target GeneOtchere, Abena A. 05 1900 (has links)
<p> Kaiso is a unique member of the BTB/POZ (Broad complex, Tramtrak, Bric à brac,/Pox virus and zinc finger) zinc finger family of transcription factors with established roles in development and tumourigenesis. Kaiso was originally identified as a novel binding partner of the Armadillo catenin p120^ctn, a cytosolic co-factor and regulator of the cell-cell adhesion molecule and tumor suppressor E-cadherin. In addition to their roles in cell adhesion, the multifunctional Armadillo catenins also regulate gene expression, thus providing at least two mechanisms for their contribution to tumourigenesis. The discovery of a novel interaction between p120^ctn and the transcription factor Kaiso was therefore consistent with gene regulatory roles for Armadillo catenins. Interestingly, Kaiso represses transcription via a sequence-specific DNA binding site (TCCTGCnA) as well as through methylated CpG di-nucleotides, and one role of nuclear p120^ctn is to inhibit Kaiso DNA-binding and transcriptional repression. We recently identified sequence-specific Kaiso binding sites in a subset of Wnt/β-catenin/TCF tumour-associated target genes, and here we present data
characterizing cyclin D1 as a putative Kaiso target gene.</p> <p> Kaiso binds the cyclin D1 promoter in vitro and in vivo, and artificial promoter assays revealed that Kaiso overexpression results in the repression of a cyclin D1 promoter luciferase reporter. Since cyclin D1 is highly amplified in ~50% of human breast tumours, and a cancer profiling array demonstrated that Kaiso is misexpressed in ~40% of human breast tumours, we hypothesized that Kaiso represses and regulates cyclin D1 expression to inhibit breast tumourigenesis. In fact, examination of Kaiso expression in human breast cell lines demonstrated that cyclin D1 mRNA levels were upregulated in Kaiso-depleted cells. My studies further revealed that methylation-dependent Kaiso-DNA binding may contribute to Kaiso's transcriptional repression of the cyclin D1 promoter. We also determined that Kaiso inhibits, while p120^ctn activates, β-catenin-mediated activation of the cyclin D1 promoter. These findings further support a role for Kaiso and p120^ctn in breast tumourigenesis via their modulation of the canonical Wnt signaling pathway which is highly implicated in human tumourigenesis. Together these findings support our hypothesis that Kaiso regulates cyclin D1 expression. However, further
studies are required to elucidate the mechanism employed by Kaiso to elicit cyclin D1
repression and to examine how this activity may contribute to breast tumourigenesis.</p> / Thesis / Master of Science (MSc)
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Growth Hormone and Nutritional Regulation of Insulin-Like Growth Factor-I Gene ExpressionWang, Ying 30 December 2005 (has links)
The objectives of this research were to characterize insulin-like growth factor-I (IGF-I) gene expression in cattle, to determine how IGF-I gene expression is affected by nutritional intake and growth hormone (GH) in cattle, and to identify the regulatory DNA region that mediates GH stimulation of IGF-I gene expression. It was found that transcription of the IGF-I gene in cattle was initiated from both exon 1 and exon 2, generating class 1 and class 2 IGF-I mRNA, respectively. Both classes of IGF-I mRNA appeared to be ubiquitously expressed, with the highest level in liver and with class 1 being more abundant than class 2 in all tissues examined. Class 1 IGF-I mRNA may be also translated more efficiently than class 2 IGF-I mRNA. Liver expression of IGF-I mRNA was decreased (P < 0.01) by food deprivation in cattle, and this decrease was due to an equivalent decrease in both classes of IGF-I mRNA. Liver expression of IGF-I mRNA was increased (P < 0.01) by GH, and this increase resulted mainly from increased expression of class 2 IGF-I mRNA. Using cotransfection analyses, a ~700 bp chromosomal region ~75 kb 5' from the first exon of the human IGF-I gene was found to enhance reporter gene expression in the presence of constitutively active signal transducer and activator of transcription 5 (STAT5) proteins, transcription factors that are known to be essential for GH-increased IGF-I gene expression. This 700 bp DNA region contains two STAT5-binding sites that appear to be conserved in mammals including cattle. Electrophoretic mobility shift assays and cotransfection analyses confirmed their ability to bind to STAT5 proteins and to mediate STAT5 activation of gene expression, respectively. Chromatin immunoprecipitation assays indicated that overexpressed constitutively active STAT5b protein bound to the chromosomal region containing these two STAT5-binding sites in Hep G2 cells, and this binding was associated with increased expression of IGF-I mRNA. These two STAT5-binding sites were also able to mediate GH-induced STAT5 activation of gene expression in reconstituted GH-responsive cells. These results together suggest that the distal DNA region that contains two STAT5-binding sites may mediate GH-induced STAT5 activation of IGF-I gene transcription in vivo. / Ph. D.
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Characterization of Transcriptional and Post-transcriptional Regulation of lin-42/Period During Post-embryonic Development of C. elegansJames, Tracy 23 October 2012 (has links)
Period, which is broadly conserved in metazoans, regulates circadian timing of neurophysiology as well as cell fate specification. Studies in mouse and humans indicate that period functions as a tumor suppressor and controls adult stem cell differentiation. However, regulation of period function in developmental pathways has not been characterized and appears to be different from its regulation and function in circadian pathways. lin-42 is the Caenorhabditis elegans ortholog of period and has both circadian and developmental timing functions. During post-embryonic larval development, cyclic expression and function of lin-42 controls stage-specific and reiterative cell fate choices of a subset of epidermal stem cells called seam cells. We are studying lin-42 regulation of seam cell fate during C. elegans larval development as a model for understanding the mechanisms of period regulation of adult stem cell fate in mammals.
This dissertation describes the research undertaken to characterize the cis-regulatory elements and the trans-regulatory factors that control lin-42 expression. We used direct molecular interaction assays (Electrophoretic Mobility Shift Assay, EMSA) (Chapter 2) followed by an RNA interference (RNAi)-based genetic screen (Chapter 3) to identify lin-42 transcriptional regulators. Using the EMSA, we identified three 50 to 100 base pair regions (binding regions, BR1-3) in the lin-42 5â noncoding sequences that were bound with specificity by C. elegans nuclear proteins. These binding regions represent putative cis-regulatory elements that may serve as transcription factor binding sites (TFBSs). We attempted to identify by mass spectrometry the proteins that bind to the BR sequences. We also used Phylogenetic Footprinting and bioinformatics screens to identify candidate C. elegans transcription factors (TFs) that may bind to putative TFBSs within the BR sequences. Using an RNAi-based screen, we tested the candidate TF genes for potential genetic interactions with lin-42. We identified ZTF-16, a member of the Hunchback/Ikaros zinc-finger transcription factor family, as a potential lin-42 activator and, using quantitative real-time PCR, confirmed that ztf-16 mutation results in down-regulation and loss of cycling expression of lin-42. We further determined that loss of ztf-16 results in seam cell development defects that phenocopy lin-42 loss-of-function, thus validating ZTF-16 as a transcriptional activator of lin-42. / Ph. D.
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Transcriptional Regulation of the Glycogen Phosphorylase-2 Gene in <I>Dictyostelium discoideum</i>Warner, Nikita 25 September 1999 (has links)
The expression of the <I>glycogen phosphorylase- 2</I> gene (<I>gp2</I>) is initiated during early development and regulated by the extracellular morphogens cAMP and Differentiation Inducing Factor (DIF-1) [1-3]. Glycogen phosphorylase- 2 catalyzes the breakdown of glycogen reserves in developing cells to generate glucose precursors required for the synthesis of the end products of differentiation [4-6]. Thus, the expression of <I>gp2</I> is a significant event for cellular differentiation. The sequence of the <I>gp2</I> promoter, like other <I>Dictyostelium</I> promoters, has an AT-rich bias (88%) [7]. Previous deletional analyses of the promoter provided a map of the regions that contained transcriptional regulatory elements. The regions thus identified contained either "TAAAAATGGA" or C-rich repeat sequences [2]. These regions were dissected further by site-directed mutagenesis (SDM) to better define the physical boundaries of the regulatory elements. It was shown that the mutation of either one of the C-rich repeats resulted in a dramatic drop of about 95% in reporter gene levels. These data strongly suggested that both the C-rich repeats of <I>gp2</I> functioned as transcriptional regulatory elements. I have identified and purified a factor called TF2 that demonstrates a high specificity for a C-rich transcriptional regulatory element, the 5' C box. TF2 was first detected with electrophoretic mobility shift assays of DEAE chromatographic fractions of cell-free extracts. The specificity of TF2 for the 5' C box was tested by competition analysis using six other oligonucleotides. Purification of TF2 was achieved by ion-exchange chromatography, DNA affinity chromatography, gel filtration chromatography, and preparative SDS-PAGE. SDS-PAGE analysis indicated an apparent subunit molecular weight of 28 kDa. The apparent molecular weight of the native protein as estimated by gel filtration was about 53 kDa. This suggested that TF2 binds gp2 as a homodimer. A cDNA clone of the tf2 gene was provided by the Japanese <I>Dictyostelium</I> cDNA project. This allowed me to synthesize probes for Southern and Northern blot analyses. Southern blot analysis indicated that there is only one form of the <I>tf2</I> gene. Northern analysis showed little or no expression of <I>tf2</I> in undifferentiated cells. During development <I>tf2</I> expression increases up to a maximum at 8 h, then decreases in later stages. Attempts to disrupt the gene suggest that <I>tf2</I> mutation may be lethal. / Ph. D.
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