Browsing by Subject "Gene Silencing"
Now showing 1 - 6 of 6
- Results Per Page
- Sort Options
Item Communal Cell Death and P53 Mediated Transcriptional Control in Drosophila Melanogaster(2011-08-10) Link, Nichole Lynn; Abrams, John M.Apoptosis is essential for all metazoan development. The key component that functions in apoptosis, the apoptosome, is a molecular machine that initiates caspase activation and is conserved throughout the animal kingdom. Drosophila strains that are mutated for genes encoding the apoptosome show pronounced defects in programmed cell death (PCD). Using a characteristic phenotype associated with mosaic animals, we conducted a screen in Drosophila to discover new regulators or effectors of the apoptosome. Using this model, we also discovered a unique communal form of cell death where large regions of epithelial cells are eliminated within minutes. We also produced 'saturation tile' arrays by digital optical chemistry for an unbiased sampling of transcriptional activity in the Drosophila genome. We found that the scope of unannotated transcriptional activity is extensive and widespread. A dominant population of noncanonical transcripts was stress-responsive and required p53, a master regulator of conventional stress-responsive target genes in vertebrates and invertebrates. This prompted us to examine stimulus dependent activity surrounding a single p53 enhancer in our tiled region. Through genetic analyses, we showed that this enhancer coordinates stimulus dependent induction of multiple genes spanning over 300kb throughout the Reaper region. Surprisingly, this same enhancer regulated a gene positioned across the centromere at distances over 20Mb and also controlled at least one gene mapping to a different chromosome. Chromosome conformation capture analyses placed this enhancer in close proximity to these distant targets in vivo through specific DNA looping and these interactions were influenced by p53. Therefore, a single p53 enhancer is necessary and sufficient for long range, multigenic regulation in cis and in trans.Item DNA methylation: a two-edged sword for embryogenesis and cancer(2001-02-08) Cox, Rody P.Item Establishing a Dual-Reporter Mouse Model to Monitor INK4A/ARF Regulation in Vivo(2015-08-24) Sung, Caroline Yeh-Chien; Amatruda, James F.; Skapek, Stephen X.; Johnson, Jane E.; Mendelson, Carole R.The INK4B-ARF-INK4A locus on human chromosome 9p21 (and on mouse chromosome 4) encodes three linked, yet functionally distinctive, gene products that coordinate signaling events in both normal development and disease states. INK4 proteins exert their function as gate-keepers for cell cycle progression by inhibiting D-type cyclin-dependent kinases (CDKs), which are responsible for the phosphorylation of the retinoblastoma (RB) protein and consequent G1 exit. The structurally distinct ARF protein (p14ARF in human and p19Arf in mouse), however, activates another major tumor suppressor, TP53, via direct inhibition of its negative regulator, murine double minute 2 (MDM2). The identification of these separate but interacting effector pathways has highlighted the importance of this locus in the initiation and progression of tumorigenesis. Indeed, loss of expression of these gene products, either by deletion or gene silencing, has been linked to many forms of adult and pediatric cancer. To gain a better understanding of how the two gene products of the Cdkn2a locus, p16Ink4a and p19Arf, are regulated in vivo, I generated a BAC reporter mouse by replacing the coding sequence of each gene with a unique fluorescent reporter, allowing us to monitor the coordinate and independent regulation of the two genes at single-cell resolution. Here, I report the generation of the dual-reporter mouse model, functional validation in response to physiological processes previously associated with Cdkn2a expression as well as the novel discovery of Arf promoter activation in the central nervous system under normal conditions. The use of this transgenic reporter system will further allow for the identification of cues conferring Ink4a/Arf locus regulation during normal development as well as disease pathogenesis.Item Regulatory Mechanisms of the MicroRNA Pathway(2017-10-25) Golden, Ryan Joshua; Mendelson, Carole R.; Mendell, Joshua T.; Russell, David W.; Liu, QinghuaMicroRNAs (miRNAs) associate with members of the Argonaute protein family and downregulate partially complementary messenger RNAs (mRNAs) (1). miRNA activity is tightly regulated during development and in normal physiologic settings, while gain or loss of these control mechanisms can contribute to disease (2-4). To identify new mechanisms that regulate the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening (5, 6) coupled with a fluorescent miRNA pathway reporter. These experiments revealed an unanticipated role for the ANKRD52-PPP6C serine/threonine phosphatase complex as a critical regulator of miRNA activity in human cells. Loss of this complex significantly impaired global miRNA function. Genetic and biochemical studies revealed that phosphorylation of Argonaute2 (AGO2) on a set of highly conserved serine residues, S824-S834, blocks target mRNA engagement. Constitutive activity of the ANKRD52-PPP6C complex is necessary to remove these inhibitory phosphates and thereby allow miRNA-mediated silencing. A genome-wide CRISPR-Cas9 suppressor screen performed in ANKRD52-/- cells identified CSNK1A1 as the inhibitory AGO2 kinase that phosphorylates these sites. Together, these findings reveal a previously uncharacterized AGO2 phosphorylation cycle, uncovering a major mechanism through which the miRNA pathway is regulated and highlighting the power of iterative CRISPR-Cas9 screening for the dissection of biological pathways directly in human cells.Item [Southwestern News](2005-07-31) McKenzie, Aline; Siegfried, AmandaItem Transcriptional Gene Silencing in Mammalian Cells by MicroRNAs That Target Gene Promoters(2011-08-10) Younger, Scott Thomas; Corey, David R.A rich history exists for RNA-based regulation of gene transcription. It was reported more than a decade ago that RNA is capable of inducing DNA methylation and transcriptional gene silencing in plants. It was subsequently shown that small RNAs are involved in the establishment of heterochromatic regions of the yeast genome. More recently it has been demonstrated that small duplex RNAs designed to be complementary to gene promoters are potent regulators of gene transcription in mammalian cells. Potent and robust transcriptional regulation by designed small RNAs suggests the existence of endogenous mechanisms that facilitate recognition of gene promoters by small RNAs in mammalian cells. microRNAs (miRNAs) are endogenous small RNAs that regulate gene expression post transcriptionally through base complementarity to target sequences within 3’ UTRs of mRNA transcripts. In this body of work I test the hypothesis that miRNAs can also recognize sequences within gene promoters using two alternative approaches. In the first approach I computationally evaluate the potential for miRNAs to recognize gene promoters by performing a genome-wide survey of putative miRNA target sites within promoter sequences. In the second approach I use the well characterized human progesterone receptor (PR) gene as a model to experimentally validate that miRNAs possess the ability to regulate transcription in a cell culture system. Upon completion of this work I found that gene promoters are significantly enriched for miRNA target sites. Furthermore, the frequency of miRNA target sites within promoter sequences is comparable to their frequency within 3’ UTRs. I experimentally screened multiple miRNAs predicted to target the PR gene promoter, identified several that were capable of inhibiting transcription of the PR gene, and characterize the mechanism of transcriptional silencing. miRNAs have been understood to regulate gene expression at the post transcriptional level through recognition of 3’ UTRs within mRNA transcripts. My study extends miRNA function to recognition of sequences within gene promoters. Sequence specific recognition of gene promoters by miRNAs may complement protein transcription factors. In addition, the ability of small RNAs to rapidly evolve specificity for new sequences would have evolutionary advantages.