Genetics and Genomics Commons^™

Mechanism Of Translation Inhibition By Type Ii Gnat Toxin Atat2, Stepan V Ovchinnikov, Dmitry Bikmetov, Alexei Livenskyi, Marina Serebryakova, Brendan Wilcox, Kyle Mangano, Dmitrii I Shiriaev, Ilya A Osterman, Petr V Sergiev, Sergei Borukhov, Nora Vazquez-Laslop, Alexander S Mankin, Konstantin Severinov, Svetlana Dubiley

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Type II toxin-antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin-antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin-antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 …

Model-Based Analysis Of Chromatin Interactions From Dcas9-Based Capture-3c-Seq., Yong Chen, Yunfei Wang, Xin Liu, Jian Xu, Michael Q Zhang

College of Science & Mathematics Departmental Research

Deciphering long-range chromatin interactions is critical for understanding temporal and tissue-specific gene expression regulated by cis- and trans-acting factors. By combining the chromosome conformation capture (3C) and biotinylated dCas9 system, we previously established a method CAPTURE-3C-seq to unbiasedly identify high-resolution and locus-specific long-range DNA interactions. Here we present the statistical model and a flexible pipeline, C3S, for analysing CAPTURE-3C-seq or similar experimental data from raw sequencing reads to significantly interacting chromatin loci. C3S provides all steps for data processing, quality control and result illustration. It can automatically define the bin size based on the binding peak of the dCas9-targeted regions. …

Relative Model Fit Does Not Predict Topological Accuracy In Single-Gene Protein Phylogenetics, Stephanie J. Spielman

College of Science & Mathematics Departmental Research

It is regarded as best practice in phylogenetic reconstruction to perform relative model selection to determine an appropriate evolutionary model for the data. This procedure ranks a set of candidate models according to their goodness of fit to the data, commonly using an information theoretic criterion. Users then specify the best-ranking model for inference. Although it is often assumed that better-fitting models translate to increase accuracy, recent studies have shown that the specific model employed may not substantially affect inferences. We examine whether there is a systematic relationship between relative model fit and topological inference accuracy in protein phylogenetics, using …

Multiplexed Capture Of Spatial Configuration And Temporal Dynamics Of Locus-Specific 3d Chromatin By Biotinylated Dcas9., Xin Liu, Yong Chen, Yuannyu Zhang, Yuxuan Liu, Nan Liu, Giovanni A Botten, Hui Cao, Stuart H Orkin, Michael Q Zhang, Jian Xu

College of Science & Mathematics Departmental Research

The spatiotemporal control of 3D genome is fundamental for gene regulation, yet it remains challenging to profile high-resolution chromatin structure at cis-regulatory elements (CREs). Using C-terminally biotinylated dCas9, endogenous biotin ligases, and pooled sgRNAs, we describe the dCas9-based CAPTURE method for multiplexed analysis of locus-specific chromatin interactions. The redesigned system allows for quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling comparisons across CREs within and between gene clusters. Multiplexed analyses of the spatiotemporal configuration of erythroid super-enhancers and promoter-centric interactions reveal organizational principles of genome structure and function.

Geographic Life History Differences Predict Genomic Divergence Better Than Mitochondrial Barcodes Or Phenotype, Daniel P. Duran, Robert A. Laroche, Harlan M. Gough, Rodger A. Gwiazdowski, Charles B. Knisley, David P. Herrmann, Stephen J. Roman, Scott P. Egan

School of Earth & Environment Departmental Research

Species diversity can be inferred using multiple data types, however, results based on genetic data can be at odds with patterns of phenotypic variation. Tiger beetles of the Cicindelidia politula (LeConte, 1875) species complex have been taxonomically problematic due to extreme phenotypic variation within and between populations. To better understand the biology and taxonomy of this group, we used mtDNA genealogies and multilocus nuclear analyses of 34,921 SNPs to elucidate its evolutionary history and evaluate the validity of phenotypically circumscribed species and subspecies. Genetic analyses recovered two divergent species that are also ecologically distinct, based on adult life history. These …

Increased Interactions And Engulfment Of Dendrites By Microglia Precede Purkinje Cell Degeneration In A Mouse Model Of Niemann Pick Type-C., Larisa Kavetsky, Kayla K Green, Bridget R Boyle, Fawad A K Yousufzai, Zachary M Padron, Sierra E Melli, Victoria L Kuhnel, Harriet M Jackson, Rosa E Blanco, Gareth R Howell, Ileana Soto Reyes

College of Science & Mathematics Departmental Research

Niemann Pick Type-C disease (NPC) is an inherited lysosomal storage disease (LSD) caused by pathogenic variants in the Npc1 or Npc2 genes that lead to the accumulation of cholesterol and lipids in lysosomes. NPC1 deficiency causes neurodegeneration, dementia and early death. Cerebellar Purkinje cells (PCs) are particularly hypersensitive to NPC1 deficiency and degenerate earlier than other neurons in the brain. Activation of microglia is an important contributor to PCs degeneration in NPC. However, the mechanisms by which activated microglia promote PCs degeneration in NPC are not completely understood. Here, we are demonstrating that in the Npc1nmf164 mouse cerebellum, microglia …

Chromatin Digestion By The Chemotherapeutic Agent Bleomycin Produces Nucleosome And Transcription Factor Footprinting Patterns Similar To Micrococcal Nuclease, Joshua Michael Stolz

Theses and Dissertations

Bleomycin (BLM), a glycopeptide antibiotic commonly used in chemotherapeutic treatments, has been shown to produce single and double stranded DNA breaks. Subsequent analysis of DNA fragmentation patterns has demonstrated preferential digestion of chromatin in the TSS of active genes and the ability to produce nucleosome-sized fragments within intact chromatin. Nucleosome positioning plays a critical role in the regulation of gene activation. Currently, micrococcal nuclease (MNase) is used as the standard for mapping the position of nucleosomes in the genome. In order to identify whether BLM can be used as an effective nucleosome-mapping agent, BLM was used to digest chromatin in …

Meox2 Haploinsufficiency Accelerates Axonal Degeneration In Dba/2j Glaucoma, Rebecca A Buchanan, Kate E Foley, Keating W Pepper, Alaina M Reagan, Kelly J Keezer, Amanda A Hewes, Cory A Diemler, Christoph Preuss, Ileana Soto Reyes, Simon W M John, Gareth R Howell

College of Science & Mathematics Departmental Research

Purpose: Glaucoma is a complex disease with major risk factors including advancing age and increased intraocular pressure (IOP). Dissecting these earliest events will likely identify new avenues for therapeutics. Previously, we performed transcriptional profiling in DBA/2J (D2) mice, a widely used mouse model relevant to glaucoma. Here, we use these data to identify and test regulators of early gene expression changes in DBA/2J glaucoma.

Methods: Upstream regulator analysis (URA) in Ingenuity Pathway Analysis was performed to identify potential master regulators of differentially expressed genes. The function of one putative regulator, mesenchyme homeobox 2 (Meox2), was tested using a combination of …

Characterization Of Human Dutpase, Shawna Marie Rotoli

Graduate School of Biomedical Sciences Theses and Dissertations

Deoxyuridine nucleotidyl transferase (dUTPase) is an enzyme found in all organisms that have thymine as a component of DNA. It catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate thus precluding the buildup of dUTP pools as well as providing the substrate, dUMP, for the de novo synthesis of thymidylate. In Homo sapiens, there are four isoforms: mitochondrial (mDut), nuclear (nDut), variant 3 and variant 4. This work is largely focused on nDut. Using structural and MS analyses of recombinant dUTPase constructs, an intermolecular disulfide bridge between cysteine-3 of each nDut monomer was discovered. It was found that these two …

The Yeast Protein Mam33 Functions In The Assembly Of The Mitochondrial Ribosome, Gabrielle A Hillman, Michael F Henry

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Mitochondrial ribosomes are functionally specialized for the synthesis of several essential inner membrane proteins of the respiratory chain. Although remarkable progress has been made toward understanding the structure of mitoribosomes, the pathways and factors that facilitate their biogenesis remain largely unknown. The long unstructured domains of unassembled ribosomal proteins are highly prone to misfolding and often require dedicated chaperones to prevent aggregation. To date, chaperones that ensure safe delivery to the assembling ribosome have not been identified in the mitochondrion. In this study, a respiratory synthetic lethality screen revealed a role for an evolutionarily conserved mitochondrial matrix protein called Mam33 …

Fishermp: Fully Parallel Algorithm For Detecting Combinatorial Motifs From Large Chip-Seq Datasets., Shaoqiang Zhang, Ying Liang, Xiangyun Wang, Zhengchang Su, Yong Chen

College of Science & Mathematics Departmental Research

Detecting binding motifs of combinatorial transcription factors (TFs) from chromatin immunoprecipitation sequencing (ChIP-seq) experiments is an important and challenging computational problem for understanding gene regulations. Although a number of motif-finding algorithms have been presented, most are either time consuming or have sub-optimal accuracy for processing large-scale datasets. In this article, we present a fully parallelized algorithm for detecting combinatorial motifs from ChIP-seq datasets by using Fisher combined method and OpenMP parallel design. Large scale validations on both synthetic data and 350 ChIP-seq datasets from the ENCODE database showed that FisherMP has not only super speeds on large datasets, but also …

The Effect Of Intracellular Zinc Levels In Escherichia Coli On Metallo-Β-Lactamase Activity, Jill Thompson

Graduate School of Biomedical Sciences Theses and Dissertations

Antibiotic resistance is a consequence of evolution via natural selection and poses a serious threat to the world's population. Escherichia coli, and other bacterial species, have acquired genes encoding Metallo-β-Lactamases (MBLs). MBLs are zinc-dependent enzymes that degrade most β-lactam antibiotics, and have emerged as important antibacterial resistance determinants in multidrug resistant pathogens. There are several different families of MBLs, and the major clinically relevant ones are Verona Integrated-encoded Metallo-β-Lactamases (VIMs), Imipenemases (IMPs), and New Delhi Metallo-β-Lactamases (NDMs). These enzymes confer high level resistance to penicillins, cephalosporins, and carbapenems, and are not inhibited by FDA-approved β-lactamase inhibitors. Variants of NDM have …

Yeast Mitochondrial Protein Pet111p Binds Directly To Two Distinct Targets In Cox2 Mrna, Suggesting A Mechanism Of Translational Activation, Julia L Jones, Katharina B Hofmann, Andrew T Cowan, Dmitry Temiakov, Patrick Cramer, Michael Anikin

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in …

Times Of Action And Evolutionary Conservation Of Heterochronic Genes, Maria Ivanova, Eric G. Moss

Rowan-Virtua Research Day

Specific genes called heterochronic genes control the timing and sequence of developmental events during larval stages of C. elegans. Mutations in heterochronic genes can cause skipping or reiteration of cell fates associated with certain larval stages. lin-14 and lin-28 are two well-studied heterochronic genes. LIN-14 acts during the first larval stage (L1) and controls events of the L1 and L2 stages, LIN-28 acts during the L2 stage and controls its events.

Enhancing Face Validity Of Mouse Models Of Alzheimer's Disease With Natural Genetic Variation., Kristen D Onos, Asli Uyar, Kelly J Keezer, Harriet M Jackson, Christoph Preuss, Casey J Acklin, Rita O'Rourke, Rebecca Buchanan, Travis L Cossette, Stacey J Sukoff Rizzo, Ileana Soto Reyes, Gregory W Carter, Gareth R Howell

College of Science & Mathematics Departmental Research

Classical laboratory strains show limited genetic diversity and do not harness natural genetic variation. Mouse models relevant to Alzheimer's disease (AD) have largely been developed using these classical laboratory strains, such as C57BL/6J (B6), and this has likely contributed to the failure of translation of findings from mice to the clinic. Therefore, here we test the potential for natural genetic variation to enhance the translatability of AD mouse models. Two widely used AD-relevant transgenes, APPswe and PS1de9 (APP/PS1), were backcrossed from B6 to three wild-derived strains CAST/EiJ, WSB/EiJ, PWK/PhJ, representative of three Mus musculus subspecies. These new AD strains were …

A Dedicated Chaperone Mediates The Safe Transfer Of Mitoribosomal Proteins To Their Site Of Assembly, Gabrielle Ashley Hillman

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondrial ribosomes are functionally specialized for the synthesis of several essential inner membrane proteins of the respiratory chain. While remarkable progress has recently been made towards understanding the structure of mitoribosomes, the unique pathways and factors that facilitate their biogenesis remain largely unknown. This dissertation defines the physiological role of an evolutionarily conserved yeast protein called Mam33 in mitochondrial ribosome assembly. The biomedical relevance of this finding stems from the fact that mutations or changes in its expression of the human ortholog p32 result in mitochondrial dysfunction. In human patients, bi-allelic mutations cause severe multisystemic defects in mitochondrial energy metabolism, …

Cyclin C: The Story Of A Non-Cycling Cyclin, Jan Jezek, Daniel G J Smethurst, David C Stieg, Z A C Kiss, Sara E Hanley, Vidyaramanan Ganesan, Kai-Ti Chang, Katrina F Cooper, Randy Strich

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The class I cyclin family is a well-studied group of structurally conserved proteins that interact with their associated cyclin-dependent kinases (Cdks) to regulate different stages of cell cycle progression depending on their oscillating expression levels. However, the role of class II cyclins, which primarily act as transcription factors and whose expression remains constant throughout the cell cycle, is less well understood. As a classic example of a transcriptional cyclin, cyclin C forms a regulatory sub-complex with its partner kinase Cdk8 and two accessory subunits Med12 and Med13 called the Cdk8-dependent kinase module (CKM). The CKM reversibly associates with the multi-subunit …

Gain-Of-Function Experiments With Bacteriophage Lambda Uncover Residues Under Diversifying Selection In Nature, Rohan Maddamsetti, Daniel T. Johnson, Stephanie J. Spielman, Katherine L. Petrie, Debora S. Marks, Justin R. Meyer

College of Science & Mathematics Departmental Research

Viral gain‐of‐function mutations frequently evolve during laboratory experiments. Whether the specific mutations that evolve in the lab also evolve in nature and whether they have the same impact on evolution in the real world is unknown. We studied a model virus, bacteriophage λ, that repeatedly evolves to exploit a new host receptor under typical laboratory conditions. Here, we demonstrate that two residues of λ’s J protein are required for the new function. In natural λ variants, these amino acid sites are highly diverse and evolve at high rates. Insertions and deletions at these locations are associated with phylogenetic patterns indicative …

N-Terminal Domain Of Human Uracil Dna Glycosylase (Hung2) Promotes Targeting To Uracil Sites Adjacent To Ssdna-Dsdna Junctions, Brian P Weiser, Gaddiel Rodriguez, Philip A Cole, James T Stivers

Rowan-Virtua School of Osteopathic Medicine Departmental Research

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias …

Inhibition Of Ribosome Biogenesis Through Genetic And Chemical Approaches, Leonid Anikin

Graduate School of Biomedical Sciences Theses and Dissertations

In order to maintain the ability to generate proteins, proliferating cells must continuously generate ribosomes, designating up to 80% of their energy to ribosome biogenesis (RBG). RBG involves transcription of rDNA by RNA polymerases I (Pol I) and III (Pol III), expression of approximately 80 ribosomal proteins, and assembly of these components in a process referred to as ribosome maturation. During maturation, the Pol I transcribed 47S pre-rRNA undergoes a number of processing events, while simultaneously interacting with processing factors and ribosomal proteins that drive pre-ribosome assembly. Inhibition of RBG has become one of the pursued targets for cancer therapy …

Insight Into Translational Activation In Yeast Mitochondria, Julia Lynn Jones

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondrial function depends on over a thousand proteins, of which the majority are nuclear DNA-encoded and approximately one percent are mitochondrial DNA-encoded. The mitochondrial DNA of Saccharomyces cerevisiae contains eight protein-encoding genes, seven of which are required for proper function of the respiratory complexes and one encodes a ribosomal protein. The bigenomic nature of the oxidative phosphorylation complexes requires coordinated expression and regulation from both the nuclear and the mitochondrial genomes. It is currently unclear how this regulatory network operates. However, it is thought that nuclear genome-encoded messengers localized to the mitochondria aid in this coordination.

A family of proteins …

Snf1 Cooperates With The Cwi Mapk Pathway To Mediate The Degradation Of Med13 Following Oxidative Stress, Stephen D Willis, David C Stieg, Kai Li Ong, Ravina Shah, Alexandra K. Strich, Julianne H Grose, Katrina F Cooper

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Eukaryotic cells, when faced with unfavorable environmental conditions, mount either pro-survival or pro-death programs. The conserved cyclin C-Cdk8 kinase plays a key role in this decision. Both are members of the Cdk8 kinase module that, along with Med12 and Med13, associate with the core Mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which releases cyclin C into the cytoplasm to promote mitochondrial fission and programmed cell death. The SCFGrr1 ubiquitin ligase mediates Med13 degradation dependent on the cell wall integrity pathway, MAPK Slt2. Here we show that the AMP kinase Snf1 activates a second …

The Zinc Transporter Zipt-7.1 Regulates Sperm Activation In Nematodes, Yanmei Zhao, Chieh-Hsiang Tan, Amber Krauchunas, Andrea Scharf, Nicholas Dietrich, Kurt Warnhoff, Zhiheng Yuan, Marina Druzhinina, Sam Guoping Gu, Long Miao, Andrew Singson, Ronald E Ellis, Kerry Kornfeld

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Sperm activation is a fascinating example of cell differentiation, in which immotile spermatids undergo a rapid and dramatic transition to become mature, motile sperm. Because the sperm nucleus is transcriptionally silent, this transition does not involve transcriptional changes. Although Caenorhabditis elegans is a leading model for studies of sperm activation, the mechanisms by which signaling pathways induce this transformation remain poorly characterized. Here we show that a conserved transmembrane zinc transporter, ZIPT-7.1, regulates the induction of sperm activation in Caenorhabditis nematodes. The zipt-7.1 mutant hermaphrodites cannot self-fertilize, and males reproduce poorly, because mutant spermatids are defective in responding to activating …

Acetic Acid Induces Sch9p-Dependent Translocation Of Isc1p From The Endoplasmic Reticulum Into Mitochondria, António Rego, Katrina F Cooper, Justin Snider, Yusuf A Hannun, Vítor Costa, Manuela Côrte-Real, Susana R Chaves

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Changes in sphingolipid metabolism have been linked to modulation of cell fate in both yeast and mammalian cells. We previously assessed the role of sphingolipids in cell death regulation using a well characterized yeast model of acetic acid-induced regulated cell death, finding that Isc1p, inositol phosphosphingolipid phospholipase C, plays a pro-death role in this process. Indeed, isc1∆ mutants exhibited a higher resistance to acetic acid associated with reduced mitochondrial alterations. Here, we show that Isc1p is regulated by Sch9p under acetic acid stress, since both single and double mutants lacking Isc1p or/and Sch9p have the same resistant phenotype, and SCH9 …

Till Death Do Us Part: The Marriage Of Autophagy And Apoptosis., Katrina F Cooper

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Autophagy is a widely conserved catabolic process that is necessary for maintaining cellular homeostasis under normal physiological conditions and driving the cell to switch back to this status quo under times of starvation, hypoxia, and oxidative stress. The potential similarities and differences between basal autophagy and stimulus-induced autophagy are still largely unknown. Both act by clearing aberrant or unnecessary cytoplasmic material, such as misfolded proteins, supernumerary and defective organelles. The relationship between reactive oxygen species (ROS) and autophagy is complex. Cellular ROS is predominantly derived from mitochondria. Autophagy is triggered by this event, and by clearing the defective organelles effectively, …

Integrated Transcriptomic Analysis Of Trichosporon Asahii Uncovers The Core Genes And Pathways Of Fluconazole Resistance., Haitao Li, Congmin Wang, Yong Chen, Shaoqiang Zhang, Rongya Yang

College of Science & Mathematics Departmental Research

Trichosporon asahii (T. asahii) has emerged as a dangerous pathogen that causes rare but life-threatening infections. Its resistance to certain antifungal agents makes it difficult to treat, especially for patients undergoing long-term antibiotic therapy. In this study, we performed a series of fluconazole (FLC) perturbation experiments for two T. asahii strains, a clinical isolate stain CBS 2479 (T2) and an environmental isolate strain CBS 8904 (T8), to uncover potential genes and pathways involved in FLC resistance. We achieved 10 transcriptomes of T2 and T8 that were based on dose and time series of FLC perturbations. Systematic comparisons of the transcriptomes …

Mechanistic Insights Into The Regulation Of Mitochondrial Fission By Cyclin C, Vidyaramanan Ganesan, Katrina F Cooper, Randy Strich

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Cyclin C is a component of the mediator complex of RNA polymerase II that localizes to the nucleus under normal conditions. In response to stress, cyclin C translocates to the cytosol and mitochondria and mediates stress‐induced mitochondrial fission and apoptosis. The molecular mechanisms by which cyclin C induces mitochondrial fission are unknown. Using in vitro experimental approaches, we sought to investigate the mechanistic basis of cyclin C mediated mitochondrial fission.

Mechanism Of Transcription Anti-Termination In Human Mitochondria., Hauke S Hillen, Andrey V Parshin, Karen Agaronyan, Yaroslav I Morozov, James J Graber, Aleksandar Chernev, Kathrin Schwinghammer, Henning Urlaub, Michael Anikin, Patrick Cramer, Dmitry Temiakov

Rowan-Virtua School of Osteopathic Medicine Departmental Research

In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream "sliding clamp," providing high processivity to the EC. …

In Situ Capture Of Chromatin Interactions By Biotinylated Dcas9., Xin Liu, Yuannyu Zhang, Yong Chen, Mushan Li, Feng Zhou, Kailong Li, Hui Cao, Min Ni, Yuxuan Liu, Zhimin Gu, Kathryn E Dickerson, Shiqi Xie, Gary C Hon, Zhenyu Xuan, Michael Q Zhang, Zhen Shao, Jian Xu

College of Science & Mathematics Departmental Research

Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin …

Molecular Mechanisms Of Dna Replication Initiation In Hpvs With Genetic Variations Leading To Cellular Carcinogenesis, Gulden Yilmaz

Graduate School of Biomedical Sciences Theses and Dissertations

Human papillomaviruses are a vast family of double-stranded DNA viruses containing non-carcinogenic and carcinogenic types, whose crucial differences remain unknown, except for the difference in the frequency of DNA replication. The human papillomavirus (HPV) E2 protein regulates the initiation of viral DNA replication and transcription. Its recognition and binding to four 12 bp palindromic sequences in the viral origin is essential for its function. Little is known about the DNA binding mechanism of the E2 protein found in HPV types that have low risk for oncogenicity (low-risk) as well as the roles of various elements of the individual binding sites. …