Genetic Processes Commons^™

A Substitution In Cgmp-Dependent Protein Kinase 1 Associated With Aortic Disease Induces An Active Conformation In The Absence Of Cgmp, Matthew H Chan, Sahar Aminzai, Tingfei Hu, Amatya Taran, Sheng Li, Choel Kim, Renate B Pilz, Darren E Casteel

Faculty, Staff and Students Publications

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and …

Aav-Crispr Gene Editing Is Negated By Pre-Existing Immunity To Cas9, Ang Li, Mark R Tanner, Ciaran M Lee, Ayrea E Hurley, Marco De Giorgi, Kelsey E Jarrett, Timothy H Davis, Alexandria M Doerfler, Gang Bao, Christine Beeton, William R Lagor

Faculty, Staff and Students Publications

Adeno-associated viral (AAV) vectors are a leading candidate for the delivery of CRISPR-Cas9 for therapeutic genome editing in vivo. However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR-Cas9. Although efficient genome editing occurred in mouse liver with pre-existing SaCas9 immunity, this was accompanied by …

Integrating Mouse And Human Genetic Data To Move Beyond Gwas And Identify Causal Genes In Cholesterol Metabolism, Zhonggang Li, James A Votava, Gregory J M Zajac, Jenny N Nguyen, Fernanda B Leyva Jaimes, Sophia M Ly, Jacqueline A Brinkman, Marco De Giorgi, Sushma Kaul, Cara L Green, Samantha L St Clair, Sabrina L Belisle, Julia M Rios, David W Nelson, Mary G Sorci-Thomas, William R Lagor, Dudley W Lamming, Chi-Liang Eric Yen, Brian W Parks

Faculty, Staff and Students Publications

Identifying the causal gene(s) that connects genetic variation to a phenotype is a challenging problem in genome-wide association studies (GWASs). Here, we develop a systematic approach that integrates mouse liver co-expression networks with human lipid GWAS data to identify regulators of cholesterol and lipid metabolism. Through our approach, we identified 48 genes showing replication in mice and associated with plasma lipid traits in humans and six genes on the X chromosome. Among these 54 genes, 25 have no previously identified role in lipid metabolism. Based on functional studies and integration with additional human lipid GWAS datasets, we pinpoint Sestrin1 as …

Provider Attitudes And Practice Patterns For Direct-Acting Antiviral Therapy For Patients With Hepatocellular Carcinoma, Nicole E Rich, Ju Dong Yang, Ponni V Perumalswami, Naim Alkhouri, Whitney Jackson, Neehar D Parikh, Neil Mehta, Reena Salgia, Andres Duarte-Rojo, Laura Kulik, Mina Rakoski, Adnan Said, Omobonike Oloruntoba, George N Ioannou, Maarouf A Hoteit, Andrew M Moon, Amol S Rangnekar, Sheila L Eswaran, Elizabeth Zheng, Janice H Jou, James Hanje, Anjana Pillai, Ruben Hernaez, Robert Wong, Steven Scaglione, Hrishikesh Samant, Devika Kapuria, Shaun Chandna, Russell Rosenblatt, Veeral Ajmera, Catherine T Frenette, Sanjaya K Satapathy, Parvez Mantry, Prasun Jalal, Binu V John, Oren K Fix, Michael Leise, Christina C Lindenmeyer, Avegail Flores, Nayan Patel, Z Gordon Jiang, Nyan Latt, Renumathy Dhanasekaran, Mobolaji Odewole, Sofia Kagan, Jorge A Marrero, Amit G Singal

Faculty, Staff and Students Publications

BACKGROUND & AIMS: Direct-acting antivirals (DAAs) are effective against hepatitis C virus and sustained virologic response is associated with reduced incidence of hepatocellular carcinoma (HCC). However, there is controversy over the use of DAAs in patients with active or treated HCC and uncertainty about optimal management of these patients. We aimed to characterize attitudes and practice patterns of hepatology practitioners in the United States regarding the use of DAAs in patients with HCC.

METHODS: We conducted a survey of hepatology providers at 47 tertiary care centers in 25 states. Surveys were sent to 476 providers and we received 279 responses …

The Sineb1 Element In The Long Non-Coding Rna Malat1 Is Necessary For Tdp-43 Proteostasis, Tuan M Nguyen, Elena B Kabotyanski, Lucas C Reineke, Jiaofang Shao, Feng Xiong, Joo-Hyung Lee, Julien Dubrulle, Hannah Johnson, Fabio Stossi, Phoebe S Tsoi, Kyoung-Jae Choi, Alexander G Ellis, Na Zhao, Jin Cao, Oluwatoyosi Adewunmi, Josephine C Ferreon, Allan Chris M Ferreon, Joel R Neilson, Michael A Mancini, Xi Chen, Jongchan Kim, Li Ma, Wenbo Li, Jeffrey M Rosen

Faculty, Staff and Students Publications

Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the …

Single-Nuclei Rna-Seq On Human Retinal Tissue Provides Improved Transcriptome Profiling, Qingnan Liang, Rachayata Dharmat, Leah Owen, Akbar Shakoor, Yumei Li, Sangbae Kim, Albert Vitale, Ivana Kim, Denise Morgan, Shaoheng Liang, Nathaniel Wu, Ken Chen, Margaret M Deangelis, Rui Chen

Faculty, Staff and Students Publications

Single-cell RNA-seq is a powerful tool in decoding the heterogeneity in complex tissues by generating transcriptomic profiles of the individual cell. Here, we report a single-nuclei RNA-seq (snRNA-seq) transcriptomic study on human retinal tissue, which is composed of multiple cell types with distinct functions. Six samples from three healthy donors are profiled and high-quality RNA-seq data is obtained for 5873 single nuclei. All major retinal cell types are observed and marker genes for each cell type are identified. The gene expression of the macular and peripheral retina is compared to each other at cell-type level. Furthermore, our dataset shows an …

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 …

Persistent Stress-Induced Neuroplastic Changes In The Locus Coeruleus/Norepinephrine System, Olga Borodovitsyna, Neal Joshi, Daniel Chandler

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Neural plasticity plays a critical role in mediating short- and long-term brain responses to environmental stimuli. A major effector of plasticity throughout many regions of the brain is stress. Activation of the locus coeruleus (LC) is a critical step in mediating the neuroendocrine and behavioral limbs of the stress response. During stressor exposure, activation of the hypothalamic-pituitary-adrenal axis promotes release of corticotropin-releasing factor in LC, where its signaling promotes a number of physiological and cellular changes. While the acute effects of stress on LC physiology have been described, its long-term effects are less clear. This review will describe how stress …

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. …

Assembly And Structure Of Human Mitochondrial Transcription Initiation Complex, Yaroslav Morozov

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondria play a key role in energy metabolism in nearly all eukaryotic cells. Apart from that, they participate in many other biochemical pathways, such as amino acid and nucleotide metabolism, and biosynthesis of cholesterol, heme, and iron-sulfur clusters. Mitochondria are also involved in cell signaling, apoptosis and regulation of cell cycle. Abnormalities of mitochondrial morphology and function are often associated with tumor progression, neurodegeneration and aging.

Mitochondria are unique organelles in mammalian cells because they have their own genomic DNA, transcription and translation systems. Various mutations and defects in mitochondrial gene expression lead to severe diseases that include various mitochondrial …

Cohort Of Birth Modifies The Association Between Fto Genotype And Bmi, James Niels Rosenquist, Steven F. Lehrer, A. James O'Malley, Alan M. Zaslavsky, Jordan W. Smoller, Nicholas A. Christakis

Dartmouth Scholarship

A substantial body of research has explored the relative roles of genetic and environmental factors on phenotype expression in humans. Recent research has also sought to identify gene-environment (or g-by-e) interactions, with mixed success. One potential reason for these mixed results may relate to the fact that genetic effects might be modified by changes in the environment over time. For example, the noted rise of obesity in the United States in the latter part of the 20th century might reflect an interaction between genetic variation and changing environmental conditions that together affect the penetrance of genetic influences. To evaluate this …

Acute Hypersensitivity Of Pluripotent Testicular Cancer-Derived Embryonal Carcinoma To Low-Dose 5-Aza Deoxycytidine Is Associated With Global Dna Damage-Associated P53 Activation, Anti-Pluripotency And Dna Demethylation, Bijesh K. Biswal, Maroun J. Beyrouthy, Mary P. Hever-Jardine, David Armstrong, Craig R. Tomlinson, Brock C. Christensen, Carmen J. Marsit, Michael J. Spinella

Dartmouth Scholarship

Human embryonal carcinoma (EC) cells are the stem cells of nonseminoma testicular germ cells tumors (TGCTs) and share remarkable similarities to human embryonic stem (ES) cells. In prior work we found that EC cells are hypersensitive to low nanomolar doses of 5-aza deoxycytidine (5-aza) and that this hypersensitivity partially depended on unusually high levels of the DNA methyltransferase, DNMT3B. We show here that low-dose 5-aza treatment results in DNA damage and induction of p53 in NT2/D1 cells. In addition, low-dose 5-aza results in global and gene specific promoter DNA hypomethylation. Low- dose 5-aza induces a p53 transcriptional signature distinct from …

Aging And Environmental Exposures Alter Tissue-Specific Dna Methylation Dependent Upon Cpg Island Context, Brock C. Christensen, E Andres Houseman, Carmen J. Marsit, Shichun Zheng, Margaret R. Wrensch, Joseph L. Wiemels, Heather H. Nelson, Margaret R. Karagas

Dartmouth Scholarship

Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variations in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1,413 autosomal CpG loci …

Retinoid X Receptor And Peroxisome Proliferator-Activated Receptor-Gamma Agonists Cooperate To Inhibit Matrix Metalloproteinase Gene Expression, Peter S. Burrage, Adam C. Schmucker, Yanqing Ren, Michael B. Sporn, Constance E. Brinckerhoff

Dartmouth Scholarship

We recently described the ability of retinoid X receptor (RXR) ligand LG100268 (LG268) to inhibit interleukin-1-beta (IL-1-β)-driven matrix metalloproteinase-1 (MMP-1) and MMP-13 gene expression in SW-1353 chondrosarcoma cells. Other investigators have demonstrated similar effects in chondrocytes treated with rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPARγ), for which RXR is an obligate dimerization partner. The goals of this study were to evaluate the inhibition of IL-1--induced expression of MMP-1andMMP-13 by combinatorial treatment with RXR and PPAR  ligands and to investigate the molecular mechanisms of this inhibition.

A Novel Runx2 Missense Mutation Predicted To Disrupt Dna Binding Causes Cleidocranial Dysplasia In A Large Chinese Family With Hyperplastic Nails, Shaohua Tang, Qiyu Xu, Xueqin Xu, Jicheng Du, Xuemei Yang, Yusheng Jiang, Xiaoqin Wang, Nancy Speck, Taosheng Huang

Dartmouth Scholarship

Background: Cleidocranial dysplasia (CCD) is a dominantly inherited disease characterized by hypoplastic or absent clavicles, large fontanels, dental dysplasia, and delayed skeletal development. The purpose of this study is to investigate the genetic basis of Chinese family with CCD.

Methods: Here, a large Chinese family with CCD and hyperplastic nails was recruited. The clinical features displayed a significant intrafamilial variation. We sequenced the coding region of the RUNX2 gene for the mutation and phenotype analysis.

Results: The family carries a c.T407C (p.L136P) mutation in the DNA- and CBFβ-binding Runt domain of RUNX2. Based on the crystal structure, we predict this …

Direct Inhibition Of Cdk9 Blocks Hiv-1 Replication Without Preventing T Cell Activation In Primary Human Peripheral Blood Lymphocytes, Dominic Salerno, Muneer G Hasham, Renee Marshall Demarest, Judit Garriga, Alexander Y Tsygankov, Xavier Graña

Rowan-Virtua School of Osteopathic Medicine Departmental Research

HIV-1 transcription is essential for the virus replication cycle. HIV-1 Tat is a viral transactivator that strongly stimulates the processivity of RNA polymerase II (RNAPII) via recruitment of the cyclin T1/CDK9 positive transcription elongation factor, which phosphorylates the C-terminal domain (CTD) of RNAPII. Consistently, HIV-1 replication in transformed cells is very sensitive to direct CDK9 inhibition. Thus, CDK9 could be a potential target for anti-HIV-1 therapy. A clearer understanding of the requirements for CDK9 activity in primary human T cells is needed to assess whether the CDK9-dependent step in HIV-1 transcription can be targeted clinically. We have investigated the effects …