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Articles 1 - 30 of 888

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

A Chromosome Folding Intermediate At The Condensin-To-Cohesin Transition During Telophase, Kristin Abramo, Anne-Laure Valton, Sergey V. Venev, Hakan Ozadam, A. Nicole Fox, Job Dekker Jun 2019

A Chromosome Folding Intermediate At The Condensin-To-Cohesin Transition During Telophase, Kristin Abramo, Anne-Laure Valton, Sergey V. Venev, Hakan Ozadam, A. Nicole Fox, Job Dekker

University of Massachusetts Medical School Faculty Publications

Chromosome folding is extensively modulated as cells progress through the cell cycle. During mitosis, condensin complexes fold chromosomes in helically arranged nested loop arrays. In interphase, the cohesin complex generates loops that can be stalled at CTCF sites leading to positioned loops and topologically associating domains (TADs), while a separate process of compartmentalization drives the spatial segregation of active and inactive chromatin domains. We used synchronized cell cultures to determine how the mitotic chromosome conformation is transformed into the interphase state. Using Hi-C, chromatin binding assays, and immunofluorescence we show that by telophase condensin-mediated loops are lost and a transient ...


Ste5 Membrane Localization Allows Mapk Pathway Signaling In Trans Between Kinases On Separate Scaffold Molecules, Rachel E. Lamson, Matthew J. Winters, Peter M. Pryciak Jun 2019

Ste5 Membrane Localization Allows Mapk Pathway Signaling In Trans Between Kinases On Separate Scaffold Molecules, Rachel E. Lamson, Matthew J. Winters, Peter M. Pryciak

University of Massachusetts Medical School Faculty Publications

The MAP kinase cascade is a ubiquitous eukaryotic signaling module that can be controlled by a diverse group of scaffold proteins. In budding yeast, activation of the mating MAP kinase cascade involves regulated membrane recruitment of the archetypal scaffold protein Ste5. This event promotes activation of the first kinase, but it also enhances subsequent signal propagation through the remainder of the cascade. By studying this latter effect, we find that membrane recruitment promotes signaling in trans between kinases on separate Ste5 molecules. First, trans signaling requires all Ste5 domains that mediate membrane recruitment, including both protein-binding and membrane-binding domains. Second ...


Trim5alpha Restricts Flavivirus Replication By Targeting The Viral Protease For Proteasomal Degradation, Abhilash I. Chiramel, Nicholas R. Meyerson, Kristin L. Mcnally, Rebecca M. Broeckel, Vanessa R. Montoya, Omayra Mendez-Solis, Shelly J. Robertson, Gail L. Sturdevant, Kirk J. Lubick, Vinod Nair, Brian H. Youseff, Robin M. Ireland, Catharine M. Bosio, Kyusik Kim, Jeremy Luban, Vanessa M. Hirsch, R. Travis Taylor, Fadila Bouamr, Sara L. Sawyer, Sonja M. Best Jun 2019

Trim5alpha Restricts Flavivirus Replication By Targeting The Viral Protease For Proteasomal Degradation, Abhilash I. Chiramel, Nicholas R. Meyerson, Kristin L. Mcnally, Rebecca M. Broeckel, Vanessa R. Montoya, Omayra Mendez-Solis, Shelly J. Robertson, Gail L. Sturdevant, Kirk J. Lubick, Vinod Nair, Brian H. Youseff, Robin M. Ireland, Catharine M. Bosio, Kyusik Kim, Jeremy Luban, Vanessa M. Hirsch, R. Travis Taylor, Fadila Bouamr, Sara L. Sawyer, Sonja M. Best

Program in Molecular Medicine Publications and Presentations

Tripartite motif-containing protein 5alpha (TRIM5alpha) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5alpha is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we show that both human and rhesus macaque TRIM5alpha suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5alpha-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. TRIM5alpha suppresses replication by binding to the viral protease NS2B/3 to promote its K48-linked ubiquitination and ...


Smooth Muscle Cell-Specific Tmem16a Deletion Does Not Alter Ca2+ Signaling, Uterine Contraction, Gestation Length Or Litter Size In Micedagger, Mingzi Qu, Ping Lu, Karl D. Bellve, Kevin E. Fogarty, Lawrence M. Lifshitz, Fangxiong Shi, Ronghua Zhuge Jun 2019

Smooth Muscle Cell-Specific Tmem16a Deletion Does Not Alter Ca2+ Signaling, Uterine Contraction, Gestation Length Or Litter Size In Micedagger, Mingzi Qu, Ping Lu, Karl D. Bellve, Kevin E. Fogarty, Lawrence M. Lifshitz, Fangxiong Shi, Ronghua Zhuge

Program in Molecular Medicine Publications and Presentations

Ion channels in myometrial cells play critical roles in spontaneous and agonist-induced uterine contraction during the menstrual cycle, pregnancy maintenance and parturition; thus identifying the genes of ion channels in these cells and determining their roles are essential to understanding the biology of reproduction. Previous studies with in vitro functional and pharmacological approaches have produced controversial results regarding the presence and role of TMEM16A Ca2+-activated Cl- channels in myometrial cells. To unambiguously determine the function of this channel in these cells, we employed a genetic approach by using smooth muscle cell-specific TMEM16A deletion (i.e. TMEM16ASMKO) mice. We found ...


Yeast Sirtuin Family Members Maintain Transcription Homeostasis To Ensure Genome Stability, Jessica L. Feldman, Craig L. Peterson Jun 2019

Yeast Sirtuin Family Members Maintain Transcription Homeostasis To Ensure Genome Stability, Jessica L. Feldman, Craig L. Peterson

Program in Molecular Medicine Publications and Presentations

The mammalian sirtuin, SIRT6, is a key tumor suppressor that maintains genome stability and regulates transcription, though how SIRT6 family members control genome stability is unclear. Here, we use multiple genome-wide approaches to demonstrate that the yeast SIRT6 homologs, Hst3 and Hst4, prevent genome instability by tuning levels of both coding and noncoding transcription. While nascent RNAs are elevated in the absence of Hst3 and Hst4, a global impact on steady-state mRNAs is masked by the nuclear exosome, indicating that sirtuins and the exosome provide two levels of regulation to maintain transcription homeostasis. We find that, in the absence of ...


Adipocyte Acly Facilitates Dietary Carbohydrate Handling To Maintain Metabolic Homeostasis In Females, Sully Fernandez, John M. Viola, Annmarie Torres, Martina Wallace, Sophie Trefely, Steven Zhao, Hayley C. Affronti, Jivani M. Gengatharan, David A. Guertin, Nathaniel W. Snyder, Christian M. Metallo, Kathryn E. Wellen May 2019

Adipocyte Acly Facilitates Dietary Carbohydrate Handling To Maintain Metabolic Homeostasis In Females, Sully Fernandez, John M. Viola, Annmarie Torres, Martina Wallace, Sophie Trefely, Steven Zhao, Hayley C. Affronti, Jivani M. Gengatharan, David A. Guertin, Nathaniel W. Snyder, Christian M. Metallo, Kathryn E. Wellen

Open Access Articles

Sugars and refined carbohydrates are major components of the modern diet. ATP-citrate lyase (ACLY) is upregulated in adipocytes in response to carbohydrate consumption and generates acetyl-coenzyme A (CoA) for both lipid synthesis and acetylation reactions. Here, we investigate the role of ACLY in the metabolic and transcriptional responses to carbohydrates in adipocytes and unexpectedly uncover a sexually dimorphic function in maintaining systemic metabolic homeostasis. When fed a high-sucrose diet, Acly(FAT-/-) females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas Acly(FAT-/-) males have only mild metabolic phenotypes. We find that ACLY is ...


An Order-To-Disorder Structural Switch Activates The Foxm1 Transcription Factor, Aimee H. Marceau, Caileen M. Brison, Santrupti Nerli, Heather E. Arsenault, Andrew C. Mcshan, Eefei Chen, Hsiau-Wei Lee, Jennifer A. Benanti, Nikolaos G. Sgourakis, Seth M. Rubin May 2019

An Order-To-Disorder Structural Switch Activates The Foxm1 Transcription Factor, Aimee H. Marceau, Caileen M. Brison, Santrupti Nerli, Heather E. Arsenault, Andrew C. Mcshan, Eefei Chen, Hsiau-Wei Lee, Jennifer A. Benanti, Nikolaos G. Sgourakis, Seth M. Rubin

Open Access Articles

Intrinsically disordered transcription factor transactivation domains (TADs) function through structural plasticity, adopting ordered conformations when bound to transcriptional co-regulators. Many transcription factors contain a negative regulatory domain (NRD) that suppresses recruitment of transcriptional machinery through autoregulation of the TAD. We report the solution structure of an autoinhibited NRD-TAD complex within FoxM1, a critical activator of mitotic gene expression. We observe that while both the FoxM1 NRD and TAD are primarily intrinsically disordered domains, they associate and adopt a structured conformation. We identify how Plk1 and Cdk kinases cooperate to phosphorylate FoxM1, which releases the TAD into a disordered conformation that ...


Serum Deprivation Of Mesenchymal Stem Cells Improves Exosome Activity And Alters Lipid And Protein Composition, Reka A. Haraszti, Rachael Miller, Michelle L. Dubuke, Andrew H. Coles, Marie C. Didiot, Dimas Echeverria, Matteo Stoppato, Yves Y. Sere, John D. Leszyk, Julia F. Alterman, Bruno M. D. C. Godinho, Matthew R. Hassler, Rachel Wollacott, Yan Wang, Scott A. Shaffer, Neil Aronin, Anastasia Khvorova May 2019

Serum Deprivation Of Mesenchymal Stem Cells Improves Exosome Activity And Alters Lipid And Protein Composition, Reka A. Haraszti, Rachael Miller, Michelle L. Dubuke, Andrew H. Coles, Marie C. Didiot, Dimas Echeverria, Matteo Stoppato, Yves Y. Sere, John D. Leszyk, Julia F. Alterman, Bruno M. D. C. Godinho, Matthew R. Hassler, Rachel Wollacott, Yan Wang, Scott A. Shaffer, Neil Aronin, Anastasia Khvorova

Open Access Articles

Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome-mediated delivery of small interfering RNAs (siRNAs). This information is used to create effective artificial exosomes. We show that serum-deprived mesenchymal stem cells produce exosomes up to 22-fold more effective at delivering siRNAs to neurons than exosomes derived from control cells. Proteinase treatment of exosomes stops siRNA transfer, indicating that surface proteins on exosomes are involved in trafficking. Proteomic and lipidomic analyses show that exosomes derived in ...


Enhanced Cas12a Editing In Mammalian Cells And Zebrafish, Pengpeng Liu, Kevin Luk, Masahiro Shin, Feston Idrizi, Samantha F. Kwok, Benjamin P. Roscoe, Esther Mintzer, Sneha Suresh, Kyle Morrison, Josias B. Frazao, Mehmet Fatih Bolukbasi, Karthikeyan Ponnienselvan, Jeremy Luban, Lihua Julie Zhu, Nathan D. Lawson, Scot A. Wolfe May 2019

Enhanced Cas12a Editing In Mammalian Cells And Zebrafish, Pengpeng Liu, Kevin Luk, Masahiro Shin, Feston Idrizi, Samantha F. Kwok, Benjamin P. Roscoe, Esther Mintzer, Sneha Suresh, Kyle Morrison, Josias B. Frazao, Mehmet Fatih Bolukbasi, Karthikeyan Ponnienselvan, Jeremy Luban, Lihua Julie Zhu, Nathan D. Lawson, Scot A. Wolfe

Open Access Articles

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the ...


Brown Fat Organogenesis And Maintenance Requires Akt1 And Akt2, Joan Sanchez-Gurmaches, Camila Martinez Calejman, Su Myung Jung, Huawei Li, David A. Guertin May 2019

Brown Fat Organogenesis And Maintenance Requires Akt1 And Akt2, Joan Sanchez-Gurmaches, Camila Martinez Calejman, Su Myung Jung, Huawei Li, David A. Guertin

Open Access Articles

OBJECTIVE: Understanding the signaling mechanisms that control brown adipose tissue (BAT) development is relevant to understanding energy homeostasis and obesity. The AKT kinases are insulin effectors with critical in vivo functions in adipocytes; however, their role in adipocyte development remains poorly understood. The goal of this study was to investigate AKT function in BAT development.

METHODS: We conditionally deleted Akt1 and Akt2 either individually or together with Myf5-Cre, which targets early mesenchymal precursors that give rise to brown adipocytes. Because Myf5-Cre also targets skeletal muscle and some white adipocyte lineages, comparisons were made between AKT function in BAT versus white ...


Regulation Of The Drosophila Imd Pathway By Signaling Amyloids, Anni Kleino, Neal S. Silverman May 2019

Regulation Of The Drosophila Imd Pathway By Signaling Amyloids, Anni Kleino, Neal S. Silverman

Open Access Articles

Fruit flies elicit effective defense responses against numerous microbes. The responses against Gram-negative bacteria are mediated by the Imd pathway, an evolutionarily conserved NF-kappaB pathway recognizing meso-diaminopimelic acid (DAP)-type peptidoglycan from bacterial cell walls. Several reviews already provide a detailed view of ligand recognition and signal transduction during Imd signaling, but the formation and regulation of the signaling complex immediately downstream of the peptidoglycan-sensing receptors is still elusive. In this review, we focus on the formation of the Imd amyloidal signaling center and post-translational modifications in the assembly and disassembly of the Imd signaling complex.


Ouabain Enhances Cell-Cell Adhesion Mediated By Beta1 Subunits Of The Na(+),K(+)-Atpase In Cho Fibroblasts, Claudia Andrea Vilchis-Nestor, Maria Luisa Roldan, Angelina Leonardi, Juan G. Navea, Teresita Padilla-Benavides, Liora Shoshani Apr 2019

Ouabain Enhances Cell-Cell Adhesion Mediated By Beta1 Subunits Of The Na(+),K(+)-Atpase In Cho Fibroblasts, Claudia Andrea Vilchis-Nestor, Maria Luisa Roldan, Angelina Leonardi, Juan G. Navea, Teresita Padilla-Benavides, Liora Shoshani

Open Access Articles

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na(+),K(+)-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine beta1 subunit become adhesive, and those homotypic interactions amongst beta1 subunits of the Na(+),K(+)-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the alpha subunit of the Na(+),K(+)-ATPase, inhibits the pump activity and induces ...


Exercise Rescues Gene Pathways Involved In Vascular Expansion And Promotes Functional Angiogenesis In Subcutaneous White Adipose Tissue, So Yun Min, Heather Learnard, Shashi Kant, Olga Gaelikman, Raziel Rojas-Rodriguez, Tiffany Desouza, Anand Desai, John F. Keaney Jr., Silvia Corvera, Siobhan M. Craige Apr 2019

Exercise Rescues Gene Pathways Involved In Vascular Expansion And Promotes Functional Angiogenesis In Subcutaneous White Adipose Tissue, So Yun Min, Heather Learnard, Shashi Kant, Olga Gaelikman, Raziel Rojas-Rodriguez, Tiffany Desouza, Anand Desai, John F. Keaney Jr., Silvia Corvera, Siobhan M. Craige

Open Access Articles

Exercise mitigates chronic diseases such as diabetes, cardiovascular diseases, and obesity; however, the molecular mechanisms governing protection from these diseases are not completely understood. Here we demonstrate that exercise rescues metabolically compromised high fat diet (HFD) fed mice, and reprograms subcutaneous white adipose tissue (scWAT). Using transcriptomic profiling, scWAT was analyzed for HFD gene expression changes that were rescued by exercise. Gene networks involved in vascularization were identified as prominent targets of exercise, which led us to investigate the vasculature architecture and endothelial phenotype. Vascular density in scWAT was found to be compromised in HFD, and exercise rescued this defect ...


Crispr-Sonic: Targeted Somatic Oncogene Knock-In Enables Rapid In Vivo Cancer Modeling, Haiwei Mou, Deniz M. Ozata, Jordan L. Smith, Ankur Sheel, Suet-Yan Kwan, Soren Hough, Alper Kucukural, Zachary Kennedy, Yueying Cao, Wen Xue Apr 2019

Crispr-Sonic: Targeted Somatic Oncogene Knock-In Enables Rapid In Vivo Cancer Modeling, Haiwei Mou, Deniz M. Ozata, Jordan L. Smith, Ankur Sheel, Suet-Yan Kwan, Soren Hough, Alper Kucukural, Zachary Kennedy, Yueying Cao, Wen Xue

RNA Therapeutics Institute Publications

CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3'-UTR of mouse beta-actin, allowing co-expression of reporter genes or oncogenes from the beta-actin promoter. We showed that knock-in of oncogenic Ras and ...


The Nua4 Acetyltransferase And Histone H4 Acetylation Promote Replication Recovery After Topoisomerase I-Poisoning, Chiaki Noguchi, Tanu Singh, Melissa A. Ziegler, Jasmine D. Peake, Lyne Khair, Ana Aza, Toru M. Nakamura, Eishi Noguchi Apr 2019

The Nua4 Acetyltransferase And Histone H4 Acetylation Promote Replication Recovery After Topoisomerase I-Poisoning, Chiaki Noguchi, Tanu Singh, Melissa A. Ziegler, Jasmine D. Peake, Lyne Khair, Ana Aza, Toru M. Nakamura, Eishi Noguchi

Open Access Articles

BACKGROUND: Histone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe.

RESULTS: Downregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the ...


The Erk Mapk Pathway Is Essential For Skeletal Development And Homeostasis, Jung-Min Kim, Yeon-Suk Yang, Kwang Hwan Park, Hwanhee Oh, Matthew B. Greenblatt, Jae-Hyuck Shim Apr 2019

The Erk Mapk Pathway Is Essential For Skeletal Development And Homeostasis, Jung-Min Kim, Yeon-Suk Yang, Kwang Hwan Park, Hwanhee Oh, Matthew B. Greenblatt, Jae-Hyuck Shim

Open Access Articles

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that function as key signal transducers of a wide spectrum of extracellular stimuli, including growth factors and pro-inflammatory cytokines. Dysregulation of the extracellular signal-regulated kinase (ERK) MAPK pathway is associated with human skeletal abnormalities including Noonan syndrome, neurofibromatosis type 1, and cardiofaciocutaneous syndrome. Here, we demonstrate that ERK activation in osteoprogenitors is required for bone formation during skeletal development and homeostasis. Deletion of Mek1 and Mek2, kinases upstream of ERK MAPK, in osteoprogenitors (Mek1(Osx)Mek2(-/-)), resulted in severe osteopenia and cleidocranial dysplasia (CCD), similar to that seen in humans ...


The Er-Localized Protein Dfcp1 Modulates Er-Lipid Droplet Contact Formation, Dongfang Li, Yan G. Zhao, Di Li, Hongyu Zhao, Jie Huang, Guangyan Miao, Du Feng, Pingsheng Liu, Dong Li, Hong Zhang Apr 2019

The Er-Localized Protein Dfcp1 Modulates Er-Lipid Droplet Contact Formation, Dongfang Li, Yan G. Zhao, Di Li, Hongyu Zhao, Jie Huang, Guangyan Miao, Du Feng, Pingsheng Liu, Dong Li, Hong Zhang

Open Access Articles

Very little is known about the spatiotemporal generation of lipid droplets (LDs) from the endoplasmic reticulum (ER) and the factors that mediate ER-LD contacts for LD growth. Using super-resolution grazing incidence structured illumination microscopy (GI-SIM) live-cell imaging, we reveal that upon LD induction, the ER-localized protein DFCP1 redistributes to nascent puncta on the ER, whose formation depends on triglyceride synthesis. These structures move along the ER and fuse to form expanding LDs. Fusion and expansion of DFCP1-labeled nascent structures is controlled by BSCL2. BSCL2 depletion causes accumulation of nascent DFCP1 structures. DFCP1 overexpression increases LD size and enhances ER-LD contacts ...


Transient Kinetic Analysis Of Swr1c-Catalyzed H2a.Z Deposition Unravels The Impact Of Nucleosome Dynamics And The Asymmetry Of Histone Exchange, Raushan K. Singh, Jiayi Fan, Nathan Gioacchini, Shinya Watanabe, Osman Bilsel, Craig L. Peterson Apr 2019

Transient Kinetic Analysis Of Swr1c-Catalyzed H2a.Z Deposition Unravels The Impact Of Nucleosome Dynamics And The Asymmetry Of Histone Exchange, Raushan K. Singh, Jiayi Fan, Nathan Gioacchini, Shinya Watanabe, Osman Bilsel, Craig L. Peterson

Open Access Articles

The SWR1C chromatin remodeling enzyme catalyzes ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes such as transcription and DNA repair. Here, we investigate the transient kinetic mechanism of the histone exchange reaction, employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady-state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATP-dependent replacement of nucleosomal dimers, promoted ...


Transfer Rna Genes Affect Chromosome Structure And Function Via Local Effects, Omar Hamdani, Tsung-Han S. Hsieh, Oliver J. Rando, Rohinton T. Kamakaka Apr 2019

Transfer Rna Genes Affect Chromosome Structure And Function Via Local Effects, Omar Hamdani, Tsung-Han S. Hsieh, Oliver J. Rando, Rohinton T. Kamakaka

Open Access Articles

The genome is packaged and organized in an ordered, non-random manner and specific chromatin segments contact nuclear substructures to mediate this organization. Transfer RNA genes (tDNAs) are binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the role of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacks any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromatin architecture or chromosome tethering and mobility. However, loss of ...


Measuring The Reproducibility And Quality Of Hi-C Data, Galip Gurkan Yardimci, Hakan Ozadam, Bryan R. Lajoie, Ye Zhan, Job Dekker, William S. Noble Mar 2019

Measuring The Reproducibility And Quality Of Hi-C Data, Galip Gurkan Yardimci, Hakan Ozadam, Bryan R. Lajoie, Ye Zhan, Job Dekker, William S. Noble

Open Access Articles

BACKGROUND: Hi-C is currently the most widely used assay to investigate the 3D organization of the genome and to study its role in gene regulation, DNA replication, and disease. However, Hi-C experiments are costly to perform and involve multiple complex experimental steps; thus, accurate methods for measuring the quality and reproducibility of Hi-C data are essential to determine whether the output should be used further in a study.

RESULTS: Using real and simulated data, we profile the performance of several recently proposed methods for assessing reproducibility of population Hi-C data, including HiCRep, GenomeDISCO, HiC-Spector, and QuASAR-Rep. By explicitly controlling noise ...


Optimization Of Ribosome Profiling Using Low-Input Brain Tissue From Fragile X Syndrome Model Mice, Botao Liu, Gemma Molinaro, Huan Shu, Emily E. Stackpole, Kimberly M. Huber, Joel D. Richter Mar 2019

Optimization Of Ribosome Profiling Using Low-Input Brain Tissue From Fragile X Syndrome Model Mice, Botao Liu, Gemma Molinaro, Huan Shu, Emily E. Stackpole, Kimberly M. Huber, Joel D. Richter

Open Access Articles

Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases including fragile X syndrome. In order to capture subtle but biologically significant differences in translation in these disorders, a robust technique is required. One powerful tool to study translational control is ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease (RNase) digestion by ribosomes. However, this approach has been mainly applied to rapidly dividing cells where translation is active and large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and gene ...


Genome-Wide Prediction Of Synthetic Rescue Mediators Of Resistance To Targeted And Immunotherapy, Avinash Das Sahu, Olga Ponomarova, Eytan Ruppin Mar 2019

Genome-Wide Prediction Of Synthetic Rescue Mediators Of Resistance To Targeted And Immunotherapy, Avinash Das Sahu, Olga Ponomarova, Eytan Ruppin

Open Access Articles

Most patients with advanced cancer eventually acquire resistance to targeted therapies, spurring extensive efforts to identify molecular events mediating therapy resistance. Many of these events involve synthetic rescue (SR) interactions, where the reduction in cancer cell viability caused by targeted gene inactivation is rescued by an adaptive alteration of another gene (the rescuer). Here, we perform a genome-wide in silico prediction of SR rescuer genes by analyzing tumor transcriptomics and survival data of 10,000 TCGA cancer patients. Predicted SR interactions are validated in new experimental screens. We show that SR interactions can successfully predict cancer patients' response and emerging ...


Anti-Drug Antibody Responses Impair Prophylaxis Mediated By Aav-Delivered Hiv-1 Broadly Neutralizing Antibodies, Matthew R. Gardner, Ina Fetzer, Lisa M. Kattenhorn, Meredith E. Davis-Gardner, Amber S. Zhou, Barnett Alfant, Jesse A. Weber, Hema R. Kondur, Jose M. Martinez-Navio, Sebastian P. Fuchs, Ronald C. Desrosiers, Guangping Gao, Jeffrey D. Lifson, Michael Farzan Mar 2019

Anti-Drug Antibody Responses Impair Prophylaxis Mediated By Aav-Delivered Hiv-1 Broadly Neutralizing Antibodies, Matthew R. Gardner, Ina Fetzer, Lisa M. Kattenhorn, Meredith E. Davis-Gardner, Amber S. Zhou, Barnett Alfant, Jesse A. Weber, Hema R. Kondur, Jose M. Martinez-Navio, Sebastian P. Fuchs, Ronald C. Desrosiers, Guangping Gao, Jeffrey D. Lifson, Michael Farzan

Open Access Articles

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly ...


Mutations In The Glycosyltransferase Domain Of Glt8d1 Are Associated With Familial Amyotrophic Lateral Sclerosis, Johnathan Cooper-Knock, John E. Landers, Pamela J. Shaw Feb 2019

Mutations In The Glycosyltransferase Domain Of Glt8d1 Are Associated With Familial Amyotrophic Lateral Sclerosis, Johnathan Cooper-Knock, John E. Landers, Pamela J. Shaw

Open Access Articles

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder without effective neuroprotective therapy. Known genetic variants impair pathways, including RNA processing, axonal transport, and protein homeostasis. We report ALS-causing mutations within the gene encoding the glycosyltransferase GLT8D1. Exome sequencing in an autosomal-dominant ALS pedigree identified p.R92C mutations in GLT8D1, which co-segregate with disease. Sequencing of local and international cohorts demonstrated significant ALS association in the same exon, including additional rare deleterious mutations in conserved amino acids. Mutations are associated with the substrate binding site, and both R92C and G78W changes impair GLT8D1 enzyme activity. Mutated GLT8D1 exhibits in vitro ...


Diverse Lipid Conjugates For Functional Extra-Hepatic Sirna Delivery In Vivo, Annabelle Biscans, Andrew H. Coles, Reka A. Haraszti, Dimas Echeverria, Matthew R. Hassler, Maire F. Osborn, Anastasia Khvorova Feb 2019

Diverse Lipid Conjugates For Functional Extra-Hepatic Sirna Delivery In Vivo, Annabelle Biscans, Andrew H. Coles, Reka A. Haraszti, Dimas Echeverria, Matthew R. Hassler, Maire F. Osborn, Anastasia Khvorova

RNA Therapeutics Institute Publications

Small interfering RNA (siRNA)-based therapies are proving to be efficient for treating liver-associated disorders. However, extra-hepatic delivery remains challenging, limiting therapeutic siRNA utility. We synthesized a panel of fifteen lipid-conjugated siRNAs and systematically evaluated the impact of conjugate on siRNA tissue distribution and efficacy. Generally, conjugate hydrophobicity defines the degree of clearance and the liver-to-kidney distribution profile. In addition to primary clearance tissues, several conjugates achieve significant siRNA accumulation in muscle, lung, heart, adrenal glands and fat. Oligonucleotide distribution to extra-hepatic tissues with some conjugates was significantly higher than with cholesterol, a well studied conjugate, suggesting that altering conjugate ...


Hydrophobicity Drives The Systemic Distribution Of Lipid-Conjugated Sirnas Via Lipid Transport Pathways, Maire F. Osborn, Andrew H. Coles, Annabelle Biscans, Reka A. Haraszti, Loic Roux, Sarah M. Davis, Socheata Ly, Dimas Echeverria, Matthew R. Hassler, Bruno M. D. C. Godinho, Mehran Nikan, Anastasia Khvorova Feb 2019

Hydrophobicity Drives The Systemic Distribution Of Lipid-Conjugated Sirnas Via Lipid Transport Pathways, Maire F. Osborn, Andrew H. Coles, Annabelle Biscans, Reka A. Haraszti, Loic Roux, Sarah M. Davis, Socheata Ly, Dimas Echeverria, Matthew R. Hassler, Bruno M. D. C. Godinho, Mehran Nikan, Anastasia Khvorova

RNA Therapeutics Institute Publications

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in ...


Thioredoxin Modulates Protein Arginine Deiminase 4 (Pad4)-Catalyzed Citrullination, Mitesh Nagar, Ronak Tilvawala, Paul R. Thompson Feb 2019

Thioredoxin Modulates Protein Arginine Deiminase 4 (Pad4)-Catalyzed Citrullination, Mitesh Nagar, Ronak Tilvawala, Paul R. Thompson

Open Access Articles

Protein citrullination is a post-translational modification catalyzed by the protein arginine deiminases (PADs). This modification plays a crucial role in the pathophysiology of numerous autoimmune disorders including RA. Recently, there has been a growing interest in investigating physiological regulators of PAD activity to understand the primary cause of the associated disorders. Apart from calcium, it is well-documented that a reducing environment activates the PADs. Although the concentration of thioredoxin (hTRX), an oxidoreductase that maintains the cellular reducing environment, is elevated in RA patients, its contribution toward RA progression or PAD activity has not been explored. Herein, we demonstrate that hTRX ...


Rac1 Activity Is Modulated By Huntingtin And Dysregulated In Models Of Huntington's Disease, Adelaide Tousley, Anastasia Khvorova, Neil Aronin, Kimberly B. Kegel-Gleason Feb 2019

Rac1 Activity Is Modulated By Huntingtin And Dysregulated In Models Of Huntington's Disease, Adelaide Tousley, Anastasia Khvorova, Neil Aronin, Kimberly B. Kegel-Gleason

RNA Therapeutics Institute Publications

BACKGROUND: Previous studies suggest that Huntingtin, the protein mutated in Huntington's disease (HD), is required for actin based changes in cell morphology, and undergoes stimulus induced targeting to plasma membranes where it interacts with phospholipids involved in cell signaling. The small GTPase Rac1 is a downstream target of growth factor stimulation and PI 3-kinase activity and is critical for actin dependent membrane remodeling.

OBJECTIVE: To determine if Rac1 activity is impaired in HD or regulated by normal Huntingtin.

METHODS: Analyses were performed in differentiated control and HD human stem cells and HD Q140/Q140 knock-in mice. Biochemical methods included ...


Hypomorphic Mutations Of Trip11 Cause Odontochondrodysplasia, Anika Wehrle, John A. Follit, Gregory J. Pazour, Andrea Superti-Furga, Martin Lowe, Ekkehart Lausch Feb 2019

Hypomorphic Mutations Of Trip11 Cause Odontochondrodysplasia, Anika Wehrle, John A. Follit, Gregory J. Pazour, Andrea Superti-Furga, Martin Lowe, Ekkehart Lausch

Open Access Articles

Odontochondrodysplasia (ODCD) is an unresolved genetic disorder of skeletal and dental development. Here, we show that ODCD is caused by hypomorphic TRIP11 mutations, and we identify ODCD as the nonlethal counterpart to achondrogenesis 1A (ACG1A), the known null phenotype in humans. TRIP11 encodes Golgi-associated microtubule-binding protein 210 (GMAP-210), an essential tether protein of the Golgi apparatus that physically interacts with intraflagellar transport 20 (IFT20), a component of the ciliary intraflagellar transport complex B. This association and extraskeletal disease manifestations in ODCD point to a cilium-dependent pathogenesis. However, our functional studies in patient-derived primary cells clearly support a Golgi-based disease mechanism ...


Bridging From Intramuscular To Limb Perfusion Delivery Of Raav: Optimization In A Non-Human Primate Study, Alisha Gruntman, Gwladys Gernoux, Qiushi Tang, Guo-Jie Ye, Dave R. Knop, Gensheng Wang, Janet Benson, Kristen E. Coleman, Allison M. Keeler, Christian Mueller, Louis G. Chicoine, Jeffrey D. Chulay, Terence R. Flotte Feb 2019

Bridging From Intramuscular To Limb Perfusion Delivery Of Raav: Optimization In A Non-Human Primate Study, Alisha Gruntman, Gwladys Gernoux, Qiushi Tang, Guo-Jie Ye, Dave R. Knop, Gensheng Wang, Janet Benson, Kristen E. Coleman, Allison M. Keeler, Christian Mueller, Louis G. Chicoine, Jeffrey D. Chulay, Terence R. Flotte

Open Access Articles

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development ...