HMGN proteins play roles in DNA repair and gene expression in mammalian cells

2004 ◽  
Vol 32 (6) ◽  
pp. 918-919 ◽  
Author(s):  
K.L. West
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Knockout Mice ◽  
Mammalian Cells ◽  
High Mobility ◽  
Genetically Engineered ◽  
Expression In Mammalian Cells ◽  
Transcription And Replication

HMGN (high-mobility-group N) family members are vertebrate proteins that unfold chromatin and promote transcription and replication of chromatin templates in vitro. However, their precise roles in vivo have been elusive until recently. This paper summarizes recent advances from studies of Hmgn1 knockout mice and genetically engineered cell lines that are beginning to reveal the diverse roles that HMGN proteins play in DNA repair and transcription within mammalian cells.

scholarly journals Indirect regulation of HMGB1 release by gasdermin D

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Allen Volchuk ◽  
Anna Ye ◽  
Leon Chi ◽  
Benjamin E. Steinberg ◽  
Neil M. Goldenberg
Keyword(s):  
Bone Marrow ◽  
Knockout Mice ◽  
Extracellular Space ◽  
High Mobility ◽  
High Mobility Group ◽  
Inflammasome Activation ◽  
Inflammatory Stimuli ◽  
Lps Treatment

Abstract The protein high-mobility group box 1 (HMGB1) is released into the extracellular space in response to many inflammatory stimuli, where it is a potent signaling molecule. Although research has focused on downstream HMGB1 signaling, the means by which HMGB1 exits the cell is controversial. Here we demonstrate that HMGB1 is not released from bone marrow-derived macrophages (BMDM) after lipopolysaccharide (LPS) treatment. We also explore whether HMGB1 is released via the pore-forming protein gasdermin D after inflammasome activation, as is the case for IL-1β. HMGB1 is only released under conditions that cause cell lysis (pyroptosis). When pyroptosis is prevented, HMGB1 is not released, despite inflammasome activation and IL-1β secretion. During endotoxemia, gasdermin D knockout mice secrete HMGB1 normally, yet secretion of IL-1β is completely blocked. Together, these data demonstrate that in vitro HMGB1 release after inflammasome activation occurs after cellular rupture, which is probably inflammasome-independent in vivo.

scholarly journals A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay

2000 ◽  
Vol 74 (5) ◽  
pp. 2372-2382 ◽  
Author(s):  
Toni Cathomen ◽  
Delphine Collete ◽  
Matthew D. Weitzman
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Target Site ◽  
Yeast Genome ◽  
Chimeric Proteins ◽  
Adeno Associated Virus ◽  
Amino Terminal ◽  
N Terminus

ABSTRACT The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and inSaccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.

scholarly journals How are base excision DNA repair pathways deployed in vivo?

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 279 ◽  
Author(s):  
Upasna Thapar ◽  
Bruce Demple
Keyword(s):  
Dna Repair ◽  
Base Excision Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Biochemical Level ◽  
Repair Pathways ◽  
Base Excision ◽  
Base Excision Dna Repair

Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them.

scholarly journals TbKAP6, a Mitochondrial HMG Box-Containing Protein in Trypanosoma brucei, Is the First Trypanosomatid Kinetoplast-Associated Protein Essential for Kinetoplast DNA Replication and Maintenance

2014 ◽  
Vol 13 (7) ◽  
pp. 919-932 ◽  
Author(s):  
Jianyang Wang ◽  
Valeria Pappas-Brown ◽  
Paul T. Englund ◽  
Robert E. Jensen
Keyword(s):  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
High Mobility ◽  
Kinetoplast Dna ◽  
Content Type ◽  
Hmg Box ◽  
Mitochondrial Nucleoids ◽  
Topo Ii

ABSTRACT Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, is a giant planar network of catenated minicircles and maxicircles. In vivo kDNA is organized as a highly condensed nucleoprotein disk. So far, in Trypanosoma brucei , proteins involved in the maintenance of the kDNA condensed structure remain poorly characterized. In Crithidia fasciculata , some small basic histone H1-like k inetoplast- a ssociated p roteins (CfKAP) have been shown to condense isolated kDNA networks in vitro . High-mobility group (HMG) box-containing proteins, such as mitochondrial transcription factor A (TFAM) in mammalian cells and Abf2 in the budding yeast, have been shown essential for the packaging of mitochondrial DNA (mtDNA) into mitochondrial nucleoids, remodeling of mitochondrial nucleoids, gene expression, and maintenance of mtDNA. Here, we report that TbKAP6, a mitochondrial HMG box-containing protein, is essential for parasite cell viability and involved in kDNA replication and maintenance. The RNA interference (RNAi) depletion of TbKAP6 stopped cell growth. Replication of both minicircles and maxicircles was inhibited. RNAi or overexpression of TbKAP6 resulted in the disorganization, shrinkage, and loss of kDNA. Minicircle release, the first step in kDNA replication, was inhibited immediately after induction of RNAi, but it quickly increased 3-fold upon overexpression of TbKAP6. Since the release of covalently closed minicircles is mediated by a type II topoisomerase (topo II), we examined the potential interactions between TbKAP6 and topo II. Recombinant TbKAP6 (rTbKAP6) promotes the topo II-mediated decatenation of kDNA. rTbKAP6 can condense isolated kDNA networks in vitro . These results indicate that TbKAP6 is involved in the replication and maintenance of kDNA.

scholarly journals Enzyme mechanism-based, oxidative DNA–protein cross-links formed with DNA polymerase β in vivo

2015 ◽  
Vol 112 (28) ◽  
pp. 8602-8607 ◽  
Author(s):  
Jason L. Quiñones ◽  
Upasna Thapar ◽  
Kefei Yu ◽  
Qingming Fang ◽  
Robert William Sobol ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Polymerase ◽  
Mammalian Cells ◽  
Proteasome Inhibition ◽  
Enzyme Mechanism ◽  
Dna Polymerase Β ◽  
Cross Links ◽  
Base Excision

Free radical attack on the C1′ position of DNA deoxyribose generates the oxidized abasic (AP) site 2-deoxyribonolactone (dL). Upon encountering dL, AP lyase enzymes such as DNA polymerase β (Polβ) form dead-end, covalent intermediates in vitro during attempted DNA repair. However, the conditions that lead to the in vivo formation of such DNA–protein cross-links (DPC), and their impact on cellular functions, have remained unknown. We adapted an immuno-slot blot approach to detect oxidative Polβ-DPC in vivo. Treatment of mammalian cells with genotoxic oxidants that generate dL in DNA led to the formation of Polβ-DPC in vivo. In a dose-dependent fashion, Polβ-DPC were detected in MDA-MB-231 human cells treated with the antitumor drug tirapazamine (TPZ; much more Polβ-DPC under 1% O2 than under 21% O2) and even more robustly with the “chemical nuclease” 1,10-copper-ortho-phenanthroline, Cu(OP)2. Mouse embryonic fibroblasts challenged with TPZ or Cu(OP)2 also incurred Polβ-DPC. Nonoxidative agents did not generate Polβ-DPC. The cross-linking in vivo was clearly a result of the base excision DNA repair pathway: oxidative Polβ-DPC depended on the Ape1 AP endonuclease, which generates the Polβ lyase substrate, and they required the essential lysine-72 in the Polβ lyase active site. Oxidative Polβ-DPC had an unexpectedly short half-life (∼30 min) in both human and mouse cells, and their removal was dependent on the proteasome. Proteasome inhibition under Cu(OP)2 treatment was significantly more cytotoxic to cells expressing wild-type Polβ than to cells with the lyase-defective form. That observation underscores the genotoxic potential of oxidative Polβ-DPC and the biological pressure to repair them.

scholarly journals Cas9-Assisted Biological Containment of a Genetically Engineered Human Commensal Bacterium and Genetic Elements

2021 ◽  
Author(s):  
Naoki Hayashi ◽  
Yong Lai ◽  
Mark Mimee ◽  
Timothy K Lu
Keyword(s):  
Gene Expression ◽  
Gene Cassette ◽  
Genetically Engineered ◽  
Commensal Bacterium ◽  
Human Gut ◽  
Genetic Elements ◽  
Thymidine Auxotrophy ◽  
Engineered Bacteria

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Biosensing bacteria can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination for at least 10 days. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.

scholarly journals Precise tuning of gene expression output levels in mammalian cells

2018 ◽  
Author(s):  
Yale S. Michaels ◽  
Mike B. Barnkob ◽  
Hector Barbosa ◽  
Toni A. Baeumler ◽  
Mary K. Thompson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
High Throughput Sequencing ◽  
Regulation Of Gene Expression ◽  
Antigen Expression ◽  
Control Of Gene Expression ◽  
Precise Control ◽  
Mouse Melanoma Model

ABSTRACTPrecise, analogue regulation of gene expression is critical for development, homeostasis and regeneration in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity, while RNA interference (RNAi) can lead to pervasive off-target effects and unpredictable levels of repression. Here we report on a method for the precise control of gene expression levels in mammalian cells based on engineered, synthetic microRNA response elements (MREs). To develop this system, we established a high-throughput sequencing approach for measuring the efficacy of thousands of miR-17 MRE variants. This allowed us to create a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to control the expression of user specified genes. To demonstrate the value of this technology, we used a panel of miSFITs to tune the expression of a peptide antigen in a mouse melanoma model. This analysis revealed that antigen expression level is a key determinant of the anti-tumour immune response in vitro and in vivo. miSFITs are a powerful tool for modulating gene expression output levels with applications in research and cellular engineering.

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