Medicine and Health Sciences Commons^™

Extrachromosomal Dna (Ecdna): An Origin Of Tumor Heterogeneity, Genomic Remodeling, And Drug Resistance., Lauren T Pecorino, Roel G W Verhaak, Anton Henssen, Paul S Mischel

Faculty Research 2022

The genome of cancer cells contains circular extrachromosomal DNA (ecDNA) elements not found in normal cells. Analysis of clinical samples reveal they are common in most cancers and their presence indicates poor prognosis. They often contain enhancers and driver oncogenes that are highly expressed. The circular ecDNA topology leads to an open chromatin conformation and generates new gene regulatory interactions, including with distal enhancers. The absence of centromeres leads to random distribution of ecDNAs during cell division and genes encoded on them are transmitted in a non-mendelian manner. ecDNA can integrate into and exit from chromosomal DNA. The numbers of …

Detection Of Progression Or Regression Of Breast Cancer By Circulating Tumor Dna (Ctdna), Ujjwal Karki, Bipin Ghimire, Emma Herrman, Siddhartha Yadav, Mohammad Muhsin Chisti

Conference Presentation Abstracts

Circulating tumor DNA (ctDNA) are short DNA sequences shed by tumor cells into the systemic circulation. Studies have shown potential utility of the test to predict relapse or recurrence following treatment in solid tumors, but sensitivity and specificity have varied widely, ranging from 19-100% and 80-100% respectively, in breast cancer specifically. Moreover, literature describing the utility of monitoring dynamic changes in ctDNA trends is limited. We aim to evaluate the correlation between ctDNA test, both single test as well as dynamic trends in value over time, with imaging findings.

A Novel, Likely Pathogenic Variant In Ubtf-Related Neurodegeneration With Brain Atrophy Is Associated With A Severe Divergent Neurodevelopmental Phenotype, Rory J Tinker, Tiffany Guess, David C Rinker, Jonathan H Sheehan, Daniel Lubarsky, Binu Porath, Mackenzie Mosera, Ping Mayo, Emily Solem, Laura A Lee, Asha Sharam, Jennifer Brault

2020-Current year OA Pubs

BACKGROUND: A de novo, pathogenic, missense variant in UBTF, c.628G>A p.Glu210Lys, has been described as the cause of an emerging neurodegenerative disorder, Childhood-Onset Neurodegeneration with Brain Atrophy (CONDBA). The p.Glu210Lys alteration yields a positively charged stretch of three lysine residues. Functional studies confirmed this change results in a stronger interaction with negatively charged DNA and gain-of-function activity when compared to the wild-type sequence. The CONDBA phenotype reported in association with p.Glu210Lys consists of normal early-neurodevelopment followed by progressive motor, cognitive, and behavioral regression in early-to-middle childhood.

METHODS AND RESULTS: The current proband presented at 9 months of age with …

Lipid Surfaces And Glutamate Anions Enhance Formation Of Dynamic Biomolecular Condensates Containing Bacterial Cell Division Protein Ftsz And Its Dna-Bound Regulator Slma, Gianfranco Paccione, Miguel Á Robles-Ramos, Carlos Alfonso, Marta Sobrinos-Sanguino, William Margolin, Silvia Zorrilla, Begoña Monterroso, Germán Rivas

Journal Articles

Dynamic biomolecular condensates formed by liquid-liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the …

A Quantitative Metric Of Pioneer Activity Reveals That Hnf4a Has Stronger In Vivo Pioneer Activity Than Foxa1, Jeffrey L Hansen, Barak A Cohen

2020-Current year OA Pubs

BACKGROUND: We and others have suggested that pioneer activity - a transcription factor's (TF's) ability to bind and open inaccessible loci - is not a qualitative trait limited to a select class of pioneer TFs. We hypothesize that most TFs display pioneering activity that depends on the TF concentration and the motif content at their target loci.

RESULTS: Here, we present a quantitative in vivo measure of pioneer activity that captures the relative difference in a TF's ability to bind accessible versus inaccessible DNA. The metric is based on experiments that use CUT&Tag to measure the binding of doxycycline-inducible TFs. …

Overlapping And Distinct Functions Of The Paralogous Pagr Regulators Of Bacillus Anthracis, Ileana D Corsi, Theresa M Koehler

Journal Articles

The Bacillus anthracis pagA gene, encoding the protective antigen component of anthrax toxin, is part of a bicistronic operon on pXO1 that codes for its own repressor, PagR1. In addition to the pagAR1 operon, PagR1 regulates sap and eag, two chromosome genes encoding components of the surface layer, a mounting structure for surface proteins involved in virulence. Genomic studies have revealed a PagR1 paralog, PagR2, encoded by a gene on pXO2. The amino acid sequences of the paralogues are 71% identical and show similarity to the ArsR family of transcription regulators. We determined that the expression of either rPagR1 …

Integrated Dna Copy Number And Expression Profiling Identifies Igf1r As A Prognostic Biomarker In Pediatric Osteosarcoma., Aaron M Taylor, Jiayi M Sun, Alexander Yu, Horatiu Voicu, Jianhe Shen, Donald A Barkauskas, Timothy J Triche, Julie M Gastier-Foster, Tsz-Kwong Man, Ching C Lau

Faculty Research 2022

Osteosarcoma is a primary malignant bone tumor arising from bone-forming mesenchymal cells in children and adolescents. Despite efforts to understand the biology of the disease and identify novel therapeutics, the survival of osteosarcoma patients remains dismal. We have concurrently profiled the copy number and gene expression of 226 osteosarcoma samples as part of the Strategic Partnering to Evaluate Cancer Signatures (SPECS) initiative. Our results demonstrate the heterogeneous landscape of osteosarcoma in younger populations by showing the presence of genome-wide copy number abnormalities occurring both recurrently among samples and in a high frequency. Insulin growth factor receptor 1 (IGF1R) is a …

A Nickase Cas9 Gene-Drive System Promotes Super-Mendelian Inheritance In Drosophila, Víctor López Del Amo, Sara Sanz Juste, Valentino M Gantz

Journal Articles

CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that …

Nanog Prion-Like Assembly Mediates Dna Bridging To Facilitate Chromatin Reorganization And Activation Of Pluripotency, Kyoung-Jae Choi, My Diem Quan, Chuangye Qi, Joo-Hyung Lee, Phoebe S Tsoi, Mahla Zahabiyon, Aleksandar Bajic, Liya Hu, B V Venkataram Prasad, Shih-Chu Jeff Liao, Wenbo Li, Allan Chris M Ferreon, Josephine C Ferreon

Journal Articles

Human NANOG expression resets stem cells to ground-state pluripotency. Here we identify the unique features of human NANOG that relate to its dose-sensitive function as a master transcription factor. NANOG is largely disordered, with a C-terminal prion-like domain that phase-transitions to gel-like condensates. Full-length NANOG readily forms higher-order oligomers at low nanomolar concentrations, orders of magnitude lower than typical amyloids. Using single-molecule Förster resonance energy transfer and fluorescence cross-correlation techniques, we show that NANOG oligomerization is essential for bridging DNA elements in vitro. Using chromatin immunoprecipitation sequencing and Hi-C 3.0 in cells, we validate that NANOG prion-like domain assembly is …

Svanna: Efficient And Accurate Pathogenicity Prediction Of Coding And Regulatory Structural Variants In Long-Read Genome Sequencing., Daniel Danis, Julius O B Jacobsen, Parithi Balachandran, Qihui Zhu, Feyza Yilmaz, Justin Reese, Matthias Haimel, Gholson J Lyon, Ingo Helbig, Christopher J Mungall, Christine R Beck, Charles Lee, Damian Smedley, Peter N Robinson

Faculty Research 2022

Structural variants (SVs) are implicated in the etiology of Mendelian diseases but have been systematically underascertained owing to sequencing technology limitations. Long-read sequencing enables comprehensive detection of SVs, but approaches for prioritization of candidate SVs are needed. Structural variant Annotation and analysis (SvAnna) assesses all classes of SVs and their intersection with transcripts and regulatory sequences, relating predicted effects on gene function with clinical phenotype data. SvAnna places 87% of deleterious SVs in the top ten ranks. The interpretable prioritizations offered by SvAnna will facilitate the widespread adoption of long-read sequencing in diagnostic genomics. SvAnna is available at https://github.com/TheJacksonLaboratory/SvAnn a …

Efficient Targeted Transgenesis Of Large Donor Dna Into Multiple Mouse Genetic Backgrounds Using Bacteriophage Bxb1 Integrase., Benjamin E. Low, Vishnu Hosur, Simon Lesbirel, Michael V. Wiles

Faculty Research 2022

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free …

Mechanisms Of Mitochondrial Promoter Recognition In Humans And Other Mammalian Species, Angelica Zamudio-Ochoa, Yaroslav I Morozov, Azadeh Sarfallah, Michael Anikin, Dmitry Temiakov

Department of Biochemistry and Molecular Biology Faculty Papers

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for > 50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of …

Selective Elimination Of Pluripotent Stem Cells By Pikfyve Specific Inhibitors., Arup R Chakraborty, Alex Vassilev, Sushil K Jaiswal, Constandina E O'Connell, John F Ahrens, Barbara S Mallon, Martin Pera, Melvin L Depamphilis

Faculty Research 2022

Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their …

Mako: A Graph-Based Pattern Growth Approach To Detect Complex Structural Variants., Jiadong Lin, Xiaofei Yang, Walter Kosters, Tun Xu, Yanyan Jia, Songbo Wang, Qihui Zhu, Mallory Ryan, Li Guo, Chengsheng Zhang, The Human Genome Structural Variation Consortium, Charles Lee, Scott E Devine, Evan E Eichler, Kai Ye

Faculty Research 2022

Complex structural variants (CSVs) are genomic alterations that have more than two breakpoints and are considered as the simultaneous occurrence of simple structural variants. However, detecting the compounded mutational signals of CSVs is challenging through a commonly used model-match strategy. As a result, there has been limited progress for CSV discovery compared with simple structural variants. Here, we systematically analyzed the multi-breakpoint connection feature of CSVs, and proposed Mako, utilizing a bottom-up guided model-free strategy, to detect CSVs from paired-end short-read sequencing. Specifically, we implemented a graph-based pattern growth approach, where the graph depicts potential breakpoint connections, and pattern growth …

Utility Of Blood As The Clinical Specimen For The Diagnosis Of Ocular Toxoplasmosis Using Uracil Dna Glycosylase-Supplemented Loop-Mediated Isothermal Amplification And Real-Time Polymerase Chain Reaction Assays Based On Rep-529 Sequence And B1 Gene, Bahman Rahimi Esboei, Shirzad Fallahi, Mohammad Zarei, Bahram Kazemi, Mehdi Mohebali, Saeedeh Shojaee, Parisa Mousavi, Aref Teimouri, Raziyeh Mahmoudzadeh, Mirataollah Salabati, Hossein Keshavarz Valian

Wills Eye Hospital Papers

Background: Ocular infection with Toxoplasma gondii is a major preventable cause of blindness, especially in young people. The aim of the present study was to assess detection rate of T. gondii DNA in blood samples of clinically diagnosed of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) and real-time quantitative PCR (qPCR) based on REP-529 and B1.

Methods: One hundred and seventeen patients with clinically diagnosed ocular toxoplasmosis (OT) were participated in the study as well as 200 control patients. Peripheral blood samples were assessed using UDG-LAMP and qPCR techniques targeting REP-529 and B1.

Results: Detection limits of …

The Sequence Dependent Nanoscale Structure Of Cenp-A Nucleosomes, Tommy Stormberg, Yuri L. Lyubchenko

Journal Articles: Pharmaceutical Sciences

CENP-A is a histone variant found in high abundance at the centromere in humans. At the centromere, this histone variant replaces the histone H3 found throughout the bulk chromatin. Additionally, the centromere comprises tandem repeats of α-satellite DNA, which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here, we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an α-satellite motif and characterized their positioning and wrapping efficiency. We …

Nanorings To Probe Mechanical Stress Of Single-Stranded Dna Mediated By The Dna Duplex, Karen Zagorski, Tommy Stormberg, Mohtadin Hashemi, Anatoly B. Kolomeisky, Yuri L. Lyubchenko

Journal Articles: Pharmaceutical Sciences

The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic …

Crispr-Krispr: A Method To Identify On-Target And Random Insertion Of Donor Dnas And Their Characterization In Knock-In Mice, Masayuki Tanaka, Keiko Yokoyama, Hideki Hayashi, Sanae Isaki, Kanae Kitatani, Ting Wang, Hisako Kawata, Hideyuki Matsuzawa, Channabasavaiah B. Gurumurthy, Hiromi Miura, Masato Ohtsuka

Journal Articles: Pharmacology & Experimental Neuroscience

CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding.

Expression Variation Of Ogg1 And Hprt Gene And Dna Damage In Arsenic Exposed Industrial Workers, Zertashia Akram, Ishrat Mahjabeen, Muhammad Umair, Muhammad Fahim, Mahmood Akhter Kayani, Lulu Fatima, Malik Waqar Ahmad, Sarwat Jahan, Tayyaba Afsar, Ali Almajwal, Nawaf W. Alruwaili, Iftikhar Ali Khawar, Suhail Razak

2020-Current year OA Pubs

Arsenic exposure alters redox balance, induces DNA damage, and deregulates many genes. OGG1 gene involved in base repair mechanism, for excision of 8-oxoguanine (8-oxoG) from DNA formed as a result of accumulation of ROS in cell. HPRT gene encode transferase enzymes involved in purine recycling mechanism. The main focus of the study was to evaluate the expression variation in HPRT, OGG1 gene expression, and DNA damage of industrial workers. Blood samples of 300 occupational workers were collected from welding, brick kiln, furniture, pesticide, and paint industry (n = 60/industry) to evaluate the expression variation in HPRT, OGG1 gene expression, and …

Detection Of Progression Or Regression Of Gynecologic Cancers By Circulating Tumor Dna (Ctdna), Bipin Ghimire, Ujjwal Karki, Emma Herrman, Mohammad Muhsin Chisti

Conference Presentation Abstracts

Objectives The use of post-operative circulating tumor DNA (ctDNA) to detect cancer recurrence has been reported in various studies but the literature describing variable changes in ctDNA is limited. The objective of this study is to describe the utility of single and serial ctDNA values in detecting the progression or regression of gynecological cancers.

Methods This is a retrospective observational study including nineteen patients, aged >=18 years who had the ctDNA test completed at hematology/oncology clinic of William Beaumont – Royal Oak and Troy Hospitals, Michigan, USA.

Results Among the nineteen patients, fifteen had breast, three had ovarian, and one …