205 results on '"Paul A. Khavari"'
Search Results
2. Structural modularity of the XIST ribonucleoprotein complex
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Zhipeng Lu, Jimmy K. Guo, Yuning Wei, Diana R. Dou, Brian Zarnegar, Qing Ma, Rui Li, Yang Zhao, Fan Liu, Hani Choudhry, Paul A. Khavari, and Howard Y. Chang
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Science - Abstract
The long noncoding RNA XIST plays a central role in sex-specific gene expression in humans by silencing one of two X chromosomes in female cells. Here the authors show that higher order secondary structure creates the modular domain structure of XIST ribonucleoprotein complex and spatial separation of functions.
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- 2020
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3. Genome-wide meta-analysis identifies eight new susceptibility loci for cutaneous squamous cell carcinoma
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Kavita Y. Sarin, Yuan Lin, Roxana Daneshjou, Andrey Ziyatdinov, Gudmar Thorleifsson, Adam Rubin, Luba M. Pardo, Wenting Wu, Paul A. Khavari, Andre Uitterlinden, Tamar Nijsten, Amanda E. Toland, Jon H. Olafsson, Bardur Sigurgeirsson, Kristin Thorisdottir, Eric Jorgensen, Alice S. Whittemore, Peter Kraft, Simon N. Stacey, Kari Stefansson, Maryam M. Asgari, and Jiali Han
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Science - Abstract
The authors perform a meta-analysis of cutaneous squamous cell carcinoma, identifying causal variants within skin-specific regulatory elements.
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- 2020
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4. KRAS regulation by small non-coding RNAs and SNARE proteins
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Yonglu Che, Zurab Siprashvili, Joanna R. Kovalski, Tiffany Jiang, Glenn Wozniak, Lara Elcavage, and Paul A. Khavari
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Science - Abstract
Small nucleolar RNAs, SNORD50A/B are frequently deleted in cancers and can inhibit KRAS but by unclear means. Here, the authors show that KRAS trafficking and enrichment on the plasma membrane is mediated by SNARE vesicle transport proteins and that SNORD50A/B compete with these proteins to antagonise these effects.
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- 2019
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5. High-Efficiency Gene Transfer and Pharmacologic Selection of Genetically Engineered Human Keratinocytes
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Helen Deng, Keith A. Choate, Qun Lin, and Paul A. Khavari
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Biology (General) ,QH301-705.5 - Abstract
Low efficiencies of gene transfer to somatic cells have frustrated therapeutic gene delivery efforts in a wide array of tissues including the skin. Production of populations of keratinocytes in which all cells contain the desired therapeutic gene may be important in future genetic therapies. This may be the case in disorders such as epidermolysis bullosa and ichthyosis, where a failure to correct the vast majority of cells within tissue could perpetuate central disease features such as skin fragility and defective barrier function. We have refined retroviral gene transfer parameters to achieve significant improvements in gene delivery efficiencies to human keratinocytes compared to those previously reported. We have also generated retroviral vectors that allow rapid pharmacologic selection of human keratinocytes without interfering with the potential of these cells to regenerate epidermis in vivo—we determined that blasticidin is superior to the commonly used neomycin. The combined capabilities for efficient retroviral gene transfer and effective pharmacologic selection allow production of entirely engineered populations of human keratinocytes for use in future efforts to achieve effective cutaneous gene delivery.
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- 1998
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6. Targeted Proteomic Approaches for Proteome-Wide Characterizations of the AMP-Binding Capacities of Kinases
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Weili Miao, Jiekai Yin, Douglas F. Porter, Xiaogang Jiang, Paul A. Khavari, and Yinsheng Wang
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Proteomics ,Adenosine Triphosphate ,Proteome ,Tandem Mass Spectrometry ,General Chemistry ,AMP-Activated Protein Kinases ,Peptides ,Biochemistry ,Adenosine Monophosphate ,Chromatography, Liquid - Abstract
Kinases play important roles in cell signaling, and adenosine monophosphate (AMP) is known to modulate cellular energy homeostasis through AMP-activated protein kinase (AMPK). Here, we explored novel AMP-binding kinases by employing a desthiobiotin-conjugated AMP acyl-phosphate probe to enrich efficiently AMP-binding proteins. Together with a parallel-reaction monitoring-based targeted proteomic approach, we uncovered 195 candidate AMP-binding kinases. We also enriched desthiobiotin-labeled peptides from adenine nucleotide-binding sites of kinases and analyzed them using LC-MS/MS in the multiple-reaction monitoring mode, which resulted in the identification of 44 peptides derived from 43 kinases displaying comparable or better binding affinities toward AMP relative to adenosine triphosphate (ATP). Moreover, our proteomic data revealed a potential involvement of AMP in the MAPK pathway through binding directly to the relevant kinases, especially MEK2 and MEK3. Together, we revealed the AMP-binding capacities of a large number of kinases, and our work built a strong foundation for understanding how AMP functions as a second messenger to modulate cell signaling.
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- 2023
7. Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation
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Ava C Carter, Jin Xu, Meagan Y Nakamoto, Yuning Wei, Brian J Zarnegar, Quanming Shi, James P Broughton, Ryan C Ransom, Ankit Salhotra, Surya D Nagaraja, Rui Li, Diana R Dou, Kathryn E Yost, Seung-Woo Cho, Anil Mistry, Michael T Longaker, Paul A Khavari, Robert T Batey, Deborah S Wuttke, and Howard Y Chang
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Xist ,long noncoding RNA ,transposon ,chromatin ,epigenetic ,gene silencing ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.
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- 2020
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8. PROBER identifies proteins associated with programmable sequence-specific DNA in living cells
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Smarajit Mondal, Muthukumar Ramanathan, Weili Miao, Robin M. Meyers, Deepti Rao, Vanessa Lopez-Pajares, Zurab Siprashvili, David L. Reynolds, Douglas F. Porter, Ian Ferguson, Poornima Neela, Yang Zhao, Lindsey M. Meservey, Margaret Guo, Yen-Yu Yang, Lin Li, Yinsheng Wang, and Paul A. Khavari
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Biotinylation ,DNA ,Cell Biology ,Molecular Biology ,Biochemistry ,Chromatin ,Article ,Plasmids ,Transcription Factors ,Biotechnology - Abstract
DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.
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- 2022
9. Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site.
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Miguel Mata, Steven Neben, Karim Majzoub, Jan Carette, Muthukumar Ramanathan, Paul A Khavari, and Peter Sarnow
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5' end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.
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- 2019
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10. Supplementary Figures from Mek1 Alters Epidermal Growth and Differentiation
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Paul A. Khavari, Phillip A. Dumesic, and Florence A. Scholl
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Supplementary Figures from Mek1 Alters Epidermal Growth and Differentiation
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- 2023
11. Supplementary Information, Figure 1, Table 1 from A Laminin-Collagen Complex Drives Human Epidermal Carcinogenesis through Phosphoinositol-3-Kinase Activation
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M. Peter Marinkovich, Paul A. Khavari, Susana Ortiz-Urda, Clara N. Dey, Dallas P. Veitch, Basil A.J. Horst, Ngon T. Nguyen, Noriyasu Sakai, and Elizabeth A. Waterman
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Supplementary Information, Figure 1, Table 1 from A Laminin-Collagen Complex Drives Human Epidermal Carcinogenesis through Phosphoinositol-3-Kinase Activation
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- 2023
12. Supplementary Figures 1-6 from Tumor Necrosis Factor Receptor 1/c-Jun-NH2-Kinase Signaling Promotes Human Neoplasia
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Paul A. Khavari, Shiying Tao, Todd W. Ridky, Amy E. Adams, and Jennifer Y. Zhang
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Supplementary Figures 1-6 from Tumor Necrosis Factor Receptor 1/c-Jun-NH2-Kinase Signaling Promotes Human Neoplasia
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- 2023
13. Data from Tumor Necrosis Factor Receptor 1/c-Jun-NH2-Kinase Signaling Promotes Human Neoplasia
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Paul A. Khavari, Shiying Tao, Todd W. Ridky, Amy E. Adams, and Jennifer Y. Zhang
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The tumor necrosis factor α receptor (TNFR1) activates downstream effectors that include the mitogen-activated protein kinase kinase 7 (MKK7)/c-Jun-NH2-kinase (JNK)/activator protein 1 (AP1) cascade. Here, we report that JNK is activated in a majority of spontaneous human squamous cell carcinomas (SCC). JNK pathway induction bypassed cell cycle restraints induced by oncogenic Ras and cooperated with Ras to convert normal human epidermis into tumors indistinguishable from SCC, confirming its oncogenic potency in human tissue. Inhibiting MKK7, JNK, and AP1 as well as TNFR1 itself using genetic, pharmacologic, or antibody-mediated approaches abolished invasive human epidermal neoplasia in a tumor cell autonomous fashion. The TNFR1/MKK7/JNK/AP1 cascade thus promotes human neoplasia and represents a potential therapeutic target for human epithelial cancers. [Cancer Res 2007;67(8):3827–34]
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- 2023
14. Supplementary Figure Legends 1-6 from Tumor Necrosis Factor Receptor 1/c-Jun-NH2-Kinase Signaling Promotes Human Neoplasia
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Paul A. Khavari, Shiying Tao, Todd W. Ridky, Amy E. Adams, and Jennifer Y. Zhang
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Supplementary Figure Legends 1-6 from Tumor Necrosis Factor Receptor 1/c-Jun-NH2-Kinase Signaling Promotes Human Neoplasia
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- 2023
15. Legends to Supplementary Figures from Mek1 Alters Epidermal Growth and Differentiation
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Paul A. Khavari, Phillip A. Dumesic, and Florence A. Scholl
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Legends to Supplementary Figures from Mek1 Alters Epidermal Growth and Differentiation
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- 2023
16. Analyzing RNA‐Protein Interactions by Cross‐Link Rates and CLIP‐seq Libraries
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Douglas F. Porter, Raghav M. Garg, Robin M. Meyers, Weili Miao, Luca Ducoli, Brian J. Zarnegar, and Paul A. Khavari
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Medical Laboratory Technology ,General Immunology and Microbiology ,General Neuroscience ,Health Informatics ,General Pharmacology, Toxicology and Pharmaceutics ,General Biochemistry, Genetics and Molecular Biology - Published
- 2023
17. Glucose modulates transcription factor dimerization to enable tissue differentiation
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Vanessa Lopez-Pajares, Aparna Bhaduri, Yang Zhao, Gayatri Gowrishankar, Laura Donohue, Margaret G. Guo, Zurab Siprashvili, Weili Miao, Duy T. Nguyen, Albert M. Li, Ronald L. Shanderson, Robin M. Meyers, Angela Guerrero, Andrew L. Ji, Omar S. Garcia, Shiying Tao, Lindsey M. Meservey, Xue Yang, Sanjiv S. Gambhir, Jiangbin Ye, and Paul A. Khavari
- Abstract
Glucose is a universal energy currency for living organisms, however, its non-energetic functions in processes such as differentiation are undefined. In epidermis, differentiating cells exhibit dynamic changes in gene expression1–4driven by specific transcription factors (TFs)5–9. The interplay between such TFs and biomolecules that also change in this process is not understood. Metabolomic analyses revealed that increased intracellular glucose accompanies differentiation of epidermal keratinocytes. This elevation also occurred in differentiating cells from other tissues and was verified in epidermal tissue engineered with glucose sensors, which detected a glucose gradient that peaked in the outermost differentiated layers. Free glucose accumulation, unaccompanied by its increased metabolism, was essential for epidermal differentiation and required GLUT1, GLUT3, and SGLT1 transporters. Glucose affinity chromatography and azido-glucose click chemistry uncovered glucose binding to diverse regulatory proteins, including the IRF6 TF, whose epidermal knockout confirmed its requirement in glucose-dependent differentiation. Direct glucose binding enabled IRF6 dimerization, DNA binding, genomic localization, and induction of IRF6 target genes, including essential pro-differentiation TFsGRHL1, GRHL3, HOPXandPRDM1. The IRF6R84Cmutant found in undifferentiated cancers was unable to bind glucose. These data identify a new role for glucose as a gradient morphogen that modulates protein multimerization in cellular differentiation.
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- 2022
18. Mutant collagen COL11A1 enhances cancerous invasion
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Angela Mah, Yonglu Che, L. Elcavage, Tomas Bencomo, Zurab Siprashvili, Paul A. Khavari, Sumaira Z. Aasi, Rajani M Shenoy, and Carolyn S. Lee
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Cancer Research ,Skin Neoplasms ,Cell ,Mutant ,Biology ,Collagen Type XI ,medicine.disease_cause ,Protein Structure, Secondary ,Article ,Mice ,Stroma ,In vivo ,Exome Sequencing ,Genetics ,medicine ,Animals ,Humans ,Neoplasm Invasiveness ,Molecular Biology ,Gene ,Cancer ,Integrin beta1 ,medicine.disease ,Cell biology ,medicine.anatomical_structure ,Case-Control Studies ,Mutation ,Carcinoma, Squamous Cell ,Female ,Carcinogenesis ,Neoplasm Transplantation ,Function (biology) - Abstract
Collagens are the most abundant proteins in the body and comprise the basement membranes and stroma through which cancerous invasion occurs; however, a pro-neoplastic function for mutant collagens is undefined. Here we identify COL11A1 mutations in 66 of 100 cutaneous squamous cell carcinomas (cSCCs), the second most common U.S. cancer, concentrated in a triple helical region known to produce trans-dominant collagens. Analysis of COL11A1 and other collagen genes found that they are mutated across common epithelial malignancies. Knockout of mutant COL11A1 impairs cSCC tumorigenesis in vivo. Compared to otherwise genetically identical COL11A1 wild-type tissue, gene-edited mutant COL11A1 skin is characterized by induction of β1 integrin targets and accelerated neoplastic invasion. In mosaic tissue, mutant COL11A1 cells enhanced invasion by neighboring wild-type cells. These results suggest that specific collagens are commonly mutated in cancer and that mutant collagens may accelerate this process.
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- 2021
19. Spatially resolved clonal copy number alterations in benign and malignant tissue
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Andrew Erickson, Mengxiao He, Emelie Berglund, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Linda Kvastad, Alma Andersson, Ludvig Bergenstråhle, Joseph Bergenstråhle, Ludvig Larsson, Leire Alonso Galicia, Alia Shamikh, Elisa Basmaci, Teresita Díaz De Ståhl, Timothy Rajakumar, Dimitrios Doultsinos, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie C. Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundeberg, Research Program in Systems Oncology, HUSLAB, Department of Pathology, Helsinki University Hospital Area, University of Helsinki, and Digital Precision Cancer Medicine (iCAN)
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Male ,Spatial Analysis ,Multidisciplinary ,DNA Copy Number Variations ,MUTATIONS ,Genome, Human ,Prostate ,Prostatic Neoplasms ,Genomics ,GENE ,Models, Biological ,Genomic Instability ,PROSTATE-CANCER ,Clone Cells ,Neoplasms ,Humans ,3111 Biomedicine ,Transcriptome ,Early Detection of Cancer - Abstract
Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.
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- 2022
20. Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation
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Weili Miao, Douglas F. Porter, Vanessa Lopez-Pajares, Zurab Siprashvili, Robin M. Meyers, Yunhao Bai, Duy T. Nguyen, Lisa A. Ko, Brian J. Zarnegar, Ian D. Ferguson, Matthew M. Mills, Christie E. Jilly-Rehak, Cheng-Guo Wu, Yen-Yu Yang, Jordan M. Meyers, Audrey W. Hong, David L. Reynolds, Muthukumar Ramanathan, Shiying Tao, Sizun Jiang, Ryan A. Flynn, Yinsheng Wang, Garry P. Nolan, and Paul A. Khavari
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General Biochemistry, Genetics and Molecular Biology - Abstract
Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.
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- 2022
21. Super-resolved spatial transcriptomics by deep data fusion
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Alma Andersson, Paul A. Khavari, Guy E. Boeckxstaens, James Zou, Reza Mirzazadeh, Joakim Lundeberg, Joseph Bergenstråhle, Kim Thrane, Jonas Maaskola, Xesús Abalo, Bryan He, Ludvig Larsson, Ludvig Bergenstråhle, Nathalie Stakenborg, and Andrew L. Ji
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Science & Technology ,Computer science ,business.industry ,Low resolution ,Biomedical Engineering ,Bioengineering ,Pattern recognition ,Sensor fusion ,Applied Microbiology and Biotechnology ,Transcriptome ,Generative model ,Tissue sections ,SINGLE-CELL ,Biotechnology & Applied Microbiology ,TISSUE ,Molecular Medicine ,VISUALIZATION ,CELL RNA-SEQ ,Artificial intelligence ,business ,Image resolution ,Life Sciences & Biomedicine ,Biotechnology ,GENE-EXPRESSION - Abstract
Current methods for spatial transcriptomics are limited by low spatial resolution. Here we introduce a method that integrates spatial gene expression data with histological image data from the same tissue section to infer higher-resolution expression maps. Using a deep generative model, our method characterizes the transcriptome of micrometer-scale anatomical features and can predict spatial gene expression from histology images alone. ispartof: NATURE BIOTECHNOLOGY vol:40 issue:4 pages:476-+ ispartof: location:United States status: published
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- 2022
22. Modified forms of easyCLIP
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Douglas F Porter, Raghav M Garg, Robin M. Meyers, Weili Miao, Luca Ducoli, Brian J Zarnegar, and Paul A Khavari
- Abstract
The easyCLIP protocol describes a method for both normal CLIP library construction and the absolute quantification of RNA cross-linking rates, data which could be usefully combined to analyze RNA-protein interactions. Using these cross-linking metrics, significant interactions could be defined relative to a set of random non-RBPs. The original easyCLIP protocol did not use index reads, required custom sequencing primers, and did not have an easily reproducible analysis workflow. This short paper attempts to amend these deficiencies. It also includes some additional technical experiments and investigates the usage of alternative adapters. The results here are intended to allow more options to easily perform and analyze easyCLIP.
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- 2021
23. A cis-regulatory lexicon of DNA motif combinations mediating cell-type-specific gene regulation
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Laura K.H. Donohue, Margaret G. Guo, Yang Zhao, Namyoung Jung, Rose T. Bussat, Daniel S. Kim, Poornima H. Neela, Laura N. Kellman, Omar S. Garcia, Robin M. Meyers, Russ B. Altman, and Paul A. Khavari
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Genetics ,Biochemistry, Genetics and Molecular Biology (miscellaneous) - Published
- 2022
24. The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation
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Viviana I. Risca, Michael Snyder, Anshul Kundaje, Minyi Shi, Howard Y. Chang, Mahfuza Sharmin, Paul A. Khavari, Zhixin Zhao, William J. Greenleaf, Deepti Rao, Vanessa Lopez-Pajares, Arwa Kathiria, Namyoung Jung, Laura K. Donohue, James Chappell, Daniel S Kim, Harsh Deep, David Reynolds, Adam J. Rubin, and Shin Lin
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Keratinocytes ,Computational biology ,Biology ,Skin Diseases ,DNA sequencing ,Article ,Epigenome ,Genes, Reporter ,Gene expression ,Genetics ,Humans ,Regulatory Elements, Transcriptional ,Gene ,Transcription factor ,Regulation of gene expression ,Reporter gene ,Models, Genetic ,Cell Differentiation ,Chromatin ,Gene Expression Regulation ,Motif (music) ,Neural Networks, Computer ,Epidermis ,Genome-Wide Association Study ,Transcription Factors - Abstract
Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.
- Published
- 2021
25. The spatial landscape of clonal somatic mutations in benign and malignant tissue
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Teresita Díaz de Ståhl, Firaz Tarish, Tuomas Mirtti, Elisa Basmaci, Jonas Maaskola, Dan J. Woodcock, Timothy Rajakumar, Maja Marklund, Ludvig Larsson, Ludvig Bergenstråhle, Joakim Lundeberg, Linda Kvastad, Freddie C. Hamdy, Ian G. Mills, Thomas Helleday, Richard Colling, Alastair D. Lamb, Joseph Bergenstråhle, Andrew Erickson, Alma Andersson, Mengxiao He, Anna Tanoglidi, Reza Mirzazadeh, Kim Thrane, Andrew L. Ji, Emelie Berglund, Paul A. Khavari, Niklas Schultz, and Alia Shamikh
- Subjects
Genome instability ,Transcriptome ,Somatic cell ,Cancer evolution ,Spatially resolved ,medicine ,Cancer ,Context (language use) ,Copy-number variation ,Computational biology ,Biology ,medicine.disease ,3. Good health - Abstract
Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. Here, we provide an unsupervised approach to study spatial genome integrity in situ to describe previously unidentified clonal relationships. We employed spatially resolved transcriptomics to infer spatial copy number variations in >120 000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.
- Published
- 2021
26. Genetic and genomic studies of pathogenic EXOSC2 mutations in the newly described disease SHRF implicate the autophagy pathway in disease pathogenesis
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Tao Sun, Vanessa Lopez-Pajares, Andreas Rump, Shiying Tao, Paul A. Khavari, Brian J. Zarnegar, Chenjian Li, Nataliya DiDonato, Xue Yang, Vafa Bayat, Zurab Siprashvili, Bingwei Lu, and Yang Zhao
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Male ,0301 basic medicine ,Atg1 ,Exosome complex ,Mutation, Missense ,Dwarfism ,Biology ,Exosomes ,medicine.disease_cause ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Autophagy ,Genetics ,medicine ,Animals ,Humans ,CRISPR ,Hearing Loss ,Molecular Biology ,Genetics (clinical) ,Mutation ,Exosome Multienzyme Ribonuclease Complex ,fungi ,RNA-Binding Proteins ,Genomics ,Syndrome ,General Medicine ,Microphthalmia-associated transcription factor ,Cell biology ,General Article One ,Disease Models, Animal ,HEK293 Cells ,Phenotype ,030104 developmental biology ,Essential gene ,RNA ,Drosophila ,Female ,Retinitis Pigmentosa ,030217 neurology & neurosurgery - Abstract
Missense mutations in the RNA exosome component exosome component 2 (EXOSC2), also known as ribosomal RNA-processing protein 4 (RRP4), were recently identified in two unrelated families with a novel syndrome known as Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies (SHRF, #OMIM 617763). Little is known about the mechanism of the SHRF pathogenesis. Here we have studied the effect of mutations in EXOSC2/RRP4 in patient-derived lymphoblasts, clustered regularly interspaced short palindromic repeats (CRISPR)-generated mutant fetal keratinocytes and Drosophila. We determined that human EXOSC2 is an essential gene and that the pathogenic G198D mutation prevents binding to other RNA exosome components, resulting in protein and complex instability and altered expression and/or activities of critical genes, including those in the autophagy pathway. In parallel, we generated multiple CRISPR knockouts of the fly rrp4 gene. Using these flies, as well as rrp4 mutants with Piggy Bac (PBac) transposon insertion in the 3′UTR and RNAi flies, we determined that fly rrp4 was also essential, that fly rrp4 phenotypes could be rescued by wild-type human EXOSC2 but not the pathogenic form and that fly rrp4 is critical for eye development and maintenance, muscle ultrastructure and wing vein development. We found that overexpression of the transcription factor MITF was sufficient to rescue the small eye and adult lethal phenotypes caused by rrp4 inhibition. The autophagy genes ATG1 and ATG17, which are regulated by MITF, had similar effect. Pharmacological stimulation of autophagy with rapamycin also rescued the lethality caused by rrp4 inactivation. Our results implicate defective autophagy in SHRF pathogenesis and suggest therapeutic strategies.
- Published
- 2019
27. Ras functional proximity proteomics establishes mTORC2 as new direct ras effector
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Paul A. Khavari, Ronald L. Shanderson, and Joanna R. Kovalski
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0301 basic medicine ,Neuroblastoma RAS viral oncogene homolog ,Effector ,Biology ,medicine.disease_cause ,Proteomics ,mTORC2 ,Interactome ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,proteomics ,Oncology ,030220 oncology & carcinogenesis ,CRISPR ,Research Perspective ,medicine ,KRAS ,HRAS ,Kinase activity ,BioID ,Ras - Abstract
Although oncogenic mutations in the three major Ras isoforms, KRAS, HRAS and NRAS, are present in nearly a third of human cancers, therapeutic targeting of Ras remains a challenge due to its structure and complex regulation. However, an in-depth examination of the protein interactome of oncogenic Ras may provide new insights into key regulators, effectors and other mediators of its tumorigenic functions. Previous proteomic analyses have been limited by experimental tools that fail to capture the dynamic, transient nature of Ras cellular interactions. Therefore, in a recent study, we integrated proximity-dependent biotin labeling (BioID) proteomics with CRISPR screening of identified proteins to identify Ras proximal proteins required for Ras-dependent cancer cell growth. Oncogenic Ras was proximal to proteins involved in unexpected biological processes, such as vesicular trafficking and solute transport. Critically, we identified a direct, bona fide interaction between active Ras and the mTOR Complex 2 (mTORC2) that stimulated mTORC2 kinase activity. The oncogenic Ras-mTORC2 interaction resulted in a downstream pro-proliferative transcriptional program and promoted Ras-dependent tumor growth in vivo. Here we provide additional insight into the Ras isoform-specific protein interactomes, highlighting new opportunities for unique tumor-type therapies. Finally, we discuss the active Ras-mTORC2 interaction in detail, providing a more complete understanding of the direct relationship between Ras and mTORC2. Collectively, our findings support a model wherein Ras integrates an expanded array of pro-oncogenic signals to drive tumorigenic processes, including action on mTORC2 as a direct effector of Ras-driven proliferative signals.
- Published
- 2019
28. Profiling of rotavirus 3′UTR-binding proteins reveals the ATP synthase subunit ATP5B as a host factor that supports late-stage virus replication
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Manuel R. Amieva, Siyuan Ding, Harry B. Greenberg, Muthukumar Ramanathan, Lili Ren, Bin Li, Yanhua Song, Julia Co, and Paul A. Khavari
- Subjects
Rotavirus ,0301 basic medicine ,ATP5B ,RNA-binding protein ,Genome, Viral ,Microbiology ,Biochemistry ,Viral Proteins ,03 medical and health sciences ,Humans ,Viroplasm ,3' Untranslated Regions ,Molecular Biology ,030102 biochemistry & molecular biology ,ATP synthase ,biology ,Virus Assembly ,RNA-Binding Proteins ,RNA ,RNA virus ,Cell Biology ,Mitochondrial Proton-Translocating ATPases ,biology.organism_classification ,Cell biology ,HEK293 Cells ,030104 developmental biology ,Viral replication ,Virion assembly ,biology.protein ,RNA, Viral - Abstract
Genome replication and virion assembly of segmented RNA viruses are highly coordinated events, tightly regulated by sequence and structural elements in the UTRs of viral RNA. This process is poorly defined and likely requires the participation of host proteins in concert with viral proteins. In this study, we employed a proteomics-based approach, named RNA–protein interaction detection (RaPID), to comprehensively screen for host proteins that bind to a conserved motif within the rotavirus (RV) 3′ terminus. Using this assay, we identified ATP5B, a core subunit of the mitochondrial ATP synthase, as having high affinity to the RV 3′UTR consensus sequences. During RV infection, ATP5B bound to the RV 3′UTR and co-localized with viral RNA and viroplasm. Functionally, siRNA-mediated genetic depletion of ATP5B or other ATP synthase subunits such as ATP5A1 and ATP5O reduced the production of infectious viral progeny without significant alteration of intracellular viral RNA levels or RNA translation. Chemical inhibition of ATP synthase diminished RV yield in both conventional cell culture and in human intestinal enteroids, indicating that ATP5B positively regulates late-stage RV maturation in primary intestinal epithelial cells. Collectively, our results shed light on the role of host proteins in RV genome assembly and particle formation and identify ATP5B as a novel pro-RV RNA-binding protein, contributing to our understanding of how host ATP synthases may galvanize virus growth and pathogenesis.
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- 2019
29. Methods to study RNA–protein interactions
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Douglas F. Porter, Muthukumar Ramanathan, and Paul A. Khavari
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Proteomics ,0303 health sciences ,Messenger RNA ,Rna protein ,Sequence Analysis, RNA ,Ultraviolet Rays ,Sequence analysis ,RNA-Binding Proteins ,RNA ,A protein ,Cell Biology ,Plasma protein binding ,Computational biology ,Biology ,Non-coding RNA ,Biochemistry ,Article ,03 medical and health sciences ,Formaldehyde ,Molecular Biology ,Protein Binding ,030304 developmental biology ,Biotechnology - Abstract
Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNA-protein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications.
- Published
- 2019
30. Abstract 2171: The spatial landscape of clonal somatic mutations in benign and malignant tissue
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Andrew Erickson, Emelie Berglund, Mengxiao He, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Linda Kvastad, Alma Andersson, Ludvig Bergenstråhle, Joseph Bergenstråhle, Ludvig Larsson, Alia Shamikh, Elisa Basmaci, Teresita Diaz De Ståhl, Timothy Rajakumar, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, and Joakim Lundenberg
- Subjects
Cancer Research ,Oncology - Abstract
Introduction: Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. In order to obtain spatial information of clonal genetic events, prior studies have used methods such as laser capture microdissection, which results in assessment of small regions or even single cells. These studies have an inherent bias as a limited number of regions per tissue section can be retrieved and examined. Furthermore, because investigators have selected such regions based on morphology, previous studies have limited their analyses to histologically defined tumour areas while excluding regions populated by benign cells. The possibility to perform unsupervised genome and tissue-wide analysis would therefore provide an important contribution to delineate clonal events. We sought study spatial genome integrity in situ to gain molecular insight into clonal relationships. Materials and Methods: We employed spatially resolved transcriptomics (Visium, 10x Genomics) to infer spatial copy number variations in >120 000 spatial regions across multiple organs, including three whole axial prostates and additional tissues from skin, breast and brain tumors. We used this information to deduce clonal relationships between regions harboring 5-20 cells. Results: We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue. We perform an in-depth spatial analysis of cancers that includes an unprecedented interrogation of up to 50,000 tissue domains in a single patient, and 120,000 tissue domains across 10 patients. In a prostate section, we observed that many CNVs occurred in histologically benign luminal epithelial cells, most notably in chromosomes 8 and 10. This clone constituted a region of exclusively benign acinar cells branching off a duct lined by largely copy neutral cells. The changes in these cells were shared with the nearby intermediate risk prostate cancer cells in the same tissue section. We observed similar findings in another patient’s cutaneous squamous cell carcinoma (cSCC), wherein benign squamous epithelial had alterations in chromosomes 1 and 12 that were shared with nearby cSCC. Our results suggest a model for how genomic instability arises in histo-pathologically benign tissue that may represent early events in cancer evolution. Furthermore the spatial information allowed us to identify small clonal units not evident from morphology and hence would be overlooked by pathologists. Conclusions: We present the first large-scale, comprehensive atlas of genomic evolution at high spatial resolution in prostate cancer. Our study adds an important new approach to the armamentarium of cancer molecular pathology. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for focal therapy in prostate cancer. Citation Format: Andrew Erickson, Emelie Berglund, Mengxiao He, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Linda Kvastad, Alma Andersson, Ludvig Bergenstråhle, Joseph Bergenstråhle, Ludvig Larsson, Alia Shamikh, Elisa Basmaci, Teresita Diaz De Ståhl, Timothy Rajakumar, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundenberg. The spatial landscape of clonal somatic mutations in benign and malignant tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2171.
- Published
- 2022
31. Abstract PR016: The spatial landscape of clonal somatic mutations in benign and malignant tissue
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Andrew Erickson, Emelie Berglund, Mengxiao He, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Ludvig Bergenstråhle, Linda Kvastad, Alma Andersson, Joseph Bergenstråhle, Ludvig Larsson, Alia Shamikh, Elisa Basmaci, Teresita Diaz De Ståhl, Timothy Rajakumar, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie C. Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, and Joakim Lundeberg
- Subjects
Cancer Research ,Oncology - Abstract
Introduction: Defining the transition from benign to malignant tissue is fundamental to improve early diagnosis of cancer. In order to obtain spatial information of clonal genetic events, prior studies have used methods such as laser capture microdissection, which results in assessment of small regions or even single cells. These studies have an inherent bias as a limited number of regions per tissue section can be retrieved and examined. Furthermore, because investigators have selected such regions based on morphology, previous studies have limited their analyses to histologically defined tumor areas while excluding regions populated by benign cells. The possibility to perform unsupervised genome and tissue-wide analysis would therefore provide an important contribution to delineate clonal events. We sought study spatial genome integrity in situ to gain molecular insight into clonal relationships. Materials and Methods: We employed spatially resolved transcriptomics (Visium, 10x Genomics) to infer spatial copy number variations in >120 000 spatial regions across multiple organs, including three whole axial prostates and additional tissues from skin, breast and brain tumors. We additionally performed in silico assessment of spatial copy number inference. We used this information to deduce clonal relationships between regions harboring 5-20 cells. Results: We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumors and in nearby benign tissue. We perform an in-depth spatial analysis of cancers that includes an unprecedented interrogation of up to 50,000 tissue domains in a single patient, and 120,000 tissue domains across 10 patients. In a prostate section, we observed that many CNVs occurred in histologically benign luminal epithelial cells, most notably in chromosomes 8 and 10. This clone constituted a region of exclusively benign acinar cells branching off a duct lined by largely copy neutral cells. The changes in these cells were shared with the nearby intermediate risk prostate cancer cells in the same tissue section. We observed similar findings in another patient’s cutaneous squamous cell carcinoma (cSCC), wherein benign squamous epithelial had alterations in chromosomes 1 and 12 that were shared with nearby cSCC. Our results suggest a model for how genomic instability arises in histo-pathologically benign tissue that may represent early events in cancer evolution. Furthermore the spatial information allowed us to identify small clonal units not evident from morphology and hence would be overlooked by pathologists. Conclusions: We present the first large-scale, comprehensive atlas of genomic evolution at high spatial resolution in prostate cancer. Our study adds an important new approach to the armamentarium of cancer molecular pathology. We highlight the power of an unsupervised approach to capture the molecular and spatial continuums in a tissue context and challenge the rationale for focal therapy in prostate cancer Citation Format: Andrew Erickson, Emelie Berglund, Mengxiao He, Maja Marklund, Reza Mirzazadeh, Niklas Schultz, Ludvig Bergenstråhle, Linda Kvastad, Alma Andersson, Joseph Bergenstråhle, Ludvig Larsson, Alia Shamikh, Elisa Basmaci, Teresita Diaz De Ståhl, Timothy Rajakumar, Kim Thrane, Andrew L. Ji, Paul A. Khavari, Firaz Tarish, Anna Tanoglidi, Jonas Maaskola, Richard Colling, Tuomas Mirtti, Freddie C. Hamdy, Dan J. Woodcock, Thomas Helleday, Ian G. Mills, Alastair D. Lamb, Joakim Lundeberg. The spatial landscape of clonal somatic mutations in benign and malignant tissue [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR016.
- Published
- 2022
32. Quantitative analysis of mammalian translation initiation sites by FACS‐seq
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William L Noderer, Ross J Flockhart, Aparna Bhaduri, Alexander J Diaz de Arce, Jiajing Zhang, Paul A Khavari, and Clifford L Wang
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FACS‐seq ,Kozak motif ,proteome modeling ,start codon ,translation initiation ,Biology (General) ,QH301-705.5 ,Medicine (General) ,R5-920 - Abstract
Abstract An approach combining fluorescence‐activated cell sorting and high‐throughput DNA sequencing (FACS‐seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS‐seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the −6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome‐wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.
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- 2014
- Full Text
- View/download PDF
33. Retinoic acid and BMP4 cooperate with p63 to alter chromatin dynamics during surface epithelial commitment
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Maxwell R. Mumbach, Eric J. Liaw, Samantha N. Piekos, Gautam Shankar, Anthony E. Oro, Sandra P. Melo, Howard Y. Chang, Elizaveta Bashkirova, Paul A. Khavari, Charlotte M. Rajasingh, Hanson H. Zhen, Daniel Alber, Jillian M. Pattison, Lingjie Li, Jessica L. Torkelson, Xiaomin Bao, and Adam J. Rubin
- Subjects
Keratinocytes ,0301 basic medicine ,animal structures ,Retinoic acid ,Tretinoin ,Bone Morphogenetic Protein 4 ,Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Histone H3 ,0302 clinical medicine ,Genetics ,Humans ,Epigenetics ,Cells, Cultured ,Embryonic Stem Cells ,Regulation of gene expression ,Tumor Suppressor Proteins ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Chromatin Assembly and Disassembly ,Embryonic stem cell ,Chromatin ,3. Good health ,Cell biology ,030104 developmental biology ,chemistry ,Bone morphogenetic protein 4 ,embryonic structures ,sense organs ,Epidermis ,Stem cell ,030217 neurology & neurosurgery ,Signal Transduction ,Transcription Factors - Abstract
Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1–3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6–9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.
- Published
- 2018
34. Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics
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Sophia K Longo, Andrew L. Ji, Paul A. Khavari, and Margaret Guo
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Cell ,Computational biology ,In situ hybridization ,Cell Communication ,Biology ,Article ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology ,Spatial analysis ,Genetics (clinical) ,030304 developmental biology ,0303 health sciences ,Sequence Analysis, RNA ,RNA ,Computational Biology ,Cell subpopulations ,medicine.anatomical_structure ,Single-Cell Analysis ,030217 neurology & neurosurgery ,Software - Abstract
Single-cell RNA sequencing (scRNA-seq) identifies cell subpopulations within tissue but does not capture their spatial distribution nor reveal local networks of intercellular communication acting in situ. A suite of recently developed techniques that localize RNA within tissue, including multiplexed in situ hybridization and in situ sequencing (here defined as high-plex RNA imaging) and spatial barcoding, can help address this issue. However, no method currently provides as complete a scope of the transcriptome as does scRNA-seq, underscoring the need for approaches to integrate single-cell and spatial data. Here, we review efforts to integrate scRNA-seq with spatial transcriptomics, including emerging integrative computational methods, and propose ways to effectively combine current methodologies.
- Published
- 2021
35. easyCLIP analysis of RNA-protein interactions incorporating absolute quantification
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Paul A. Khavari, Weili Miao, Maria M. Aleman, Grant A. Goda, Xue Yang, Laura K. Donohue, Daniel Dominguez, Andrew L. Ji, and Douglas F. Porter
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0301 basic medicine ,Immunoprecipitation ,Ultraviolet Rays ,Absolute quantification ,Library preparation ,Science ,General Physics and Astronomy ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Humans ,Cancer mutations ,Binding site ,Cancer ,Multidisciplinary ,Rna protein ,Binding Sites ,RNA ,RNA-Binding Proteins ,RNA sequencing ,General Chemistry ,030104 developmental biology ,030220 oncology & carcinogenesis ,Recurrent Cancer - Abstract
Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association., It is important to develop quantitative criteria to identify RNA-binding proteins and target RNAs. Here, the authors present a method to quantify cross-linking and apply it to recurrent cancer mutations in RNA-binding proteins.
- Published
- 2021
36. The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation
- Author
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Yen-Yu Yang, Ronald L. Shanderson, Muthukumar Ramanathan, Jordan M. Meyers, Paul A. Khavari, Yinsheng Wang, Catherine A. Blish, David Reynolds, Aimee Beck, Deepti Rao, Yang Zhao, Margaret Guo, Ian D Ferguson, Weili Miao, Laura K. Donohue, and Xue Yang
- Subjects
RNA viruses ,Proteomics ,Viral Diseases ,Proteome ,Coronaviruses ,viruses ,Gene Expression ,Pathogenesis ,medicine.disease_cause ,Biochemistry ,Medical Conditions ,Interferon ,Protein biosynthesis ,Biology (General) ,Pathology and laboratory medicine ,virus diseases ,Translation (biology) ,Proteases ,Medical microbiology ,Enzymes ,Cell biology ,Infectious Diseases ,Viruses ,Host-Pathogen Interactions ,SARS CoV 2 ,Pathogens ,Translation initiation complex ,Research Article ,medicine.drug ,SARS coronavirus ,QH301-705.5 ,Viral protein ,Immunology ,Quantitative proteomics ,Biology ,Microbiology ,Article ,Protein–protein interaction ,Host-Parasite Interactions ,Viral Proteins ,Virology ,Genetics ,medicine ,Humans ,Protein Interactions ,Molecular Biology ,Medicine and health sciences ,Innate immune system ,Biology and life sciences ,SARS-CoV-2 ,Organisms ,Viral pathogens ,Proteins ,COVID-19 ,Covid 19 ,RC581-607 ,Viral Replication ,Microbial pathogens ,Protein Biosynthesis ,Enzymology ,Parasitology ,Protein Translation ,Immunologic diseases. Allergy - Abstract
Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2., Author summary SARS-CoV-2 is the latest pathogenic coronavirus to emerge as a public health threat. We create a database of proximal host proteins to 17 SARS-CoV-2 viral proteins. We validate that NSP1 is proximal to the EIF3 translation initiation complex and is a potent inhibitor of translation. We also identify ORF6 antagonism of RNA-mediate innate immune signaling. We produce a database of potential host targets of the viral protease NSP5, and create a fluorescence-based assay to screen cleavage of peptide sequences. We believe that this data will be useful for identifying roles for many of the uncharacterized SARS-CoV-2 proteins and provide insights into the pathogenicity of new or emerging coronaviruses.
- Published
- 2021
37. SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity
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Ian D Ferguson, Paul A. Khavari, Weili Miao, and Muthukumar Ramanathan
- Subjects
0301 basic medicine ,Genetics ,2019-20 coronavirus outbreak ,Coronavirus disease 2019 (COVID-19) ,SARS-CoV-2 ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,fungi ,COVID-19 ,Spike Protein ,Biology ,Article ,body regions ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Infectious Diseases ,Spike Glycoprotein, Coronavirus ,Correspondence ,Humans ,Spike (database) ,Angiotensin-Converting Enzyme 2 ,030212 general & internal medicine ,skin and connective tissue diseases ,hormones, hormone substitutes, and hormone antagonists - Abstract
SARS-CoV2 being highly infectious has been particularly effective in causing widespread infection globally and more variants of SARS-CoV2 are constantly being reported with increased genomic surveillance. In particular, the focus is on mutations of Spike protein, which binds human ACE2 protein enabling SARS-CoV2 entry and infection. Here we present a rapid experimental method leveraging the speed and flexibility of Mircoscale Thermopheresis (MST) to characterize the interaction between Spike Receptor Binding Domain (RBD) and human ACE2 protein. The B.1.351 variant harboring three mutations, (E484K, N501Y, and K417N) binds the ACE2 at nearly five-fold greater affinity than the original SARS-COV-2 RBD. We also find that the B.1.1.7 variant, binds two-fold more tightly to ACE2 than the SARS-COV-2 RBD.
- Published
- 2021
38. The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation
- Author
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Viviana I. Risca, Anshul Kundaje, Daniel Sunwook Kim, William J. Greenleaf, Harsh Deep, Michael Snyder, Minyi Shi, Paul A. Khavari, Arwa Kathiria, Laura K. Donohue, Howard Y. Chang, David Reynolds, James Chappell, Zhixin Zhao, Adam J. Rubin, and Namyoung Jung
- Subjects
Regulation of gene expression ,Gene expression ,Motif (music) ,Computational biology ,Biology ,Lexicon ,Gene ,Transcription factor ,DNA sequencing ,Chromatin - Abstract
Transcription factors (TFs) bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multi-omic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep learning framework to model the cis-regulatory logic of chromatin accessibility. This identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach reveals the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code in dynamic gene regulation.HIGHLIGHTSAn integrative multi-omic resource profiling chromatin and expression dynamics across keratinocyte differentiationPredictive deep learning models of chromatin dynamics reveal a high-resolution cis-regulatory DNA motif lexicon of epidermal differentiationModel interpretation enables discovery of combinatorial cis-regulatory logic of homotypic and heterotypic motif combinationsMassively parallel reporter experiments validate temporal dynamics and cis-regulatory logic of the combinatorial motif lexicon
- Published
- 2020
39. Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation
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Paul A. Khavari, James P. Broughton, Surya Nagaraja, Meagan Y Nakamoto, Michael T. Longaker, Howard Y. Chang, Diana R. Dou, Kathryn E. Yost, Jin Xu, Ankit Salhotra, Yuning Wei, Seung Woo Cho, Ryan C. Ransom, Ava C. Carter, Rui Li, Anil Mistry, Quanming Shi, Deborah S. Wuttke, Robert T. Batey, and Brian J. Zarnegar
- Subjects
0301 basic medicine ,Xist ,transposon ,Mouse ,gene silencing ,Mice ,0302 clinical medicine ,X Chromosome Inactivation ,Biology (General) ,Dosage compensation ,General Neuroscience ,RNA-Binding Proteins ,Mouse Embryonic Stem Cells ,General Medicine ,Chromosomes and Gene Expression ,Long non-coding RNA ,Chromatin ,Cell biology ,DNA-Binding Proteins ,Host-Pathogen Interactions ,Medicine ,RNA, Viral ,Female ,RNA, Long Noncoding ,epigenetic ,Research Article ,Protein Binding ,X Chromosome ,QH301-705.5 ,Science ,Chromatin silencing ,Biology ,General Biochemistry, Genetics and Molecular Biology ,X-inactivation ,Cell Line ,03 medical and health sciences ,Dosage Compensation, Genetic ,Gene silencing ,Animals ,long noncoding RNA ,Binding Sites ,General Immunology and Microbiology ,Endogenous Retroviruses ,RNA ,030104 developmental biology ,chromatin ,XIST ,030217 neurology & neurosurgery - Abstract
The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation., eLife digest The genetic material inside cells is often packaged into thread-like structures called chromosomes. In humans, mice and other mammals, a pair of sex chromosomes determines the genetic or chromosomal sex of each individual. Those who inherit two “X” chromosomes are said to be chromosomally female, while chromosomal males have one “X” and one “Y” chromosome. This means females have twice as many copies of genes on the X chromosome as a male does, which turns out to be double the number that the body needs. To solve this problem, mammals have developed a strategy known as dosage compensation. The second X chromosome in females becomes “silent”: its DNA remains unchanged, but none of the genes are active. A long noncoding RNA molecule called Xist is responsible for switching off the extra X genes in female cells. It does this by coating the entirety of the second X chromosome. Normally, RNA molecules transmit the coded instructions in genes to the cellular machinery that manufactures proteins. “Noncoding” RNAs like Xist, however, are RNAs that have taken on different jobs inside the cell. Researchers believe that the ancestral Xist gene may have once encoded a protein but changed over time to produce only a noncoding RNA. Carter, Xu et al. therefore set out to find out how exactly this might have happened, and also how Xist might have acquired its ability to switch genes off. Initial experiments used mouse cells grown in the laboratory, in which a protein called Spen was deleted. Spen is known to help Xist silence the X chromosome. In female cells lacking Spen, the second X chromosome remained active. Other chromosomes in male and female cells also had stretches of DNA that became active upon Spen’s removal. These DNA sequences, termed endogenous retroviruses, were remnants of ancestral viral infections. In other words, Spen normally acted as an antiviral defense. Analysis of genetic sequences showed that Spen recognized endogenous retrovirus sequences resembling a key region in Xist, a region which was needed for Xist to work properly. Inserting fragments of endogenous retroviruses into a defective version of Xist lacking this region also partially restored its ability to inactivate genes, suggesting that X chromosome silencing might work by hijacking cellular defenses against viruses. That is, female cells essentially ‘pretend’ there is a viral infection on the second X chromosome by coating it with Xist (which mimics endogenous retroviruses), thus directing Spen to shut it down. This research is an important step towards understanding how female cells carry out dosage compensation in mammals. More broadly, it sheds new light on how ancient viruses may have shaped the evolution of noncoding RNAs in the human genome.
- Published
- 2020
40. Author response: Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation
- Author
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James P. Broughton, Yuning Wei, Kathryn E. Yost, Brian J. Zarnegar, Paul A. Khavari, Surya Nagaraja, Quanming Shi, Anil Mistry, Deborah S. Wuttke, Ava C. Carter, Michael T. Longaker, Rui Li, Jin Xu, Ankit Salhotra, Howard Y. Chang, Ryan C. Ransom, Robert T. Batey, Meagan Y Nakamoto, Seung Woo Cho, and Diana R. Dou
- Subjects
Gene silencing ,Endogenous retrovirus ,RNA ,Biology ,X-inactivation ,Cell biology - Published
- 2020
41. Genome-wide meta-analysis identifies eight new susceptibility loci for cutaneous squamous cell carcinoma
- Author
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Gudmar Thorleifsson, Peter Kraft, Jón Ólafsson, André G. Uitterlinden, Paul A. Khavari, Eric Jorgensen, Andrey Ziyatdinov, Kavita Y. Sarin, Luba M. Pardo, Kristin Thorisdottir, Amanda E. Toland, Yuan Lin, Adam J. Rubin, Wenting Wu, Alice S. Whittemore, Simon N. Stacey, Roxana Daneshjou, Kari Stefansson, Maryam M. Asgari, Bardur Sigurgeirsson, Jiali Han, Tamar Nijsten, Dermatology, Internal Medicine, Læknadeild (HÍ), Faculty of Medicine (UI), Heilbrigðisvísindasvið (HÍ), School of Health Sciences (UI), Háskóli Íslands, and University of Iceland
- Subjects
0301 basic medicine ,Cutaneous squamous cell carcinoma ,Skin Neoplasms ,Science ,General Physics and Astronomy ,Gene Expression ,Genome-wide association study ,Single-nucleotide polymorphism ,Biology ,Genome ,Polymorphism, Single Nucleotide ,Genome-wide association studies ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Genetic Predisposition to Disease ,lcsh:Science ,Cancer genetics ,Genetic association ,Cancer ,Krabbamein ,Genetics ,Multidisciplinary ,Molecular Sequence Annotation ,General Chemistry ,Nucleotide polymorphisms ,medicine.disease ,3. Good health ,Genome-wide meta-analysis ,stomatognathic diseases ,030104 developmental biology ,Genetic Loci ,030220 oncology & carcinogenesis ,Meta-analysis ,Susceptibility locus ,Carcinoma, Squamous Cell ,lcsh:Q ,Erfðarannsóknir ,Genome-Wide Association Study - Abstract
Publisher's version (útgefin grein), Cutaneous squamous cell carcinoma (SCC) is one of the most common cancers in the United States. Previous genome-wide association studies (GWAS) have identified 14 single nucleotide polymorphisms (SNPs) associated with cutaneous SCC. Here, we report the largest cutaneous SCC meta-analysis to date, representing six international cohorts and totaling 19,149 SCC cases and 680,049 controls. We discover eight novel loci associated with SCC, confirm all previously associated loci, and perform fine mapping of causal variants. The novel SNPs occur within skin-specific regulatory elements and implicate loci involved in cancer development, immune regulation, and keratinocyte differentiation in SCC susceptibility. © 2020, The Author(s)., We thank the research participants and employees of 23andMe for contributing to this work. In addition, we thank the participants and staff of the Nurses’ Health Study and the Health Professionals Follow-up Study, for their valuable contributions, as well as the following state cancer registries for their help: A.L., A.Z., A.R., C.A., C.O., C.T., D.E., F.L., G.A., I.D., I.L., I.N., I.A., K.Y., L.A., M.E., M.D., M.A., M.I., N.E., N.H., N.J., N.Y., N.C., N.D., O.H., O.K., O.R., P.A., R.I., S.C., T.N., T.X., V.A., W.A. and W.Y. We assume full responsibility for analyses and interpretation of these data. EQTL data described in this paper were obtained from the GTEx Portal on 06/01/2018. We thank L. Tryggvadottir and G.H. Olafsdottir of the Icelandic Cancer Registry for assistance in the ascertainment of affected individuals. We thank D. Allain, V. Klee, and M. Bernhardt for ascertainment of cases and associated clinical data. The OSU Human Genetics Sample bank provided control samples. This work was supported in part by the National Human Genome Research Institute of the National Institutes of Health (grant number R44HG006981) and in part by NIH R01 CA49449, P01 CA87969, UM1 CA186107, UM1 CA167552, R03 CA219779, K23 CA211793 (K.Y.S.), and in part by Walther Cancer Foundation (J.H.). K.Y.S. is a Damon Runyon Clinical Investigator supported (in part) by the Damon Runyon Cancer Research Foundation. The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University Rotterdam; Netherlands Organization for the Health Research and Development (ZonMw); the Research Institute for Diseases in the Elderly (RIDE); the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam.
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- 2020
42. Structural modularity of the XIST ribonucleoprotein complex
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Fan Liu, Paul A. Khavari, Brian J. Zarnegar, Rui Li, Zhipeng Lu, Howard Y. Chang, Yuning Wei, Hani Choudhry, Yang Zhao, Qing Ma, Jimmy K. Guo, and Diana R. Dou
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0301 basic medicine ,Male ,Science ,Modularity (biology) ,General Physics and Astronomy ,Computational biology ,Plasma protein binding ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,Nucleic acid secondary structure ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Pregnancy ,Formaldehyde ,Gene expression ,Animals ,Humans ,Gene Knock-In Techniques ,Conserved Sequence ,X chromosome ,Ribonucleoprotein ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Base Sequence ,Sequence Analysis, RNA ,Effector ,Adenine ,030302 biochemistry & molecular biology ,Ficusin ,Mouse Embryonic Stem Cells ,General Chemistry ,030104 developmental biology ,Cross-Linking Reagents ,Ribonucleoproteins ,RNA ,Nucleic Acid Conformation ,Data integration ,Female ,RNA, Long Noncoding ,XIST ,CRISPR-Cas Systems ,K562 Cells ,Sequence motif ,030217 neurology & neurosurgery - Abstract
Long noncoding RNAs are thought to regulate gene expression by organizing protein complexes through unclear mechanisms. XIST controls the inactivation of an entire X chromosome in female placental mammals. Here we develop and integrate several orthogonal structure-interaction methods to demonstrate that XIST RNA-protein complex folds into an evolutionarily conserved modular architecture. Chimeric RNAs and clustered protein binding in fRIP and eCLIP experiments align with long-range RNA secondary structure, revealing discrete XIST domains that interact with distinct sets of effector proteins. CRISPR-Cas9-mediated permutation of the Xist A-repeat location shows that A-repeat serves as a nucleation center for multiple Xist-associated proteins and m6A modification. Thus modular architecture plays an essential role, in addition to sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and suggests a general strategy for mechanistic studies of large ribonucleoprotein assemblies., The long noncoding RNA XIST plays a central role in sex-specific gene expression in humans by silencing one of two X chromosomes in female cells. Here the authors show that higher order secondary structure creates the modular domain structure of XIST ribonucleoprotein complex and spatial separation of functions.
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- 2019
43. Cancer-Associated Long Noncoding RNA SMRT-2 Controls Epidermal Differentiation
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Paul A. Khavari, Cody J. Aros, Dan E. Webster, Angela Mah, Vanessa Lopez-Pajares, Carolyn S. Lee, Markus Kretz, and Aparna Bhaduri
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Keratinocytes ,0301 basic medicine ,Skin Neoplasms ,Sequence analysis ,Primary Cell Culture ,RNA-Seq ,Dermatology ,Computational biology ,Biology ,Biochemistry ,Article ,03 medical and health sciences ,medicine ,Gene Knockdown Techniques ,Humans ,RNA, Small Interfering ,Molecular Biology ,Cells, Cultured ,Regulation of gene expression ,Sequence Analysis, RNA ,Gene Expression Profiling ,Cancer ,RNA ,Cell Differentiation ,Cell Biology ,medicine.disease ,Long non-coding RNA ,Gene Expression Regulation, Neoplastic ,Gene expression profiling ,030104 developmental biology ,Epidermal Cells ,Carcinoma, Squamous Cell ,RNA, Long Noncoding ,Epidermis - Published
- 2018
44. Research Techniques Made Simple: Emerging Methods to Elucidate Protein Interactions through Spatial Proximity
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Paul A. Khavari and Yonglu Che
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Proteomics ,0301 basic medicine ,Biomedical Research ,Measure (physics) ,Dermatology ,Computational biology ,Biology ,Bioinformatics ,Biochemistry ,Biophysical Phenomena ,Protein–protein interaction ,03 medical and health sciences ,Low affinity ,Simple (abstract algebra) ,Protein Interaction Mapping ,Humans ,Biotinylation ,Molecular Biology ,Extramural ,Cell Membrane ,Proteins ,DNA ,Cell Biology ,Enzymes ,030104 developmental biology ,Research Design - Abstract
Interactions between proteins are essential for fundamental cellular processes, and the diversity of such interactions enables the vast variety of functions essential for life. A persistent goal in biological research is to develop assays that can faithfully capture different types of protein interactions to allow their study. A major step forward in this direction came with a family of methods that delineates spatial proximity of proteins as an indirect measure of protein-protein interaction. A variety of enzyme- and DNA ligation-based methods measure protein co-localization in space, capturing novel interactions that were previously too transient or low affinity to be identified. Here we review some of the methods that have been successfully used to measure spatially proximal protein-protein interactions.
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- 2017
45. The functions and unique features of long intergenic non-coding RNA
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Julia D. Ransohoff, Yuning Wei, and Paul A. Khavari
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Male ,0301 basic medicine ,RNA Stability ,Computational biology ,Biology ,ENCODE ,Genome ,Article ,Epigenesis, Genetic ,Evolution, Molecular ,Open Reading Frames ,03 medical and health sciences ,Intergenic region ,Transcriptional regulation ,Animals ,Humans ,RNA, Messenger ,Molecular Biology ,Gene ,Conserved Sequence ,Models, Genetic ,RNA ,Cell Biology ,Non-coding RNA ,Chromatin ,030104 developmental biology ,Gene Expression Regulation ,Organ Specificity ,Female ,RNA, Long Noncoding - Abstract
Long intergenic non-coding RNA (lincRNA) genes have diverse features that distinguish them from mRNA-encoding genes and exercise functions such as remodelling chromatin and genome architecture, RNA stabilization and transcription regulation, including enhancer-associated activity. Some genes currently annotated as encoding lincRNAs include small open reading frames (smORFs) and encode functional peptides and thus may be more properly classified as coding RNAs. lincRNAs may broadly serve to fine-tune the expression of neighbouring genes with remarkable tissue specificity through a diversity of mechanisms, highlighting our rapidly evolving understanding of the non-coding genome.
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- 2017
46. Rapid identification of non-human sequences in high-throughput sequencing datasets.
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Aparna Bhaduri, Kun Qu, Carolyn S. Lee, Alexander Ungewickell, and Paul A. Khavari
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- 2012
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47. 093 Dissecting intercellular communication in adult human skin with single-cell and spatial transcriptomics
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Andrew L. Ji, Margaret Guo, Daniel Sunwook Kim, Adam J. Rubin, Sumaira Z. Aasi, Joakim Lundeberg, Kim Thrane, T. Hollmig, and Paul A. Khavari
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Transcriptome ,medicine.anatomical_structure ,Cell ,medicine ,Human skin ,Cell Biology ,Dermatology ,Biology ,Molecular Biology ,Biochemistry ,Intracellular ,Cell biology - Published
- 2021
48. 132 Metabolomic identification of an essential glucose-IRF6 axis in differentiation
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Paul A. Khavari, Margaret Guo, Vanessa Lopez-Pajares, Yang Zhao, Gayatri Gowrishankar, Laura K. Donohue, Aparna Bhaduri, A. Guerrero, and Sanjiv S. Gambhir
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Metabolomics ,Identification (biology) ,Cell Biology ,Dermatology ,Computational biology ,Biology ,Molecular Biology ,Biochemistry - Published
- 2021
49. Novel lincRNA SLINKY is a prognostic biomarker in kidney cancer
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Seishi Ogawa, Yukio Homma, Paul A. Khavari, Xue Gong, Okyaz Eminaga, Jonathan R. Pollack, Zurab Siprashvili, James D. Brooks, Yusuke Sato, Haruki Kume, and Zhewei Shen
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Male ,0301 basic medicine ,Oncology ,Gerontology ,Kaplan-Meier Estimate ,Heterogeneous-Nuclear Ribonucleoprotein K ,Transcriptome ,Renal cell carcinoma ,Tumor stage ,HNRNPK ,kidney cancer ,Middle Aged ,Flow Cytometry ,Prognosis ,University hospital ,Kidney Neoplasms ,3. Good health ,Gene Expression Regulation, Neoplastic ,Decision curve analysis ,Area Under Curve ,lincRNA ,biomarker ,Biomarker (medicine) ,Female ,RNA, Long Noncoding ,SLINKY ,Adult ,renal cell carcinoma ,medicine.medical_specialty ,Protein Array Analysis ,News ,Sensitivity and Specificity ,Disease-Free Survival ,03 medical and health sciences ,Internal medicine ,Biomarkers, Tumor ,medicine ,Humans ,Immunoprecipitation ,Prognostic biomarker ,Carcinoma, Renal Cell ,Aged ,Cell Proliferation ,Proportional Hazards Models ,business.industry ,medicine.disease ,030104 developmental biology ,ROC Curve ,prognostication ,business ,prognostic ,Kidney cancer ,Priority Research Paper - Abstract
// Xue Gong 1,2 , Zurab Siprashvili 3 , Okyaz Eminaga 1,4 , Zhewei Shen 2 , Yusuke Sato 5,6 , Haruki Kume 6 , Yukio Homma 6 , Seishi Ogawa 5 , Paul A. Khavari 3 , Jonathan R. Pollack 2 and James D. Brooks 1 1 Department of Urology, School of Medicine, Stanford University, Stanford, California, USA 2 Department of Pathology, School of Medicine, Stanford University, Stanford, California, USA 3 Program in Epithelial Biology, School of Medicine, Stanford University, Stanford, California, USA 4 Department of Urology, University Hospital Cologne, Cologne, Germany 5 Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan 6 Department of Urology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Correspondence to: Jonathan R. Pollack, email: // James D. Brooks, email: // Keywords : lincRNA, renal cell carcinoma, kidney cancer, biomarker, prognostication Received : November 04, 2016 Accepted : January 24, 2017 Published : February 24, 2017 Abstract Clear cell renal cell carcinomas (ccRCC) show a broad range of clinical behavior, and prognostic biomarkers are needed to stratify patients for appropriate management. We sought to determine whether long intergenic non-coding RNAs (lincRNAs) might predict patient survival. Candidate prognostic lincRNAs were identified by mining The Cancer Genome Atlas (TCGA) transcriptome (RNA-seq) data on 466 ccRCC cases (randomized into discovery and validation sets) annotated for ~21,000 lncRNAs. A previously uncharacterized lincRNA, SLINKY (Survival-predictive LINcRNA in KidneY cancer), was the top-ranked prognostic lincRNA, and validated in an independent University of Tokyo cohort ( P =0.004). In multivariable analysis, SLINKY expression predicted overall survival independent of tumor stage and grade [TCGA HR=3.5 (CI, 2.2-5.7), P < 0.001; Tokyo HR=8.4 (CI, 1.8-40.2), P = 0.007], and by decision tree, ROC and decision curve analysis, added independent prognostic value. In ccRCC cell lines, SLINKY knockdown reduced cancer cell proliferation (with cell-cycle G 1 arrest) and induced transcriptome changes enriched for cell proliferation and survival processes. Notably, the genes affected by SLINKY knockdown in cell lines were themselves prognostic and correlated with SLINKY expression in the ccRCC patient samples. From a screen for binding partners, we identified direct binding of SLINKY to Heterogeneous Nuclear Ribonucleoprotein K (HNRNPK), whose knockdown recapitulated SLINKY knockdown phenotypes. Thus, SLINKY is a robust prognostic biomarker in ccRCC, where it functions possibly together with HNRNPK in cancer cell proliferation.
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- 2017
50. easyCLIP Quantifies RNA-Protein Interactions and Characterizes Recurrent PCBP1 Mutations in Cancer
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Paul A. Khavari and Douglas F. Porter
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0303 health sciences ,Rna protein ,Immunoprecipitation ,Cell ,RNA ,A protein ,Cancer ,Computational biology ,Biology ,medicine.disease ,Cellular protein ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,medicine ,030304 developmental biology - Abstract
RNA-protein interactions mediate a host of cellular processes, underscoring the need for methods to quantify their occurrence in living cells. RNA interaction frequencies for the average cellular protein are undefined, however, and there is no quantitative threshold to define a protein as an RNA-binding protein (RBP). Ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing, an effective and widely used means of characterizing RNA-protein interactions, would particularly benefit from the capacity to quantitate the number of RNA cross-links per protein per cell. In addition, CLIP-seq methods are difficult, have high experimental failure rates and many ambiguous analytical decisions. To address these issues, the easyCLIP method was developed and used to quantify RNA-protein interactions for a panel of known RBPs as well as a spectrum of random non-RBP proteins. easyCLIP provides the advantages of good efficiency compared to current standards, a simple protocol with a very low failure rate, troubleshooting information that includes direct visualization of prepared libraries without amplification, and a new form of analysis. easyCLIP, which uses sequential on-bead ligation of 5’ and 3’ adapters tagged with different infrared dyes, classified non-RBPs as those with a per protein RNA cross-link rate of L100 RBP mutant recurrently seen in cancer quantified increased RNA binding compared to wild-type PCBP1 and suggested a potential mechanism for this RBP mutant in cancer. easyCLIP provides a simple, efficient and robust method to both obtain both traditional CLIP-seq information and to define actual RNA interaction frequencies for a given protein, enabling quantitative cross-RBP comparisons as well as insight into RBP mechanisms.
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- 2019
- Full Text
- View/download PDF
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