33,308 results on '"Chromatin immunoprecipitation"'
Search Results
2. A network of transcription factors in complex with a regulating cell cycle cyclin orchestrates fungal oxidative stress responses
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Yanze Kan, Zhangjiang He, Nemat O. Keyhani, Ning Li, Shuaishuai Huang, Xin Zhao, Pengfei Liu, Fanqin Zeng, Min Li, Zhibing Luo, and Yongjun Zhang
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Transcription factor ,Protein complex ,Oxidative stress response ,Fungal pathogen ,Chromatin immunoprecipitation ,Gene regulation ,Biology (General) ,QH301-705.5 - Abstract
Abstract Background Response to oxidative stress is universal in almost all organisms and the mitochondrial membrane protein, BbOhmm, negatively affects oxidative stress responses and virulence in the insect fungal pathogen, Beauveria bassiana. Nothing further, however, is known concerning how BbOhmm and this phenomenon is regulated. Results Three oxidative stress response regulating Zn2Cys6 transcription factors (BbOsrR1, 2, and 3) were identified and verified via chromatin immunoprecipitation (ChIP)-qPCR analysis as binding to the BbOhmm promoter region, with BbOsrR2 showing the strongest binding. Targeted gene knockout of BbOsrR1 or BbOsrR3 led to decreased BbOhmm expression and consequently increased tolerances to free radical generating compounds (H2O2 and menadione), whereas the ΔBbOsrR2 strain showed increased BbOhmm expression with concomitant decreased tolerances to these compounds. RNA and ChIP sequencing analysis revealed that BbOsrR1 directly regulated a wide range of antioxidation and transcription-associated genes, negatively affecting the expression of the BbClp1 cyclin and BbOsrR2. BbClp1 was shown to localize to the cell nucleus and negatively mediate oxidative stress responses. BbOsrR2 and BbOsrR3 were shown to feed into the Fus3-MAPK pathway in addition to regulating antioxidation and detoxification genes. Binding motifs for the three transcription factors were found to partially overlap in the promoter region of BbOhmm and other target genes. Whereas BbOsrR1 appeared to function independently, co-immunoprecipitation revealed complex formation between BbClp1, BbOsrR2, and BbOsrR3, with BbClp1 partially regulating BbOsrR2 phosphorylation. Conclusions These findings reveal a regulatory network mediated by BbOsrR1 and the formation of a BbClp1-BbOsrR2-BbOsrR3 complex that orchestrates fungal oxidative stress responses.
- Published
- 2024
- Full Text
- View/download PDF
3. A network of transcription factors in complex with a regulating cell cycle cyclin orchestrates fungal oxidative stress responses.
- Author
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Kan, Yanze, He, Zhangjiang, Keyhani, Nemat O., Li, Ning, Huang, Shuaishuai, Zhao, Xin, Liu, Pengfei, Zeng, Fanqin, Li, Min, Luo, Zhibing, and Zhang, Yongjun
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TRANSCRIPTION factors , *OXIDATIVE stress , *CELL cycle , *GENE expression , *CYCLINS - Abstract
Background: Response to oxidative stress is universal in almost all organisms and the mitochondrial membrane protein, BbOhmm, negatively affects oxidative stress responses and virulence in the insect fungal pathogen, Beauveria bassiana. Nothing further, however, is known concerning how BbOhmm and this phenomenon is regulated. Results: Three oxidative stress response regulating Zn2Cys6 transcription factors (BbOsrR1, 2, and 3) were identified and verified via chromatin immunoprecipitation (ChIP)-qPCR analysis as binding to the BbOhmm promoter region, with BbOsrR2 showing the strongest binding. Targeted gene knockout of BbOsrR1 or BbOsrR3 led to decreased BbOhmm expression and consequently increased tolerances to free radical generating compounds (H2O2 and menadione), whereas the ΔBbOsrR2 strain showed increased BbOhmm expression with concomitant decreased tolerances to these compounds. RNA and ChIP sequencing analysis revealed that BbOsrR1 directly regulated a wide range of antioxidation and transcription-associated genes, negatively affecting the expression of the BbClp1 cyclin and BbOsrR2. BbClp1 was shown to localize to the cell nucleus and negatively mediate oxidative stress responses. BbOsrR2 and BbOsrR3 were shown to feed into the Fus3-MAPK pathway in addition to regulating antioxidation and detoxification genes. Binding motifs for the three transcription factors were found to partially overlap in the promoter region of BbOhmm and other target genes. Whereas BbOsrR1 appeared to function independently, co-immunoprecipitation revealed complex formation between BbClp1, BbOsrR2, and BbOsrR3, with BbClp1 partially regulating BbOsrR2 phosphorylation. Conclusions: These findings reveal a regulatory network mediated by BbOsrR1 and the formation of a BbClp1-BbOsrR2-BbOsrR3 complex that orchestrates fungal oxidative stress responses. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
4. Nescient helix-loop-helix 1 (Nhlh1) is a novel activating transcription factor 5 (ATF5) target gene in olfactory and vomeronasal sensory neurons in mice.
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Ishii, Chiharu, Nakano, Haruo, Higashiseto, Riko, Ooki, Yusaku, Umemura, Mariko, Takahashi, Shigeru, and Takahashi, Yuji
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TRANSCRIPTION factors , *OLFACTORY receptors , *SENSORY neurons , *CHIMERIC proteins , *GENE expression , *VOMERONASAL organ , *HOMEOBOX proteins - Abstract
Activating transcription factor 5 (ATF5) is a transcription factor that belongs to the cAMP-response element-binding protein/ATF family and is essential for the differentiation and survival of sensory neurons in mouse olfactory organs. However, transcriptional target genes for ATF5 have yet to be identified. In the present study, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) experiments were performed to verify ATF5 target genes in the main olfactory epithelium and vomeronasal organ in the postnatal pups. ChIP-qPCR was conducted using hemagglutinin (HA)-tagged ATF5 knock-in olfactory organs. The results obtained demonstrated that ATF5-HA fusion proteins bound to the CCAAT/enhancer-binding protein-ATF response element (CARE) site in the enhancer region of nescient helix-loop-helix 1 (Nhlh1), a transcription factor expressed in differentiating olfactory and vomeronasal sensory neurons. Nhlh1 mRNA expression was downregulated in ATF5-deficient (ATF5−/−) olfactory organs. The LIM/homeobox protein transcription factor Lhx2 co-localized with ATF5 in the nuclei of olfactory and vomeronasal sensory neurons and bound to the homeodomain site proximal to the CARE site in the Nhlh1 gene. The CARE region of the Nhlh1 gene was enriched by the active enhancer marker, acetyl-histone H3 (Lys27). The present study identified Nhlh1 as a novel target gene for ATF5 in murine olfactory organs. ATF5 may upregulate Nhlh1 expression in concert with Lhx2, thereby promoting the differentiation of olfactory and vomeronasal sensory neurons. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
5. Protocol for establishing inducible CRISPRd system for blocking transcription factor-binding sites in human pluripotent stem cells
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Satoshi Matsui, Joseph R. Shiley, Morgan Buckley, Hee-Woong Lim, Yueh-Chiang Hu, Christopher N. Mayhew, and Makiko Iwafuchi
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Cell Biology ,Cell culture ,Cell isolation ,Flow Cytometry ,Molecular Biology ,Chromatin immunoprecipitation ,Science (General) ,Q1-390 - Abstract
Summary: Transcription factor (TF) gene knockout or knockdown experiments provide comprehensive downstream effects on gene regulation. However, distinguishing primary direct effects from secondary effects remains challenging. To assess the direct effect of TF binding events, we present a protocol for establishing a doxycycline (Dox)-inducible CRISPRd system in human pluripotent stem cells (hPSCs). We describe the steps for establishing CRISPRd host hPSCs, designing and preparing single-guide RNA (sgRNA) expression lentivirus vectors, generating CRISPRd hPSCs transduced with sgRNAs, and analyzing CRISPRd TF-block effects by chromatin immunoprecipitation (ChIP)-qPCR.For complete details on the use and execution of this protocol, please refer to Matsui et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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- 2024
- Full Text
- View/download PDF
6. A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin
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Janith A. Seneviratne, William W. H. Ho, Eleanor Glancy, and Melanie A. Eckersley-Maslin
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Bivalent chromatin ,Embryonic stem cells ,Plasticity ,Bivalency ,Chromatin immunoprecipitation ,Epigenetics ,Genetics ,QH426-470 - Abstract
Abstract Background Bivalent chromatin is an exemplar of epigenetic plasticity. This co-occurrence of active-associated H3K4me3 and inactive-associated H3K27me3 histone modifications on opposite tails of the same nucleosome occurs predominantly at promoters that are poised for future transcriptional upregulation or terminal silencing. We know little of the dynamics, resolution, and regulation of this chromatin state outside of embryonic stem cells where it was first described. This is partly due to the technical challenges distinguishing bone-fide bivalent chromatin, where both marks are on the same nucleosome, from allelic or sample heterogeneity where there is a mix of H3K4me3-only and H3K27me3-only mononucleosomes. Results Here, we present a robust and sensitive method to accurately map bivalent chromatin genome-wide, along with controls, from as little as 2 million cells. We optimized and refined the sequential ChIP protocol which uses two sequential overnight immunoprecipitation reactions to robustly purify nucleosomes that are truly bivalent and contain both H3K4me3 and H3K27me3 modifications. Our method generates high quality genome-wide maps with strong peak enrichment and low background, which can be analyzed using standard bioinformatic packages. Using this method, we detect 8,789 bivalent regions in mouse embryonic stem cells corresponding to 3,918 predominantly CpG rich and developmentally regulated gene promoters. Furthermore, profiling Dppa2/4 knockout mouse embryonic stem cells, which lose both H3K4me3 and H3K27me3 at approximately 10% of bivalent promoters, demonstrated the ability of our method to capture bivalent chromatin dynamics. Conclusions Our optimized sequential reChIP method enables high-resolution genome-wide assessment of bivalent chromatin together with all required controls in as little as 2 million cells. We share a detailed protocol and guidelines that will enable bivalent chromatin landscapes to be generated in a range of cellular contexts, greatly enhancing our understanding of bivalent chromatin and epigenetic plasticity beyond embryonic stem cells.
- Published
- 2024
- Full Text
- View/download PDF
7. Generation of an enhancer-driven gene expression viral tool specific to dentate granule cell-types through direct hippocampal injection.
- Author
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Potenza, Maria Letizia, Blankvoort, Stefan, Carvalho, Miguel M., Grimstvedt, Joachim S., Di Maria, Valentina, Moan, Kristian, Nair, Rajeevkumar Raveendran, Flatset, Marcus S., Qiangwei Zhang, Thomas, Laurent F., Pauzin, Francois P., Mazzarini Baldinotti, Rodolfo Da Silva, Quattrocolo, Giulia, Bramham, Clive R., Sætrom, Pål, Witter, Menno P., and Kentros, Clifford G.
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GENE expression ,TRANSGENE expression ,VIRAL genes ,NEURAL circuitry ,HIPPOCAMPUS (Brain) ,GENETIC vectors - Abstract
Accurate investigations of neural circuitry require specific genetic access to individual circuit elements, i.e., the myriad neuronal cell-types in the brain. However, native promoters cannot achieve this because while most genes are expressed in the brain, few are expressed in a single neuronal cell-type. We recently used enhancers, the subcomponents of the transcriptional apparatus which tell promoters when and where to express, combined with heterologous minimal promoters to increase specificity of transgene expression, an approach we call Enhancer-Driven Gene Expression (EDGE). As we discuss, EDGE is a marked improvement in specificity over native promoters, but still requires careful anatomical analysis to avoid off-target effects. In this study we present a more complete set of genomic markers from the mouse brain and characterize a novel EDGE viral vector capable of specifically driving expression in distinct subtypes of hippocampal neurons, even though it can express in other cell-types elsewhere. The advent of cell-type specific viral tools in wild-type animals provides a powerful strategy for neural circuit investigation and holds promise for studies using animal models for which transgenic tools are not available. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
8. A low-input high resolution sequential chromatin immunoprecipitation method captures genome-wide dynamics of bivalent chromatin.
- Author
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Seneviratne, Janith A., Ho, William W. H., Glancy, Eleanor, and Eckersley-Maslin, Melanie A.
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CHROMATIN , *EMBRYONIC stem cells , *IMMUNOPRECIPITATION , *EPIGENOMICS - Abstract
Background: Bivalent chromatin is an exemplar of epigenetic plasticity. This co-occurrence of active-associated H3K4me3 and inactive-associated H3K27me3 histone modifications on opposite tails of the same nucleosome occurs predominantly at promoters that are poised for future transcriptional upregulation or terminal silencing. We know little of the dynamics, resolution, and regulation of this chromatin state outside of embryonic stem cells where it was first described. This is partly due to the technical challenges distinguishing bone-fide bivalent chromatin, where both marks are on the same nucleosome, from allelic or sample heterogeneity where there is a mix of H3K4me3-only and H3K27me3-only mononucleosomes. Results: Here, we present a robust and sensitive method to accurately map bivalent chromatin genome-wide, along with controls, from as little as 2 million cells. We optimized and refined the sequential ChIP protocol which uses two sequential overnight immunoprecipitation reactions to robustly purify nucleosomes that are truly bivalent and contain both H3K4me3 and H3K27me3 modifications. Our method generates high quality genome-wide maps with strong peak enrichment and low background, which can be analyzed using standard bioinformatic packages. Using this method, we detect 8,789 bivalent regions in mouse embryonic stem cells corresponding to 3,918 predominantly CpG rich and developmentally regulated gene promoters. Furthermore, profiling Dppa2/4 knockout mouse embryonic stem cells, which lose both H3K4me3 and H3K27me3 at approximately 10% of bivalent promoters, demonstrated the ability of our method to capture bivalent chromatin dynamics. Conclusions: Our optimized sequential reChIP method enables high-resolution genome-wide assessment of bivalent chromatin together with all required controls in as little as 2 million cells. We share a detailed protocol and guidelines that will enable bivalent chromatin landscapes to be generated in a range of cellular contexts, greatly enhancing our understanding of bivalent chromatin and epigenetic plasticity beyond embryonic stem cells. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
9. Progesterone and cAMP synergistically induce SHP2 expression via PGR and CREB1 during uterine stromal decidualization.
- Author
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Zhou, Peiyi, Ouyang, Liqun, Jiang, Ting, Tian, Yingpu, Deng, Wenbo, Wang, Haibin, Kong, Shuangbo, and Lu, Zhongxian
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ENDOMETRIUM , *PROGESTERONE , *PROGESTERONE receptors , *EMBRYO implantation , *TRANSCRIPTION factors , *STROMAL cells , *MEDROXYPROGESTERONE - Abstract
Decidualization of endometrial stroma is a key step in embryo implantation and its abnormality often leads to pregnancy failure. Stromal decidualization is a very complex process that is co‐regulated by estrogen, progesterone and many local factors. The signaling protein SHP2 encoded by PTPN11 is dynamically expressed in decidualized endometrial stroma and mediates and integrates various signals to govern the decidualization. In the present study, we investigate the mechanism of PTPN11 gene transcription. Estrogen, progesterone and cAMP co‐induced decidualization of human endometrial stromal cell in vitro, but only progesterone and cAMP induced SHP2 expression. Using the luciferase reporter, we refined a region from −229 bp to +1 bp in the PTPN11 gene promoter comprising the transcriptional core regions that respond to progesterone and cAMP. Progesterone receptor (PGR) and cAMP‐responsive element‐binding protein 1 (CREB1) were predicted to be transcription factors in this core region by bioinformatic methods. The direct binding of PGR and CREB1 on the PTPN11 promoter was confirmed by electrophoretic mobility and chromatin immunoprecipitation in vitro. Knockdown of PGR and CREB1 protein significantly inhibited the expression of SHP2 induced by medroxyprogesterone acetate and cAMP. These results demonstrate that transcription factors PGR and CREB1 bind to the PTPN11 promoter to regulate the expression of SHP2 in response to decidual signals. Our results explain the transcriptional expression mechanism of SHP2 during decidualization and promote the understanding of the mechanism of decidualization of stromal cells. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
10. Protocol for generating customizable and reproducible plots of sequencing coverage data using the seqNdisplayR package
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Søren Lykke-Andersen, Jérôme O. Rouvière, Manfred Schmid, Maria Gockert, and Torben Heick Jensen
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Sequencing ,RNA-seq ,ChIP-seq ,Gene Expression ,Chromatin immunoprecipitation ,ChIP ,Science (General) ,Q1-390 - Abstract
Summary: The widespread usage of next-generation sequencing methods for functional genomics studies requires standardized tools for consistent visualization of the associated data. Here, we present seqNdisplayR, an R package for plotting standard sequencing data coverage within a genomic region of interest in a customizable and reproducible manner. We describe steps for installing software, preparing data files, choosing options, and plotting data. This tool is readily available for users with no prior experience with R through the “Shiny app” interface.For complete details on the use and execution of this protocol, please refer to Lykke-Andersen et al.,1 Gockert et al.,2 and Rouviere et al.3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
- Published
- 2024
- Full Text
- View/download PDF
11. Novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death.
- Author
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Zhan, Yongqiang, Xu, Dongwei, Tian, Yizhu, Qu, Xiaoye, Sheng, Mingwei, Lin, Yuanbang, Ke, Michael, Jiang, Longfeng, Xia, Qiang, Kaldas, Fady M, Farmer, Douglas G, and Ke, Bibo
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ALT ,alanine aminotransferase ,APAF1 ,apoptotic peptidase activating factor 1 ,ASK1 ,apoptosis signal-regulating kinase 1 ,AST ,aspartate aminotransferase ,Apoptosis ,BMM ,bone marrow-derived macrophage ,CXCL-10 ,C-X-C motif chemokine ligand 10 ,CYLD ,cyclindromatosis ,ChIP ,chromatin immunoprecipitation ,DAMP ,damage-associated molecular pattern ,DUB ,deubiquitinating enzyme ,ER ,endoplasmic reticulum ,ES ,embryonic stem ,G3BP1 ,G3BP1 ,Ras GTPase-activating protein-binding protein 1 ,GCLC ,glutamate-cysteine ligase catalytic subunit ,GCLM ,glutamate-cysteine ligase regulatory subunit ,IHC ,immunohistochemistry ,INF-β ,interferon-β ,IR ,ischaemia/reperfusion ,IRF3 ,IRF3 ,interferon regulatory factor 3 ,IRF7 ,IFN-regulating transcription factor 7 ,IRI ,ischaemia/reperfusion injury ,Innate immunity ,KO ,knockout ,LPS ,lipopolysaccharide ,Liver inflammation ,Lyz2 ,Lysozyme 2 ,MCP-1 ,monocyte chemoattractant protein 1 ,NOX2 ,NADPH oxidase 2 ,NOX4 ,NADPH oxidase 4 ,NQO1 ,NAD(P)H quinone dehydrogenase 1 ,NRF2 ,nuclear factor (erythroid-derived 2)-like 2 ,NS ,non-specific ,Necroptosis ,OASL1 ,2′ ,5′oligoadenylate synthetase-like 1 ,PAMP ,pathogen-derived molecular pattern ,RIPK3 ,receptor-interacting serine/threonine-protein kinase 3 ,ROS ,reactive oxygen species ,STING ,STING ,stimulator of interferon genes ,TBK1 ,TANK-binding kinase 1 ,TLR4 ,Toll-like receptor 4 ,TNF-α ,tumour necrosis factor-alpha ,TRX ,thioredoxin ,TSS ,transcription start sites ,TXNIP ,thioredoxin-interacting protein ,TXNIPFL/FL ,floxed TXNIP ,TXNIPM-KO ,myeloid-specific TXNIP KO ,UTR ,untranslated region ,sALT ,serum ALT ,sAST ,serum AST ,siRNA ,small interfering RNA ,Liver Disease ,Digestive Diseases ,Chronic Liver Disease and Cirrhosis ,Aetiology ,2.1 Biological and endogenous factors ,Good Health and Well Being - Abstract
Background & aimsThe stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress.MethodsA mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice.ResultsThe TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis.ConclusionsMacrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.Lay summaryLiver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.
- Published
- 2022
12. Chromatin and non-chromatin immunoprecipitations to capture protein–protein and protein-nucleic acid interactions in living cells.
- Author
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Barman, Priyanka, Kaja, Amala, Chakraborty, Pritam, and Bhaumik, Sukesh R.
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RNA-protein interactions , *CHROMATIN , *IMMUNOPRECIPITATION , *CELL physiology , *CELL analysis , *PROTEIN-protein interactions , *MESSENGER RNA - Abstract
• Analysis of the protein-nucleic acid interactions in vivo. • Analysis of the protein–protein interactions in vivo. Analysis of the protein-RNA interactions in vivo. • Analysis of the protein-RNA interactions in vivo. Proteins are expressed from genes via sequential biological processes of transcription, mRNA processing, export and translation, and play their roles in maintaining cellular functions via interactions with proteins, DNAs or RNAs. Thus, it is important to study the protein interactions during biological processes in living cells towards understanding their mechanisms-of-action in real time. Methodologies have been developed over the years to study protein interactions in vivo. One state-of-the-art approach is formaldehyde crosslinking-based immuno- or chemi-precipitation to analyze selective as well as genome/proteome-wide interactions in living cells. It is a popular and widely used methodology for cellular analysis of the protein–protein and protein-nucleic acid interactions. Here, we describe this approach to analyze protein–protein/nucleic acid interactions in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
13. Generation of an enhancer-driven gene expression viral tool specific to dentate granule cell-types through direct hippocampal injection
- Author
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Maria Letizia Potenza, Stefan Blankvoort, Miguel M. Carvalho, Joachim S. Grimstvedt, Valentina Di Maria, Kristian Moan, Rajeevkumar Raveendran Nair, Marcus S. Flatset, Qiangwei Zhang, Laurent F. Thomas, Francois P. Pauzin, Rodolfo Da Silva Mazzarini Baldinotti, Giulia Quattrocolo, Clive R. Bramham, Pål Sætrom, Menno P. Witter, and Clifford G. Kentros
- Subjects
rAAVs ,dentate gyrus ,genetic tool ,chromatin immunoprecipitation ,stereotaxic injection ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Accurate investigations of neural circuitry require specific genetic access to individual circuit elements, i.e., the myriad neuronal cell-types in the brain. However, native promoters cannot achieve this because while most genes are expressed in the brain, few are expressed in a single neuronal cell-type. We recently used enhancers, the subcomponents of the transcriptional apparatus which tell promoters when and where to express, combined with heterologous minimal promoters to increase specificity of transgene expression, an approach we call Enhancer-Driven Gene Expression (EDGE). As we discuss, EDGE is a marked improvement in specificity over native promoters, but still requires careful anatomical analysis to avoid off-target effects. In this study we present a more complete set of genomic markers from the mouse brain and characterize a novel EDGE viral vector capable of specifically driving expression in distinct subtypes of hippocampal neurons, even though it can express in other cell-types elsewhere. The advent of cell-type specific viral tools in wild-type animals provides a powerful strategy for neural circuit investigation and holds promise for studies using animal models for which transgenic tools are not available.
- Published
- 2024
- Full Text
- View/download PDF
14. ATF7ip Targets Transposable Elements for H3K9me3 Deposition to Modify CD8+ T Cell Effector and Memory Responses.
- Author
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Sin, Jun Hyung, Kashyap, Sujit, Acenas, Dante, Cortez, Jessica T, Lee, James, Marson, Alexander, Matloubian, Mehrdad, and Waterfield, Michael R
- Subjects
CD4-Positive T-Lymphocytes ,CD8-Positive T-Lymphocytes ,Cells ,Cultured ,Animals ,Mice ,Inbred C57BL ,Mice ,Knockout ,Humans ,Mice ,Listeria monocytogenes ,Receptors ,Interleukin-7 ,Histones ,Repressor Proteins ,DNA Transposable Elements ,Interleukin-2 ,Chromatin Immunoprecipitation ,Immunologic Memory ,Gene Silencing ,Gene Deletion ,Listeriosis ,Genetics ,Vaccine Related ,Aetiology ,2.1 Biological and endogenous factors ,Inflammatory and immune system ,Immunology - Abstract
CD8+ T cells are critical for the immune response to pathogens and tumors, and CD8+ T cell memory protects against repeat infections. In this study, we identify the activating transcription factor 7 interacting protein (ATF7ip) as a critical regulator of CD8+ T cell immune responses. Mice with a T cell-specific deletion of ATF7ip have a CD8+ T cell-intrinsic enhancement of Il7r expression and Il2 expression leading to enhanced effector and memory responses. Chromatin immunoprecipitation sequencing studies identified ATF7ip as a repressor of Il7r and Il2 gene expression through the deposition of the repressive histone mark H3K9me3 at the Il7r gene and Il2-Il21 intergenic region. Interestingly, ATF7ip targeted transposable elements for H3K9me3 deposition at both the IL7r locus and the Il2-Il21 intergenic region, indicating that ATF7ip silencing of transposable elements is important for regulating CD8+ T cell function. These results demonstrate a new epigenetic pathway by which IL-7R and IL-2 production are constrained in CD8+ T cells, and this may open up new avenues for modulating their production.
- Published
- 2022
15. proChIPdb: a chromatin immunoprecipitation database for prokaryotic organisms
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Decker, Katherine T, Gao, Ye, Rychel, Kevin, Bulushi, Tahani Al, Chauhan, Siddharth M, Kim, Donghyuk, Cho, Byung-Kwan, and Palsson, Bernhard O
- Subjects
Biotechnology ,Genetics ,Human Genome ,1.1 Normal biological development and functioning ,Underpinning research ,Generic health relevance ,Binding Sites ,Chromatin ,Chromatin Immunoprecipitation ,Databases ,Genetic ,Genome ,Prokaryotic Cells ,Protein Binding ,Transcription Factors ,Environmental Sciences ,Biological Sciences ,Information and Computing Sciences ,Developmental Biology - Abstract
The transcriptional regulatory network in prokaryotes controls global gene expression mostly through transcription factors (TFs), which are DNA-binding proteins. Chromatin immunoprecipitation (ChIP) with DNA sequencing methods can identify TF binding sites across the genome, providing a bottom-up, mechanistic understanding of how gene expression is regulated. ChIP provides indispensable evidence toward the goal of acquiring a comprehensive understanding of cellular adaptation and regulation, including condition-specificity. ChIP-derived data's importance and labor-intensiveness motivate its broad dissemination and reuse, which is currently an unmet need in the prokaryotic domain. To fill this gap, we present proChIPdb (prochipdb.org), an information-rich, interactive web database. This website collects public ChIP-seq/-exo data across several prokaryotes and presents them in dashboards that include curated binding sites, nucleotide-resolution genome viewers, and summary plots such as motif enrichment sequence logos. Users can search for TFs of interest or their target genes, download all data, dashboards, and visuals, and follow external links to understand regulons through biological databases and the literature. This initial release of proChIPdb covers diverse organisms, including most major TFs of Escherichia coli, and can be expanded to support regulon discovery across the prokaryotic domain.
- Published
- 2022
16. Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs
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Shen, Zeyang, Li, Rick Z, Prohaska, Thomas A, Hoeksema, Marten A, Spann, Nathan J, Tao, Jenhan, Fonseca, Gregory J, Le, Thomas, Stolze, Lindsey K, Sakai, Mashito, Romanoski, Casey E, and Glass, Christopher K
- Subjects
Biochemistry and Cell Biology ,Bioinformatics and Computational Biology ,Genetics ,Biological Sciences ,Human Genome ,Generic health relevance ,Animals ,CCAAT-Enhancer-Binding Protein-beta ,Chromatin Immunoprecipitation ,Enhancer Elements ,Genetic ,Gene Expression Regulation ,Genomics ,Humans ,K562 Cells ,Male ,Mice ,Protein Binding ,Proto-Oncogene Proteins ,Trans-Activators ,Transcription Factors ,gene regulation ,genetic variation ,transcription factors ,macrophages ,Human ,Mouse ,chromosomes ,gene expression ,genetics ,genomics ,human ,mouse ,Biological sciences ,Biomedical and clinical sciences ,Health sciences - Abstract
Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5 bp increase to >30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.
- Published
- 2022
17. Systematic identification of non-canonical transcription factor motifs
- Author
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Chumpitaz-Diaz, Luis, Samee, Md Abul Hassan, and Pollard, Katherine S
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Stem Cell Research ,Genetics ,Human Genome ,Stem Cell Research - Nonembryonic - Human ,Generic health relevance ,Binding Sites ,Chromatin Immunoprecipitation ,Computational Biology ,DNA-Binding Proteins ,Humans ,Nucleotide Motifs ,Protein Binding ,Sequence Analysis ,DNA ,Transcription Factors - Abstract
Sequence-specific transcription factors (TFs) recognize motifs of related nucleotide sequences at their DNA binding sites. Upon binding at these sites, TFs regulate critical molecular processes such as gene expression. It is widely assumed that a TF recognizes a single "canonical" motif, although recent studies have identified additional "non-canonical" motifs for some TFs. A comprehensive approach to identify non-canonical DNA binding motifs and the functional importance of those motifs' matches in the human genome is necessary for fully understanding the mechanisms of TF-regulated molecular processes in human cells. To address this need, we developed a statistical pipeline for in vitro HT-SELEX data that identifies and characterizes the distributions of non-canonical TF motifs in a stringent manner. Analyzing ~170 human TFs' HT-SELEX data, we found non-canonical motifs for 19 TFs (11%). These non-canonical motifs occur independently of the TFs' canonical motifs. Non-canonical motif occurrences in the human genome show similar evolutionary conservation to canonical motif occurrences, explain TF binding in locations without canonical motifs, and occur within gene promoters and epigenetically marked regulatory sequences in human cell lines and tissues. Our approach and collection of non-canonical motifs expand current understanding of functionally relevant DNA binding sites for human TFs.
- Published
- 2021
18. Protocol for in vitro BCR-mediated plasma cell differentiation and purification of chromatin-associated proteins.
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Ochiai, Kyoko, Shima, Hiroki, Ikura, Tsuyoshi, Franke, Marissa C, Sievert, Evelyn P, Sciammas, Roger, and Igarashi, Kazuhiko
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Plasma Cells ,Chromatin ,Animals ,Mice ,Transgenic ,Mice ,Proteins ,Receptors ,Antigen ,B-Cell ,Culture Media ,Chromatography ,Liquid ,Cell Differentiation ,Tandem Mass Spectrometry ,Antibody ,Cell Biology ,Chromatin immunoprecipitation ,Flow Cytometry/Mass Cytometry ,Gene Expression ,Immunology ,Mass Spectrometry ,Molecular Biology ,Underpinning research ,1.1 Normal biological development and functioning ,Generic health relevance - Abstract
Molecular-level understanding of plasma cell (PC) differentiation has been modeled using lipopolysaccharide (LPS) stimulation in vitro. However, this system does not involve the B-cell receptor (BCR)-a critical component of B cell biology. Here, we present a protocol for in vitro PC differentiation system dependent on BCR signaling that easily scales up for cell number-demanding applications, including protein complex purification. We describe how to set up this system and detail applications for endogenous complex purification of chromatin-associated proteins. For further details on the use and execution of this protocol, please refer to Sciammas et al. (2011) and Ochiai et al. (2018, 2020).
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- 2021
19. Histone modification analysis reveals common regulators of gene expression in liver and blood stage merozoites of Plasmodium parasites
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Ashley B. Reers, Rodriel Bautista, James McLellan, Beatriz Morales, Rolando Garza, Sebastiaan Bol, Kirsten K. Hanson, and Evelien M. Bunnik
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Malaria ,Epigenetics ,Gene regulation ,Transcriptomics ,Chromatin immunoprecipitation ,Genetics ,QH426-470 - Abstract
Abstract Gene expression in malaria parasites is subject to various layers of regulation, including histone post-translational modifications (PTMs). Gene regulatory mechanisms have been extensively studied during the main developmental stages of Plasmodium parasites inside erythrocytes, from the ring stage following invasion to the schizont stage leading up to egress. However, gene regulation in merozoites that mediate the transition from one host cell to the next is an understudied area of parasite biology. Here, we sought to characterize gene expression and the corresponding histone PTM landscape during this stage of the parasite lifecycle through RNA-seq and ChIP-seq on P. falciparum blood stage schizonts, merozoites, and rings, as well as P. berghei liver stage merozoites. In both hepatic and erythrocytic merozoites, we identified a subset of genes with a unique histone PTM profile characterized by a region of H3K4me3 depletion in their promoter. These genes were upregulated in hepatic and erythrocytic merozoites and rings, had roles in protein export, translation, and host cell remodeling, and shared a DNA motif. These results indicate that similar regulatory mechanisms may underlie merozoite formation in the liver and blood stages. We also observed that H3K4me2 was deposited in gene bodies of gene families encoding variant surface antigens in erythrocytic merozoites, which may facilitate switching of gene expression between different members of these families. Finally, H3K18me and H2K27me were uncoupled from gene expression and were enriched around the centromeres in erythrocytic schizonts and merozoites, suggesting potential roles in the maintenance of chromosomal organization during schizogony. Together, our results demonstrate that extensive changes in gene expression and histone landscape occur during the schizont-to-ring transition to facilitate productive erythrocyte infection. The dynamic remodeling of the transcriptional program in hepatic and erythrocytic merozoites makes this stage attractive as a target for novel anti-malarial drugs that may have activity against both the liver and blood stages.
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- 2023
- Full Text
- View/download PDF
20. Building an improved transcription factor-centered yeast one hybrid system to identify DNA motifs bound by protein comprehensively
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Wang Jingwen, Wang Jingxin, Zhu Ye, Zhu Yan, Liu Caozhi, Chen Yanyu, Zeng Fanli, Chen Su, and Wang Yucheng
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Chromatin immunoprecipitation ,DNA-protein interaction ,EMSA ,ERF ,High-throughput sequencing ,MEME analysis ,Botany ,QK1-989 - Abstract
Abstract Background Identification of the motifs bound by a transcription factor (TF) is important to reveal the function of TF. Previously, we built a transcription factor centered yeast one hybrid (TF-Centered Y1H) that could identify the motifs bound by a target TF. However, that method was difficult to comprehensively identify all the motifs bound by a TF. Results Here, we build an improved TF-Centered Y1H to comprehensively determine the motifs bound by a target TF. Recombination-mediated cloning in yeast was performed to construct a saturated prey library that contains 7 random base insertions. After TF-Centered Y1H screening, all the positive clones were pooled together to isolate pHIS2 vector. The insertion regions of pHIS2 were PCR amplified and the PCR product was subjected to high-throughput sequencing. The insertion sequences were then retrieved and analyzed using MEME program to identify the potential motifs bound by the TF. Using this technology, we studied the motifs bound by an ethylene-responsive factor (BpERF2) from birch. In total, 22 conserved motifs were identified, and most of them are novel cis-acting elements. Both the yeast one hybrid and electrophoretic mobility shift assay verified that the obtained motifs could be bound by BpERF2. In addition, chromatin immunoprecipitation (ChIP) study further suggested that the identified motifs can be bound by BpERF2 in cells of birch. These results together suggested that this technology is reliable and has biological significance. Conclusion This method will have wide application in DNA-protein interaction studies.
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- 2023
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21. Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments
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Ariel Caride, Jin Sung Jang, Geng-Xian Shi, Sam Lenz, Jian Zhong, Kwan Hyun Kim, Mariet Allen, Keith D. Robertson, Gianrico Farrugia, Tamas Ordog, Nilüfer Ertekin-Taner, and Jeong-Heon Lee
- Subjects
Chromatin quantification ,Chromatin immunoprecipitation ,Antibody titer ,H3K27ac ,Experimental consistency ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.
- Published
- 2023
- Full Text
- View/download PDF
22. Protocol to capture transcription factor-mediated 3D chromatin interactions using affinity tag-based BL-HiChIP
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Ruimin Ren, Yao Hua, and Heng Wang
- Subjects
Genomics ,Molecular Biology ,Chromatin Immunoprecipitation ,ChIP ,Science (General) ,Q1-390 - Abstract
Summary: Pioneer transcription factors (TFs) can directly establish higher-order chromatin interactions to instruct gene transcription. Here, we present a protocol for capturing TF-mediated 3D chromatin interactions using affinity tag-based bridge linker (BL)-Hi-chromatin immunoprecipitation (HiChIP). We describe steps for constructing FLAG-tagged TF, performing BL-HiChIP, and preparing the library. We then detail procedures for sequencing, data analysis, and quality control. This protocol has potential applications in 3D chromatin analysis centered on any specific TF in any type of cells without the need of optimal antibodies.For complete details on the use and execution of this protocol, please refer to Ren et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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- 2023
- Full Text
- View/download PDF
23. Transcription Factor MAFF (MAF Basic Leucine Zipper Transcription Factor F) Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism.
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von Scheidt, Moritz, Zhao, Yuqi, de Aguiar Vallim, Thomas, Che, Nam, Wierer, Michael, Seldin, Marcus, Franzén, Oscar, Kurt, Zeyneb, Pang, Shichao, Bongiovanni, Dario, Yamamoto, Masayuki, Edwards, Peter, Ruusalepp, Arno, Kovacic, Jason, Mann, Matthias, Björkegren, Johan, Lusis, Aldons, Yang, Xia, and Schunkert, Heribert
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atherosclerosis ,chromatin immunoprecipitation ,coronary artery disease ,inflammation ,lipopolysaccharides ,mafF transcription factor ,receptors ,LDL ,Animals ,Atherosclerosis ,Cholesterol ,DNA-Binding Proteins ,Disease Models ,Animal ,Humans ,Inflammation ,MafF Transcription Factor ,Male ,Mice ,Mice ,Knockout ,Nuclear Proteins - Abstract
BACKGROUND: Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue-specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterize central regulators and networks leading to atherosclerosis. METHODS: Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knockout models) and human (as shown by genome-wide association studies), liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models, as well as experimental studies including chromatin immunoprecipitation DNA-sequencing, chromatin immunoprecipitation mass spectrometry, overexpression, small interfering RNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. RESULTS: The transcription factor MAFF (MAF basic leucine zipper transcription factor F) interacted as a key driver of a liver network with 3 human genes at CAD genome-wide association studies loci and 11 atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor (LDLR) gene correlated with MAFF in 600 CAD patients undergoing bypass surgery (STARNET [Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task]) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of MAFF were tested in noninflammatory conditions and showed positive correlation between MAFF and LDLR in vitro and in vivo. Interestingly, after lipopolysaccharide stimulation (inflammatory conditions), an inverse correlation between MAFF and LDLR in vitro and in vivo was observed. Chromatin immunoprecipitation mass spectrometry revealed that the human CAD genome-wide association studies candidate BACH1 (BTB domain and CNC homolog 1) assists MAFF in the presence of lipopolysaccharide stimulation with respective heterodimers binding at the MAF recognition element of the LDLR promoter to transcriptionally downregulate LDLR expression. CONCLUSIONS: The transcription factor MAFF was identified as a novel central regulator of an atherosclerosis/CAD-relevant liver network. MAFF triggered context-specific expression of LDLR and other genes known to affect CAD risk. Our results suggest that MAFF is a missing link between inflammation, lipid and lipoprotein metabolism, and a possible treatment target.
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- 2021
24. Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches.
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Troutman, Ty D, Bennett, Hunter, Sakai, Mashito, Seidman, Jason S, Heinz, Sven, and Glass, Christopher K
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Cell isolation ,ChIP-seq ,Chromatin immunoprecipitation ,Flow cytometry/mass cytometry ,High-throughput screening ,Immunology ,Sequencing - Abstract
Significant advancements in understanding disease mechanisms can occur through combined analysis of next-generation sequencing datasets generated using purified cell populations. Here, we detail our optimized protocol for purification of mouse hepatic macrophages (or other liver non-parenchymal populations) suitable for use in various next-generation sequencing protocols. An alternative framework is described for sorting pre-fixed hepatic nuclei populations. This strategy has the advantage of rapidly preserving the nuclei and can facilitate success with ChIP-seq for more challenging molecules. For complete details on the use and execution of these protocols, please refer to Muse et al. (2018), Sakai et al. (2019), and Seidman et al. (2020).
- Published
- 2021
25. Inferring time series chromatin states for promoter-enhancer pairs based on Hi-C data
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Miko, Henriette, Qiu, Yunjiang, Gaertner, Bjoern, Sander, Maike, and Ohler, Uwe
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Biological Sciences ,Bioinformatics and Computational Biology ,Genetics ,1.1 Normal biological development and functioning ,Underpinning research ,Bayes Theorem ,Chromatin ,Chromosomes ,Enhancer Elements ,Genetic ,Promoter Regions ,Genetic ,Gene regulation ,Chromatin immunoprecipitation ,Histone modifications ,Hi-C ,Enhancer ,Differentiation ,Information and Computing Sciences ,Medical and Health Sciences ,Bioinformatics ,Biological sciences ,Biomedical and clinical sciences - Abstract
BackgroundCo-localized combinations of histone modifications ("chromatin states") have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points ("chromatin state trajectories") have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs.ResultsWe present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories. We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation. The code of the framework is available at https://github.com/henriettemiko/TimelessFlex .ConclusionsTimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.
- Published
- 2021
26. BAHCC1 binds H3K27me3 via a conserved BAH module to mediate gene silencing and oncogenesis
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Fan, Huitao, Lu, Jiuwei, Guo, Yiran, Li, Dongxu, Zhang, Zhi-Min, Tsai, Yi-Hsuan, Pi, Wen-Chieh, Ahn, Jeong Hyun, Gong, Weida, Xiang, Yu, Allison, David F, Geng, Huimin, He, Shenghui, Diao, Yarui, Chen, Wei-Yi, Strahl, Brian D, Cai, Ling, Song, Jikui, and Wang, Gang Greg
- Subjects
Biochemistry and Cell Biology ,Genetics ,Biological Sciences ,Human Genome ,Rare Diseases ,Pediatric Cancer ,Hematology ,Cancer ,Pediatric ,2.1 Biological and endogenous factors ,Generic health relevance ,Animals ,Carcinogenesis ,Cell Differentiation ,Cell Line ,Tumor ,Chromatin Immunoprecipitation ,Gene Expression Regulation ,Gene Silencing ,HEK293 Cells ,HeLa Cells ,Histone Code ,Histones ,Humans ,Jurkat Cells ,Leukemia ,Methylation ,Mice ,Mice ,Transgenic ,Neoplasm Transplantation ,Protein Processing ,Post-Translational ,Proteins ,Transplantation ,Heterologous ,Hela Cells ,Medical and Health Sciences ,Developmental Biology ,Agricultural biotechnology ,Bioinformatics and computational biology - Abstract
Trimethylated histone H3 lysine 27 (H3K27me3) regulates gene repression, cell-fate determination and differentiation. We report that a conserved bromo-adjacent homology (BAH) module of BAHCC1 (BAHCC1BAH) 'recognizes' H3K27me3 specifically and enforces silencing of H3K27me3-demarcated genes in mammalian cells. Biochemical, structural and integrated chromatin immunoprecipitation-sequencing-based analyses demonstrate that direct readout of H3K27me3 by BAHCC1 is achieved through a hydrophobic trimethyl-L-lysine-binding 'cage' formed by BAHCC1BAH, mediating colocalization of BAHCC1 and H3K27me3-marked genes. BAHCC1 is highly expressed in human acute leukemia and interacts with transcriptional corepressors. In leukemia, depletion of BAHCC1, or disruption of the BAHCC1BAH-H3K27me3 interaction, causes derepression of H3K27me3-targeted genes that are involved in tumor suppression and cell differentiation, leading to suppression of oncogenesis. In mice, introduction of a germline mutation at Bahcc1 to disrupt its H3K27me3 engagement causes partial postnatal lethality, supporting a role in development. This study identifies an H3K27me3-directed transduction pathway in mammals that relies on a conserved BAH 'reader'.
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- 2020
27. DiNeR: a Differential graphical model for analysis of co-regulation Network Rewiring
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Zhang, Jing, Liu, Jason, Lee, Donghoon, Lou, Shaoke, Chen, Zhanlin, Gürsoy, Gamze, and Gerstein, Mark
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Biological Sciences ,Bioinformatics and Computational Biology ,Genetics ,Cancer ,Human Genome ,Hematology ,Networking and Information Technology R&D (NITRD) ,Aetiology ,2.1 Biological and endogenous factors ,Generic health relevance ,Chromatin Immunoprecipitation ,Gene Expression Regulation ,Gene Regulatory Networks ,Genome ,Humans ,K562 Cells ,Leukemia ,Myelogenous ,Chronic ,BCR-ABL Positive ,Models ,Genetic ,Protein Binding ,Software ,Transcription Factors ,Transcription ,Genetic ,Transcription factor co-regulation network ,ENCODE ,TF dysregulation ,Network changes ,Mathematical Sciences ,Information and Computing Sciences ,Bioinformatics ,Biological sciences ,Information and computing sciences ,Mathematical sciences - Abstract
BACKGROUND:During transcription, numerous transcription factors (TFs) bind to targets in a highly coordinated manner to control the gene expression. Alterations in groups of TF-binding profiles (i.e. "co-binding changes") can affect the co-regulating associations between TFs (i.e. "rewiring the co-regulator network"). This, in turn, can potentially drive downstream expression changes, phenotypic variation, and even disease. However, quantification of co-regulatory network rewiring has not been comprehensively studied. RESULTS:To address this, we propose DiNeR, a computational method to directly construct a differential TF co-regulation network from paired disease-to-normal ChIP-seq data. Specifically, DiNeR uses a graphical model to capture the gained and lost edges in the co-regulation network. Then, it adopts a stability-based, sparsity-tuning criterion -- by sub-sampling the complete binding profiles to remove spurious edges -- to report only significant co-regulation alterations. Finally, DiNeR highlights hubs in the resultant differential network as key TFs associated with disease. We assembled genome-wide binding profiles of 104 TFs in the K562 and GM12878 cell lines, which loosely model the transition between normal and cancerous states in chronic myeloid leukemia (CML). In total, we identified 351 significantly altered TF co-regulation pairs. In particular, we found that the co-binding of the tumor suppressor BRCA1 and RNA polymerase II, a well-known transcriptional pair in healthy cells, was disrupted in tumors. Thus, DiNeR successfully extracted hub regulators and discovered well-known risk genes. CONCLUSIONS:Our method DiNeR makes it possible to quantify changes in co-regulatory networks and identify alterations to TF co-binding patterns, highlighting key disease regulators. Our method DiNeR makes it possible to quantify changes in co-regulatory networks and identify alterations to TF co-binding patterns, highlighting key disease regulators.
- Published
- 2020
28. Cohesin residency determines chromatin loop patterns.
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Costantino, Lorenzo, Hsieh, Tsung-Han S, Lamothe, Rebecca, Darzacq, Xavier, and Koshland, Douglas
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Chromatin ,Animals ,Mammals ,Saccharomyces cerevisiae ,Cell Cycle Proteins ,Chromosomal Proteins ,Non-Histone ,DNA ,Blotting ,Western ,Chromatin Immunoprecipitation ,Chromosome Segregation ,Mitosis ,S Phase ,Chromatin loops ,Micro-C ,Pds5 ,S. cerevisiae ,Wpl1 ,cohesin ,genetics ,genomics ,Genetics ,Human Genome ,1.1 Normal biological development and functioning ,Generic health relevance ,Biochemistry and Cell Biology - Abstract
The organization of chromatin into higher order structures is essential for chromosome segregation, the repair of DNA-damage, and the regulation of gene expression. Using Micro-C XL to detect chromosomal interactions, we observed the pervasive presence of cohesin-dependent loops with defined positions throughout the genome of budding yeast, as seen in mammalian cells. In early S phase, cohesin stably binds to cohesin associated regions (CARs) genome-wide. Subsequently, positioned loops accumulate with CARs at the bases of the loops. Cohesin regulators Wpl1 and Pds5 alter the levels and distribution of cohesin at CARs, changing the pattern of positioned loops. From these observations, we propose that cohesin with loop extrusion activity is stopped by preexisting CAR-bound cohesins, generating positioned loops. The patterns of loops observed in a population of wild-type and mutant cells can be explained by this mechanism, coupled with a heterogeneous residency of cohesin at CARs in individual cells.
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- 2020
29. Whole-Genome Sequencing Identifies Novel Functional Loci Associated with Lung Function in Puerto Rican Youth.
- Author
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Lee, Eunice Y, Mak, Angel CY, Hu, Donglei, Sajuthi, Satria, White, Marquitta J, Keys, Kevin L, Eckalbar, Walter, Bonser, Luke, Huntsman, Scott, Urbanek, Cydney, Eng, Celeste, Jain, Deepti, Abecasis, Gonçalo, Kang, Hyun M, Germer, Soren, Zody, Michael C, Nickerson, Deborah A, Erle, David, Ziv, Elad, Rodriguez-Santana, Jose, Seibold, Max A, and Burchard, Esteban G
- Subjects
Biomedical and Clinical Sciences ,Cardiovascular Medicine and Haematology ,Clinical Sciences ,Genetics ,Human Genome ,Biotechnology ,Pediatric ,Asthma ,Clinical Research ,Lung ,Respiratory ,Adolescent ,Black People ,Bronchi ,Case-Control Studies ,Cell Line ,Child ,Chromatin Immunoprecipitation ,Chromosome Mapping ,Chromosomes ,Human ,Pair 1 ,Chromosomes ,Human ,Pair 5 ,Esophageal Mucosa ,Female ,Forced Expiratory Volume ,Gene Expression ,Humans ,Indians ,North American ,Linkage Disequilibrium ,Male ,Membrane Proteins ,Myocytes ,Smooth Muscle ,Nasal Mucosa ,Polymorphism ,Single Nucleotide ,Puerto Rico ,Quantitative Trait Loci ,Sequence Analysis ,RNA ,White People ,Whole Genome Sequencing ,Young Adult ,admixed ,FEV1 ,RNA sequencing ,inflammatory ,TMEM9 airway epithelial cells ,Medical and Health Sciences ,Respiratory System ,Cardiovascular medicine and haematology ,Clinical sciences - Abstract
Rationale: Puerto Ricans have the highest childhood asthma prevalence in the United States (23.6%); however, the etiology is uncertain.Objectives: In this study, we sought to uncover the genetic architecture of lung function in Puerto Rican youth with and without asthma who were recruited from the island (n = 836).Methods: We used admixture-mapping and whole-genome sequencing data to discover genomic regions associated with lung function. Functional roles of the prioritized candidate SNPs were examined with chromatin immunoprecipitation sequencing, RNA sequencing, and expression quantitative trait loci data.Measurements and Main Results: We discovered a genomic region at 1q32 that was significantly associated with a 0.12-L decrease in the lung volume of exhaled air (95% confidence interval, -0.17 to -0.07; P = 6.62 × 10-8) with each allele of African ancestry. Within this region, two SNPs were expression quantitative trait loci of TMEM9 in nasal airway epithelial cells and MROH3P in esophagus mucosa. The minor alleles of these SNPs were associated with significantly decreased lung function and decreased TMEM9 gene expression. Another admixture-mapping peak was observed on chromosome 5q35.1, indicating that each Native American ancestry allele was associated with a 0.15-L increase in lung function (95% confidence interval, 0.08-0.21; P = 5.03 × 10-6). The region-based association tests identified four suggestive windows that harbored candidate rare variants associated with lung function.Conclusions: We identified common and rare genetic variants that may play a critical role in lung function among Puerto Rican youth. We independently validated an inflammatory pathway that could potentially be used to develop more targeted treatments and interventions for patients with asthma.
- Published
- 2020
30. Chromatin regulator Ahc1p co-regulates nitrogen metabolism via interactions with multiple transcription factors in Saccharomyces cerevisiae.
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Chen, Yu, Zeng, Weizhu, Yu, Shiqin, Gao, Song, and Zhou, Jingwen
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- *
TRANSCRIPTION factors , *SACCHAROMYCES cerevisiae , *NITROGEN , *GENE expression , *CHROMATIN , *METABOLISM , *EPIGENETICS - Abstract
Chromatin regulation is an important gene expression/regulation system, but little is known about how it affects nitrogen metabolism in Saccharomyces cerevisiae. A previous study demonstrated the regulatory role of the chromatin regulator Ahc1p on multiple key genes of nitrogen metabolism in S. cerevisiae , but the regulatory mechanism remains unknown. In this study, multiple key nitrogen metabolism genes directly regulated by Ahc1p were identified, and the transcription factors interacting with Ahc1p were analyzed. It was ultimately found that Ahc1p may regulate some key nitrogen metabolism genes in two ways. First, Ahc1p acts as a co-factor and is recruited with transcription factors such as Rtg3p or Gcr1p to facilitate transcription complex binding to target gene core promoters and promote transcription initiation. Second, Ahc1p binds at enhancers to promote the transcription of target genes in concert with transcription factors. This study furthers the understanding of the regulatory network of nitrogen metabolism in S. cerevisiae from an epigenetic perspective. • Ahc1p regulates multiple nitrogen metabolism genes, but the mechanism is unclear. • Ahc1p directly regulates nitrogen-related genes by binding to their promoters. • Ahc1p acts as a transcription cofactor to promote gene transcription initiation. • Ahc1p may binds at an enhancer to enhance target gene transcription. • This study furthers the understanding of nitrogen metabolism in S. cerevisiae. [ABSTRACT FROM AUTHOR]
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- 2023
- Full Text
- View/download PDF
31. Histone modification analysis reveals common regulators of gene expression in liver and blood stage merozoites of Plasmodium parasites.
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Reers, Ashley B., Bautista, Rodriel, McLellan, James, Morales, Beatriz, Garza, Rolando, Bol, Sebastiaan, Hanson, Kirsten K., and Bunnik, Evelien M.
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- *
GENE expression , *MEROZOITES , *GENE families , *GENETIC regulation , *PLASMODIUM , *GENE expression profiling - Abstract
Gene expression in malaria parasites is subject to various layers of regulation, including histone post-translational modifications (PTMs). Gene regulatory mechanisms have been extensively studied during the main developmental stages of Plasmodium parasites inside erythrocytes, from the ring stage following invasion to the schizont stage leading up to egress. However, gene regulation in merozoites that mediate the transition from one host cell to the next is an understudied area of parasite biology. Here, we sought to characterize gene expression and the corresponding histone PTM landscape during this stage of the parasite lifecycle through RNA-seq and ChIP-seq on P. falciparum blood stage schizonts, merozoites, and rings, as well as P. berghei liver stage merozoites. In both hepatic and erythrocytic merozoites, we identified a subset of genes with a unique histone PTM profile characterized by a region of H3K4me3 depletion in their promoter. These genes were upregulated in hepatic and erythrocytic merozoites and rings, had roles in protein export, translation, and host cell remodeling, and shared a DNA motif. These results indicate that similar regulatory mechanisms may underlie merozoite formation in the liver and blood stages. We also observed that H3K4me2 was deposited in gene bodies of gene families encoding variant surface antigens in erythrocytic merozoites, which may facilitate switching of gene expression between different members of these families. Finally, H3K18me and H2K27me were uncoupled from gene expression and were enriched around the centromeres in erythrocytic schizonts and merozoites, suggesting potential roles in the maintenance of chromosomal organization during schizogony. Together, our results demonstrate that extensive changes in gene expression and histone landscape occur during the schizont-to-ring transition to facilitate productive erythrocyte infection. The dynamic remodeling of the transcriptional program in hepatic and erythrocytic merozoites makes this stage attractive as a target for novel anti-malarial drugs that may have activity against both the liver and blood stages. [ABSTRACT FROM AUTHOR]
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- 2023
- Full Text
- View/download PDF
32. Application of Linear Polyacrylamide (LPA) Matrix in Cotton Chromatin Immunoprecipitation to Increase Sheared DNA Isolation Efficiency.
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AVCI, Murat Kemal, TEZCAN, Erdem, AVCI, Safiye, and CAMCI, Haluk
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POLYACRYLAMIDE , *CHROMATIN , *IMMUNOPRECIPITATION , *MATRIX isolation , *COTTON , *POLYMERS - Abstract
Linear Polyacrylamide (LPA) is one of the polymers used as a yield-enhancing agent in DNA isolation of short and small molecular weight DNA molecules. The Chromatin immunoprecipitation (ChIP) method is widely used to elucidate DNAProtein interactions. In ChIP, which consists of many successive steps, it is an important problem to experience losses due to the small size of the DNA fragments during the re-isolation of the sheared DNA fragments. In this study, the effect of using LPA matrix on the isolation of small molecular weight DNA fragments with higher efficiency in the ChIP method applied to cotton plant, which is a eukaryotic organism with high commercial value in our country and in the world, was investigated. Here, the ChIP method was adapted and applied till the DNA fragmentation process by ultrasonication and the isolation of the fragmented DNA molecules. In statistical analyzes, the concentrations of DNA isolates used at the beginning of the ChIP experiment and the DNA molecules mechanically fragmented by ultrasonication, measured by the Nanodrop, were compared in ng/µl. Experimental and statistical analyzes showed that LPA application increased DNA isolation efficiency by 0.59 times. Based on these data, it has been proven that using LPA in ChIP method with cotton will directly affect the success of the experiment and contribute to get much more efficient results. This study is unique in that it focuses on improving the yield of sheared DNA isolation in direct cotton plant-specific ChIP application. [ABSTRACT FROM AUTHOR]
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- 2023
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- View/download PDF
33. Broadly Applicable Control Approaches Improve Accuracy of ChIP-Seq Data.
- Author
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Petrie, Meghan V., He, Yiwei, Gan, Yan, Ostrow, Andrew Zachary, and Aparicio, Oscar M.
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MUTANT proteins , *PROTEIN microarrays , *DNA-protein interactions , *BINDING sites , *DNA-binding proteins - Abstract
Chromatin ImmunoPrecipitation (ChIP) is a widely used method for the analysis of protein–DNA interactions in vivo; however, ChIP has pitfalls, particularly false-positive signal enrichment that permeates the data. We have developed a new approach to control for non-specific enrichment in ChIP that involves the expression of a non-genome-binding protein targeted in the IP alongside the experimental target protein due to the sharing of epitope tags. ChIP of the protein provides a "sensor" for non-specific enrichment that can be used for the normalization of the experimental data, thereby correcting for non-specific signals and improving data quality as validated against known binding sites for several proteins that we tested, including Fkh1, Orc1, Mcm4, and Sir2. We also tested a DNA-binding mutant approach and showed that, when feasible, ChIP of a site-specific DNA-binding mutant of the target protein is likely an ideal control. These methods vastly improve our ChIP-seq results in S. cerevisiae and should be applicable in other systems. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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- View/download PDF
34. Characterization of centromeric DNA of Gossypium anomalum reveals sequence-independent enrichment dynamics of centromeric repeats.
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Ding, Wenjie, Zhu, Yuanbin, Han, Jinlei, Zhang, Hui, Xu, Zhenzhen, Khurshid, Haris, Liu, Fang, Hasterok, Robert, Shen, Xinlian, and Wang, Kai
- Abstract
Centromeres in eukaryotes are composed of highly repetitive DNAs, which evolve rapidly and are thought to achieve a favorable structure in mature centromeres. However, how the centromeric repeat evolves into an adaptive structure is largely unknown. We characterized the centromeric sequences of Gossypium anomalum through chromatin immunoprecipitation against CENH3 antibodies. We revealed that the G. anomalum centromeres contained only retrotransposon-like repeats but were depleted in long arrays of satellites. These retrotransposon-like centromeric repeats were present in the African–Asian and Australian lineage species, suggesting that they might have arisen in the common ancestor of these diploid species. Intriguingly, we observed a substantial increase and decrease in copy numbers among African–Asian and Australian lineages, respectively, for the retrotransposon-derived centromeric repeats without apparent structure or sequence variation in cotton. This result indicates that the sequence content is not a decisive aspect of the adaptive evolution of centromeric repeats or at least retrotransposon-like centromeric repeats. In addition, two active genes with potential roles in gametogenesis or flowering were identified in CENH3 nucleosome-binding regions. Our results provide new insights into the constitution of centromeric repetitive DNA and the adaptive evolution of centromeric repeats in plants. [ABSTRACT FROM AUTHOR]
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- 2023
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35. Building an improved transcription factor-centered yeast one hybrid system to identify DNA motifs bound by protein comprehensively.
- Author
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Jingwen, Wang, Jingxin, Wang, Ye, Zhu, Yan, Zhu, Caozhi, Liu, Yanyu, Chen, Fanli, Zeng, Su, Chen, and Yucheng, Wang
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- *
DNA-binding proteins , *HYBRID systems , *DNA-protein interactions , *YEAST , *MOLECULAR cloning , *ANTISENSE DNA , *DNA insertion elements - Abstract
Background: Identification of the motifs bound by a transcription factor (TF) is important to reveal the function of TF. Previously, we built a transcription factor centered yeast one hybrid (TF-Centered Y1H) that could identify the motifs bound by a target TF. However, that method was difficult to comprehensively identify all the motifs bound by a TF. Results: Here, we build an improved TF-Centered Y1H to comprehensively determine the motifs bound by a target TF. Recombination-mediated cloning in yeast was performed to construct a saturated prey library that contains 7 random base insertions. After TF-Centered Y1H screening, all the positive clones were pooled together to isolate pHIS2 vector. The insertion regions of pHIS2 were PCR amplified and the PCR product was subjected to high-throughput sequencing. The insertion sequences were then retrieved and analyzed using MEME program to identify the potential motifs bound by the TF. Using this technology, we studied the motifs bound by an ethylene-responsive factor (BpERF2) from birch. In total, 22 conserved motifs were identified, and most of them are novel cis-acting elements. Both the yeast one hybrid and electrophoretic mobility shift assay verified that the obtained motifs could be bound by BpERF2. In addition, chromatin immunoprecipitation (ChIP) study further suggested that the identified motifs can be bound by BpERF2 in cells of birch. These results together suggested that this technology is reliable and has biological significance. Conclusion: This method will have wide application in DNA-protein interaction studies. [ABSTRACT FROM AUTHOR]
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- 2023
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- View/download PDF
36. ChIP-Based Nuclear DNA Isolation for Genome Sequencing in Pyropia to Remove Cytosol and Bacterial DNA Contamination.
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Zhang, Zehao, Wang, Junhao, Zhang, Xiaoqian, Guan, Xiaowei, Gao, Tian, Mao, Yunxiang, Poetsch, Ansgar, and Wang, Dongmei
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NUCLEAR DNA ,BACTERIAL DNA ,BACTERIAL contamination ,NUCLEOTIDE sequencing ,CYTOSOL - Abstract
Contamination from cytosolic DNA (plastid and mitochondrion) and epiphytic bacteria is challenging the efficiency and accuracy of genome-wide analysis of nori-producing marine seaweed Pyropia yezoensis. Unlike bacteria and organellar DNA, Pyropia nuclear DNA is closely associated with histone proteins. In this study, we applied Chromatin Immunoprecipitation (ChIP) of histone H3 to isolate nuclear DNA, followed by high-throughput sequencing. More than 99.41% of ChIP-sequencing data were successfully aligned to the reference nuclear genome; this was remarkably higher than those from direct extraction and direct extraction data, in which 40.96% to 42.95% are from plastids. The proportion of data that were mapped to the bacterial database when using ChIP extraction was very low. Additionally, ChIP data can cover up to 89.00% of the nuclear genome, higher than direct extraction data at equal data size and comparable to the latter at equal sequencing depth. The uncovered regions from the three methods are mostly overlapping, suggesting that incomplete sequencing accounts for the missing data, rather than failed chromatin-antibody binding in the ChIP extraction method. This ChIP extraction method can successfully separate nuclear DNA from cytosolic DNA and bacterial DNA, thus overwhelmingly reducing the sequencing cost in a genome resequencing project and providing strictly purified reference data for genome assembly. The method's applicability to other macroalgae makes it a valuable contribution to the algal research community. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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- View/download PDF
37. Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.
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Caride, Ariel, Jang, Jin Sung, Shi, Geng-Xian, Lenz, Sam, Zhong, Jian, Kim, Kwan Hyun, Allen, Mariet, Robertson, Keith D., Farrugia, Gianrico, Ordog, Tamas, Ertekin-Taner, Nilüfer, and Lee, Jeong-Heon
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IMMUNOGLOBULINS , *ANTIBODY titer , *HISTONE acetylation , *CHROMATIN , *PROOF of concept , *IMMUNOPRECIPITATION - Abstract
Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
38. Role of Marek’s Disease Virus (MDV)-Encoded US3 Serine/Threonine Protein Kinase in Regulating MDV Meq and Cellular CREB Phosphorylation
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Liao, Yifei, Lupiani, Blanca, Bajwa, Kanika, Khan, Owais A, Izumiya, Yoshihiro, and Reddy, Sanjay M
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Veterinary Sciences ,Agricultural ,Veterinary and Food Sciences ,Biological Sciences ,Genetics ,Infectious Diseases ,Biotechnology ,2.1 Biological and endogenous factors ,Cancer ,Infection ,Alphaherpesvirinae ,Animals ,Cell Line ,Cell Transformation ,Viral ,Chickens ,Chromatin Immunoprecipitation ,Gene Dosage ,Gene Expression Regulation ,Viral ,HEK293 Cells ,Herpesvirus 2 ,Gallid ,Humans ,Marek Disease ,Phosphorylation ,Poultry ,Promoter Regions ,Genetic ,Protein Serine-Threonine Kinases ,Signal Transduction ,Transfection ,Viral Proteins ,CREB ,Marek's disease virus ,Meq ,US3 protein kinase ,herpesviruses ,transcription regulation ,Marek’s disease virus ,Agricultural and Veterinary Sciences ,Medical and Health Sciences ,Virology ,Agricultural ,veterinary and food sciences ,Biological sciences ,Biomedical and clinical sciences - Abstract
Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. The functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild-type MDV US3, but not kinase-dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase-dead recombinant MDV, we identified US3-responsive MDV genes during infection and found that the majority of US3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some US3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV US3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.
- Published
- 2020
39. Mesenchymal and MAPK Expression Signatures Associate with Telomerase Promoter Mutations in Multiple Cancers
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Stern, Josh Lewis, Hibshman, Grace, Hu, Kevin, Ferrara, Sarah E, Costello, James C, Kim, William, Tamayo, Pablo, Cech, Thomas R, and Huang, Franklin W
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Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Cancer ,Genetics ,2.1 Biological and endogenous factors ,Aetiology ,Generic health relevance ,Cell Line ,Tumor ,Chromatin Immunoprecipitation ,Epithelial-Mesenchymal Transition ,Extracellular Signal-Regulated MAP Kinases ,Gene Expression Profiling ,Gene Expression Regulation ,Neoplastic ,Gene Regulatory Networks ,Humans ,Mutation ,Neoplasms ,Promoter Regions ,Genetic ,Sequence Analysis ,RNA ,Small Molecule Libraries ,Telomerase ,Tumor Microenvironment ,Oncology and Carcinogenesis ,Developmental Biology ,Oncology & Carcinogenesis ,Biochemistry and cell biology ,Oncology and carcinogenesis - Abstract
In a substantial fraction of cancers TERT promoter (TERTp) mutations drive expression of the catalytic subunit of telomerase, contributing to their proliferative immortality. We conducted a pan-cancer analysis of cell lines and find a TERTp mutation expression signature dominated by epithelial-to-mesenchymal transition and MAPK signaling. These data indicate that TERTp mutants are likely to generate distinctive tumor microenvironments and intercellular interactions. Analysis of high-throughput screening tests of 546 small molecules on cell line growth indicated that TERTp mutants displayed heightened sensitivity to specific drugs, including RAS pathway inhibitors, and we found that inhibition of MEK1 and 2, key RAS/MAPK pathway effectors, inhibited TERT mRNA expression. Consistent with an enrichment of mesenchymal states in TERTp mutants, cell lines and some patient tumors displayed low expression of the central adherens junction protein E-cadherin, and we provide evidence that its expression in these cells is regulated by MEK1/2. Several mesenchymal transcription factors displayed elevated expression in TERTp mutants including ZEB1 and 2, TWIST1 and 2, and SNAI1. Of note, the developmental transcription factor SNAI2/SLUG was conspicuously elevated in a significant majority of TERTp-mutant cell lines, and knock-down experiments suggest that it promotes TERT expression. IMPLICATIONS: Cancers harboring TERT promoter mutations are often more lethal, but the basis for this higher mortality remains unknown. Our study identifies that TERTp mutants, as a class, associate with a distinct gene and protein expression signature likely to impact their biological and clinical behavior and provide new directions for investigating treatment approaches for these cancers.
- Published
- 2020
40. Epigenomic profiling of neuroblastoma cell lines
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Upton, Kristen, Modi, Apexa, Patel, Khushbu, Kendsersky, Nathan M, Conkrite, Karina L, Sussman, Robyn T, Way, Gregory P, Adams, Rebecca N, Sacks, Gregory I, Fortina, Paolo, Diskin, Sharon J, Maris, John M, and Rokita, Jo Lynne
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Biochemistry and Cell Biology ,Bioinformatics and Computational Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Cancer ,Neuroblastoma ,Pediatric ,Pediatric Research Initiative ,Biotechnology ,Pediatric Cancer ,Genetics ,Rare Diseases ,Human Genome ,Neurosciences ,Aetiology ,2.1 Biological and endogenous factors ,Cell Line ,Tumor ,Chromatin ,Chromatin Immunoprecipitation ,Epigenomics ,Gene Expression Profiling ,Histones ,Humans ,N-Myc Proto-Oncogene Protein - Abstract
Understanding the aberrant transcriptional landscape of neuroblastoma is necessary to provide insight to the underlying influences of the initiation, progression and persistence of this developmental cancer. Here, we present chromatin immunoprecipitation sequencing (ChIP-Seq) data for the oncogenic transcription factors, MYCN and MYC, as well as regulatory histone marks H3K4me1, H3K4me3, H3K27Ac, and H3K27me3 in ten commonly used human neuroblastoma-derived cell line models. In addition, for all of the profiled cell lines we provide ATAC-Seq as a measure of open chromatin. We validate specificity of global MYCN occupancy in MYCN amplified cell lines and functional redundancy of MYC occupancy in MYCN non-amplified cell lines. Finally, we show with H3K27Ac ChIP-Seq that these cell lines retain expression of key neuroblastoma super-enhancers (SE). We anticipate this dataset, coupled with available transcriptomic profiling on the same cell lines, will enable the discovery of novel gene regulatory mechanisms in neuroblastoma.
- Published
- 2020
41. Maf1‐dependent transcriptional regulation of tRNAs prevents genomic instability and is associated with extended lifespan
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Shetty, Mihir, Noguchi, Chiaki, Wilson, Sydney, Martinez, Esteban, Shiozaki, Kazuhiro, Sell, Christian, Mell, Joshua Chang, and Noguchi, Eishi
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Biochemistry and Cell Biology ,Biological Sciences ,Genetics ,Underpinning research ,1.1 Normal biological development and functioning ,Generic health relevance ,Chromatin Immunoprecipitation ,DNA Damage ,Gene Expression Regulation ,Fungal ,Genomic Instability ,Glucose ,Mechanistic Target of Rapamycin Complex 1 ,Phosphoprotein Phosphatases ,Phosphorylation ,Protein Biosynthesis ,Protein Phosphatase 2 ,RNA ,Transfer ,Rad52 DNA Repair and Recombination Protein ,Repressor Proteins ,Schizosaccharomyces ,Schizosaccharomyces pombe Proteins ,aging ,DNA damage ,DNA repair ,lifespan ,Maf1 ,RNA polymerase III ,transcription ,tRNA ,Medical and Health Sciences ,Developmental Biology ,Biological sciences ,Biomedical and clinical sciences - Abstract
Maf1 is the master repressor of RNA polymerase III responsible for transcription of tRNAs and 5S rRNAs. Maf1 is negatively regulated via phosphorylation by the mTOR pathway, which governs protein synthesis, growth control, and lifespan regulation in response to nutrient availability. Inhibiting the mTOR pathway extends lifespan in various organisms. However, the downstream effectors for the regulation of cell homeostasis that are critical to lifespan extension remain elusive. Here we show that fission yeast Maf1 is required for lifespan extension. Maf1's function in tRNA repression is inhibited by mTOR-dependent phosphorylation, whereas Maf1 is activated via dephosphorylation by protein phosphatase complexes, PP4 and PP2A. Mutational analysis reveals that Maf1 phosphorylation status influences lifespan, which is correlated with elevated tRNA and protein synthesis levels in maf1∆ cells. However, mTOR downregulation, which negates protein synthesis, fails to rescue the short lifespan of maf1∆ cells, suggesting that elevated protein synthesis is not a cause of lifespan shortening in maf1∆ cells. Interestingly, maf1∆ cells accumulate DNA damage represented by formation of Rad52 DNA damage foci and Rad52 recruitment at tRNA genes. Loss of the Rad52 DNA repair protein further exacerbates the shortened lifespan of maf1∆ cells. Strikingly, PP4 deletion alleviates DNA damage and rescues the short lifespan of maf1∆ cells even though tRNA synthesis is increased in this condition, suggesting that elevated DNA damage is the major cause of lifespan shortening in maf1∆ cells. We propose that Maf1-dependent inhibition of tRNA synthesis controls fission yeast lifespan by preventing genomic instability that arises at tRNA genes.
- Published
- 2020
42. Integrative pathway enrichment analysis of multivariate omics data.
- Author
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Paczkowska, Marta, Barenboim, Jonathan, Sintupisut, Nardnisa, Fox, Natalie S, Zhu, Helen, Abd-Rabbo, Diala, Mee, Miles W, Boutros, Paul C, PCAWG Drivers and Functional Interpretation Working Group, Reimand, Jüri, and PCAWG Consortium
- Subjects
PCAWG Drivers and Functional Interpretation Working Group ,PCAWG Consortium ,Humans ,Neoplasms ,Adenocarcinoma ,Breast Neoplasms ,RNA ,Messenger ,Prognosis ,Chromatin Immunoprecipitation ,Gene Expression Profiling ,Sequence Analysis ,RNA ,Computational Biology ,Genomics ,Signal Transduction ,Apoptosis ,Gene Dosage ,Mutation ,Databases ,Factual ,Female ,Gene Regulatory Networks ,Metabolic Networks and Pathways ,Hippo Signaling Pathway ,Protein Serine-Threonine Kinases ,Cancer ,Breast Cancer ,Stem Cell Research ,Biotechnology ,Human Genome ,Genetics ,2.1 Biological and endogenous factors ,Aetiology ,Good Health and Well Being - Abstract
Multi-omics datasets represent distinct aspects of the central dogma of molecular biology. Such high-dimensional molecular profiles pose challenges to data interpretation and hypothesis generation. ActivePathways is an integrative method that discovers significantly enriched pathways across multiple datasets using statistical data fusion, rationalizes contributing evidence and highlights associated genes. As part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumor types, we integrated genes with coding and non-coding mutations and revealed frequently mutated pathways and additional cancer genes with infrequent mutations. We also analyzed prognostic molecular pathways by integrating genomic and transcriptomic features of 1780 breast cancers and highlighted associations with immune response and anti-apoptotic signaling. Integration of ChIP-seq and RNA-seq data for master regulators of the Hippo pathway across normal human tissues identified processes of tissue regeneration and stem cell regulation. ActivePathways is a versatile method that improves systems-level understanding of cellular organization in health and disease through integration of multiple molecular datasets and pathway annotations.
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- 2020
43. Tead transcription factors differentially regulate cortical development
- Author
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Mukhtar, Tanzila, Breda, Jeremie, Grison, Alice, Karimaddini, Zahra, Grobecker, Pascal, Iber, Dagmar, Beisel, Christian, van Nimwegen, Erik, and Taylor, Verdon
- Subjects
Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Biological Sciences ,Pediatric ,Stem Cell Research - Nonembryonic - Non-Human ,Neurosciences ,Stem Cell Research ,Genetics ,Underpinning research ,1.1 Normal biological development and functioning ,Neurological ,Animals ,Cell Adhesion Molecules ,Neuronal ,Cell Line ,Cerebral Cortex ,Chromatin Immunoprecipitation ,DNA-Binding Proteins ,Extracellular Matrix Proteins ,Female ,Hippo Signaling Pathway ,Humans ,Mice ,Nerve Tissue Proteins ,Neural Stem Cells ,Organ Specificity ,Protein Serine-Threonine Kinases ,Reelin Protein ,Serine Endopeptidases ,Signal Transduction ,TEA Domain Transcription Factors ,Transcription Factors - Abstract
Neural stem cells (NSCs) generate neurons of the cerebral cortex with distinct morphologies and functions. How specific neuron production, differentiation and migration are orchestrated is unclear. Hippo signaling regulates gene expression through Tead transcription factors (TFs). We show that Hippo transcriptional coactivators Yap1/Taz and the Teads have distinct functions during cortical development. Yap1/Taz promote NSC maintenance and Satb2+ neuron production at the expense of Tbr1+ neuron generation. However, Teads have moderate effects on NSC maintenance and do not affect Satb2+ neuron differentiation. Conversely, whereas Tead2 blocks Tbr1+ neuron formation, Tead1 and Tead3 promote this early fate. In addition, we found that Hippo effectors regulate neuronal migration to the cortical plate (CP) in a reciprocal fashion, that ApoE, Dab2 and Cyr61 are Tead targets, and these contribute to neuronal fate determination and migration. Our results indicate that multifaceted Hippo signaling is pivotal in different aspects of cortical development.
- Published
- 2020
44. Transcriptomic and ChIP-sequence interrogation of EGFR signaling in HER2+ breast cancer cells reveals a dynamic chromatin landscape and S100 genes as targets
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Nava, Miguel, Dutta, Pranabananda, Zemke, Nathan R, Farias-Eisner, Robin, Vadgama, Jaydutt V, and Wu, Yanyuan
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Biochemistry and Cell Biology ,Biomedical and Clinical Sciences ,Oncology and Carcinogenesis ,Biological Sciences ,Genetics ,Cancer ,Breast Cancer ,2.1 Biological and endogenous factors ,1.1 Normal biological development and functioning ,Underpinning research ,Aetiology ,Breast Neoplasms ,Cell Line ,Tumor ,Chromatin ,Chromatin Immunoprecipitation ,Drug Resistance ,Neoplasm ,Epidermal Growth Factor ,ErbB Receptors ,Gene Expression Profiling ,HeLa Cells ,Histones ,Humans ,Nucleotide Motifs ,Receptor ,ErbB-2 ,S100 Proteins ,Signal Transduction ,Trastuzumab ,HER2 ,EGFR ,Epigenetics ,Next generation sequencing ,Hela Cells ,Receptor ,erbB-2 ,Medical Biochemistry and Metabolomics ,Genetics & Heredity ,Medical biochemistry and metabolomics - Abstract
BackgroundThe Human Epidermal Growth Factor Receptor (EGFR/HER1) can be activated by several ligands including Transforming Growth Factor alpha (TGF-α) and Epidermal Growth Factor (EGF). Following ligand binding, EGFR heterodimerizes with other HER family members, such as HER2 (human epidermal growth factor receptor-2). Previously, we showed that the EGFR is upregulated in trastuzumab resistant HER2 positive (HER2+) breast cancer cells. This study is aimed to determine the downstream effects on transcription following EGFR upregulation in HER2+ breast cancer cells.MethodsRNA-sequence and ChIP-sequence for H3K18ac and H3K27ac (Histone H3 lysine K18 and K27 acetylation) were conducted following an Epidermal Growth Factor (EGF) treatment time course in HER2+ breast cancer cells, SKBR3. The levels of several proteins of interest were confirmed by western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by western blot analysis.ResultsOver the course of 24 h, EGFR stimulation resulted in the modulation of over 4000 transcripts. Moreover, our data demonstrates that EGFR/HER2 signaling regulates the epigenome, with global H3K18ac and H3K27ac oscillating as a function of time following EGF treatment. RNA-sequence data demonstrates the activation of immediate early genes (IEGs) and delayed early genes (DEGs) within 1 h of EGF treatment. More importantly, we have identified members of the S100 (S100 Calcium Binding Protein) gene family as likely direct targets of EGFR signaling as H3K18ac, H3K27ac and pol2 (RNA polymerase II) increase near the transcription start sites of some of these genes.ConclusionsOur data suggests that S100 proteins, which act as Ca2+ sensors, could play a role in EGF induced tumor cell growth and metastasis, contribute to trastuzumab resistance and cell migration and that they are likely drug targets in HER2+ breast cancer.
- Published
- 2019
45. Investigation of the chromatin remodelling enzyme Uls1 and its interactions with Topoisomerase 2 in S. cerevisiae
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Swanston, Amy and Ferreira, Helder
- Subjects
572 ,Molecular biology ,Cell biology ,Top2 ,Uls1 ,Topoisomerase ,Chromatin remodelling ,Chromatin remodeller ,Chromatin ,DNA ,SUMO ,Yeast ,Protein modification ,Acriflavine ,Top2 poison ,Drug screen ,Genetic screen ,Chromatin immunoprecipitation ,ChIP-seq ,Bioinformatics ,QP616.D56S8 ,DNA topoisomerase II ,Adenosine triphosphatase ,Saccharomyces cerevisiae - Abstract
Acriflavine (ACF) is a Topoisomerase 2 (Top2) poison, a class of drugs which stall Top2 during its reaction cycle causing the formation of persistent DNA breaks to which Top2 remains covalently bound. Deletion of ULS1 causes sensitivity to ACF, with cells showing activation of the Rad53 DNA damage checkpoint. Uls1 is a chromatin remodelling enzyme also implicated in the regulation of levels of SUMO conjugated proteins. We show that Uls1 has both a genetic and physical interaction with Top2, with uls1Δ sensitivity to ACF being linked to Top2 activity. Analysis of Uls1 and Top2 localisation genome wide via ChIP-seq reveals areas where the two proteins co-localise, with Top2 enrichment on chromatin being altered upon deletion of ULS1. At these areas, the presence of Uls1 prevents accumulation of Top2 upon addition of ACF. Our data suggests that Uls1 is required for regulation of stalled Top2. Top2 poisons are used therapeutically as anti-cancer drugs, however these drugs have been implicated in the formation of secondary cancers due to chromosomal translocations arising during the repair of Top2 generated double strand breaks (DSB). The use of dual targeted therapies where a Top2 poison is paired with an inhibitor of another pathway that increases sensitivity to the Top2 poison allows a lower dose to be used, therefore reducing harmful side effects. Our work looked to identify Top2 poison sensitive pathways in S. cerevisiae, where non-essential and essential gene mutants were assayed for sensitivity to ACF. This allowed a comprehensive analysis of 83% of the genes in S. cerevisiae, identifying novel genes within the areas of DNA repair, DNA replication, transcription, chromatin structure, protein modification/degradation, cell division/cell cycle and cellular organisation/cytoskeleton as being important in the response to this bulky adduct.
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- 2019
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46. Identification and validation of a novel long non-coding RNA (LINC01465) in ovarian cancer.
- Author
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Malgundkar, Shika Hanif, Hassan, Nada Abdullah, Al Badi, Hala, Gupta, Ishita, Burney, Ikram A., Al Hashami, Zainab, Al Barwani, Hamida, Al Riyami, Hamad, Al Kalbani, Moza, Lakhtakia, Ritu, Okamoto, Aikou, and Tamimi, Yahya
- Subjects
LINCRNA ,OVARIAN epithelial cancer ,PSEUDOGENES ,CELL death ,TRANSCRIPTION factors ,OVARIAN cancer - Abstract
Epithelial Ovarian Cancer (EOC) is a heterogeneous disease usually diagnosed at advanced stages. Therefore, early detection is crucial for better survival. Despite the advances in ovarian research, mechanisms underlying EOC carcinogenesis are not elucidated. We performed chromatin immunoprecipitation sequencing to identify genes regulated by E2F5, a transcription factor involved in ovarian carcinogenesis. Results revealed several putative candidate genes (115 protein-coding genes, 20 lncRNAs, 6 pseudogenes, and 4 miRNAs). A literature review and bioinformatics analysis of these genes revealed a novel lncRNA candidate (LINC01465) in EOC. We validated LINC01465 by quantifying its expression in EOC cell lines and selected OVSAHO and SKOV3 as a model with high LINC01465 levels. We silenced LINC01465 and performed proliferation, wound healing, invasion, and drug resistance assays. Knocking-down LINC01465 resulted in reduced migration, suggesting potential involvement in EOC. Furthermore, to identify the significance of LINC01465 in chemoresistance, we assessed the LINC01465 levels in A2780 S cells treated with malformin, which revealed higher LINC01465 expression as compared to untreated A2780S cells implying the involvement of LINC01465 in cell death. Thus, this study unraveled the repertoire of E2F5 regulated candidate genes and suggested a putative role of LINC01465 in malformin-induced cell death in EOC. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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- View/download PDF
47. Characterization of Histone Modifications in Late-Stage Rotator Cuff Tendinopathy.
- Author
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Orchard, Kayleigh J. A., Akbar, Moeed, Crowe, Lindsay A. N., Cole, John, Millar, Neal L., and Raleigh, Stuart M.
- Subjects
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ROTATOR cuff , *TENDINOPATHY , *FOCAL adhesions , *GENETICS , *CHROMATIN , *AXONS - Abstract
The development and progression of rotator cuff tendinopathy (RCT) is multifactorial and likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in RCT, including the role of histone modification, is not well established. Using chromatin immunoprecipitation sequencing, differences in the trimethylation status of H3K4 and H3K27 histones in late-stage RCT compared to control were investigated in this study. For H3K4, 24 genomic loci were found to be significantly more trimethylated in RCT compared to control (p < 0.05), implicating genes such as DKK2, JAG2, and SMOC2 in RCT. For H3K27, 31 loci were shown to be more trimethylated (p < 0.05) in RCT compared to control, inferring a role for EPHA3, ROCK1, and DEFβ115. Furthermore, 14 loci were significantly less trimethylated (p < 0.05) in control compared to RCT, implicating EFNA5, GDF6, and GDF7. Finally, the TGFβ signaling, axon guidance, and regulation of focal adhesion assembly pathways were found to be enriched in RCT. These findings suggest that the development and progression of RCT is, at least in part, under epigenetic control, highlighting the influence of histone modifications in this disorder and paving the way to further understand the role of epigenome in RCT. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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48. AtrR Is an Essential Determinant of Azole Resistance in Aspergillus fumigatus
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Paul, Sanjoy, Stamnes, Mark, Thomas, Grace Heredge, Liu, Hong, Hagiwara, Daisuke, Gomi, Katsuya, Filler, Scott G, and Moye-Rowley, W Scott
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Rare Diseases ,Prevention ,Biotechnology ,Human Genome ,Genetics ,Emerging Infectious Diseases ,Biodefense ,Infectious Diseases ,Vaccine Related ,Antimicrobial Resistance ,Infection ,Good Health and Well Being ,Antifungal Agents ,Aspergillus fumigatus ,Azoles ,Chromatin Immunoprecipitation ,DNA ,Fungal ,Drug Resistance ,Fungal ,Electrophoretic Mobility Shift Assay ,Fungal Proteins ,Gene Deletion ,Gene Expression Profiling ,Gene Expression Regulation ,Fungal ,Mutation ,Promoter Regions ,Genetic ,Protein Binding ,Regulon ,Sequence Analysis ,DNA ,Transcription Factors ,ABC transporters ,antifungal resistance ,molecular genetics ,transcriptional regulation ,Aspergillus fumigatus ,Microbiology - Abstract
Aspergillosis associated with azole-resistant Aspergillus fumigatus has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the cyp51A gene that encodes the target of azole drugs, lanosterol α-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of cyp51A and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of cyp51A as well as that of an important membrane transporter protein gene called abcG1 In this work, we characterize two different mutant alleles of atrR, either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of atrR for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the abcG1 and cyp51A promoters. The ATRE in cyp51A was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis.IMPORTANCEAspergillus fumigatus is the major filamentous fungal pathogen in humans. Infections associated with A. fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene cyp51A as an important contributor to resistance in A. fumigatus Here, we demonstrate that the transcription factor AtrR binds to a region in the cyp51A promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of A. fumigatus.
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- 2019
49. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
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Wienert, Beeke, Wyman, Stacia K, Richardson, Christopher D, Yeh, Charles D, Akcakaya, Pinar, Porritt, Michelle J, Morlock, Michaela, Vu, Jonathan T, Kazane, Katelynn R, Watry, Hannah L, Judge, Luke M, Conklin, Bruce R, Maresca, Marcello, and Corn, Jacob E
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Biochemistry and Cell Biology ,Biological Sciences ,Stem Cell Research - Induced Pluripotent Stem Cell ,Human Genome ,Stem Cell Research - Induced Pluripotent Stem Cell - Human ,Biotechnology ,Stem Cell Research ,Regenerative Medicine ,Genetics ,Aetiology ,2.2 Factors relating to the physical environment ,Adenoviridae ,Animals ,CRISPR-Associated Protein 9 ,CRISPR-Cas Systems ,Cell Line ,Chromatin Immunoprecipitation ,Clustered Regularly Interspaced Short Palindromic Repeats ,DNA ,DNA Breaks ,Double-Stranded ,DNA Repair ,DNA Repair Enzymes ,Gene Editing ,Humans ,Induced Pluripotent Stem Cells ,K562 Cells ,MRE11 Homologue Protein ,RNA ,Guide ,Kinetoplastida ,Sequence Analysis ,DNA ,General Science & Technology - Abstract
CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.
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- 2019
50. Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells.
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Hollerer, Ina, Barker, Juliet C, Jorgensen, Victoria, Tresenrider, Amy, Dugast-Darzacq, Claire, Chan, Leon Y, Darzacq, Xavier, Tjian, Robert, Ünal, Elçin, and Brar, Gloria A
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Humans ,Histones ,Chromatin Immunoprecipitation ,Gene Expression Regulation ,Models ,Genetic ,Proto-Oncogene Proteins c-mdm2 ,Promoter Regions ,Genetic ,Gene Knockdown Techniques ,MCF-7 Cells ,CRISPR-Cas Systems ,LUTI ,MDM2 ,gene expression regulation ,transcript isoform ,uORF translation ,Genetics - Abstract
We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2 We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.
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- 2019
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