4 results on '"Iveta Gazova"'
Search Results
2. Global Analysis of Transcription Start Sites in the New Ovine Reference Genome (Oar rambouillet v1.0)
- Author
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Mazdak Salavati, Alex Caulton, Richard Clark, Iveta Gazova, Timothy P. L. Smith, Kim C. Worley, Noelle E. Cockett, Alan L. Archibald, Shannon M. Clarke, Brenda M. Murdoch, and Emily L. Clark
- Subjects
ovine ,TSS ,CAGE ,WGBS ,promoter ,enhancer ,Genetics ,QH426-470 - Abstract
The overall aim of the Ovine FAANG project is to provide a comprehensive annotation of the new highly contiguous sheep reference genome sequence (Oar rambouillet v1.0). Mapping of transcription start sites (TSS) is a key first step in understanding transcript regulation and diversity. Using 56 tissue samples collected from the reference ewe Benz2616, we have performed a global analysis of TSS and TSS-Enhancer clusters using Cap Analysis Gene Expression (CAGE) sequencing. CAGE measures RNA expression by 5′ cap-trapping and has been specifically designed to allow the characterization of TSS within promoters to single-nucleotide resolution. We have adapted an analysis pipeline that uses TagDust2 for clean-up and trimming, Bowtie2 for mapping, CAGEfightR for clustering, and the Integrative Genomics Viewer (IGV) for visualization. Mapping of CAGE tags indicated that the expression levels of CAGE tag clusters varied across tissues. Expression profiles across tissues were validated using corresponding polyA+ mRNA-Seq data from the same samples. After removal of CAGE tags with
- Published
- 2020
- Full Text
- View/download PDF
3. CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1
- Author
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Iveta Gažová, Lucas Lefevre, Stephen J. Bush, Rocio Rojo, David A. Hume, Andreas Lengeling, and Kim M. Summers
- Subjects
histone deubiquitinases ,epigenetic modifications ,THP-1 cell line ,genome editing ,macrophage ,monocyte ,Biology (General) ,QH301-705.5 - Abstract
USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.
- Published
- 2021
- Full Text
- View/download PDF
4. The Transcriptional Network That Controls Growth Arrest and Macrophage Differentiation in the Human Myeloid Leukemia Cell Line THP-1
- Author
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Iveta Gažová, Lucas Lefevre, Stephen J. Bush, Sara Clohisey, Erik Arner, Michiel de Hoon, Jessica Severin, Lucas van Duin, Robin Andersson, Andreas Lengeling, David A. Hume, and Kim M. Summers
- Subjects
macrophage ,monocyte ,THP-1 cells ,differentiation ,transcriptome ,cell cycle ,Biology (General) ,QH301-705.5 - Abstract
The response of the human acute myeloid leukemia cell line THP-1 to phorbol esters has been widely studied to test candidate leukemia therapies and as a model of cell cycle arrest and monocyte-macrophage differentiation. Here we have employed Cap Analysis of Gene Expression (CAGE) to analyze a dense time course of transcriptional regulation in THP-1 cells treated with phorbol myristate acetate (PMA) over 96 h. PMA treatment greatly reduced the numbers of cells entering S phase and also blocked cells exiting G2/M. The PMA-treated cells became adherent and expression of mature macrophage-specific genes increased progressively over the duration of the time course. Within 1–2 h PMA induced known targets of tumor protein p53 (TP53), notably CDKN1A, followed by gradual down-regulation of cell-cycle associated genes. Also within the first 2 h, PMA induced immediate early genes including transcription factor genes encoding proteins implicated in macrophage differentiation (EGR2, JUN, MAFB) and down-regulated genes for transcription factors involved in immature myeloid cell proliferation (MYB, IRF8, GFI1). The dense time course revealed that the response to PMA was not linear and progressive. Rather, network-based clustering of the time course data highlighted a sequential cascade of transient up- and down-regulated expression of genes encoding feedback regulators, as well as transcription factors associated with macrophage differentiation and their inferred target genes. CAGE also identified known and candidate novel enhancers expressed in THP-1 cells and many novel inducible genes that currently lack functional annotation and/or had no previously known function in macrophages. The time course is available on the ZENBU platform allowing comparison to FANTOM4 and FANTOM5 data.
- Published
- 2020
- Full Text
- View/download PDF
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