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Cyclin C Regulated Oxidative Stress Responsive Transcriptome in Mus Musculus Embryonic Fibroblasts

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Cyclin C Regulated Oxidative Stress Responsive Transcriptome in Mus Musculus Embryonic Fibroblasts Rowan University Rowan Digital Works School of Osteopathic Medicine Faculty Scholarship School of Osteopathic Medicine 6-5-2019 Cyclin C Regulated Oxidative Stress Responsive Transcriptome in Mus Musculus Embryonic Fibroblasts David C Stieg Rowan University Kai-Ti Chang Rowan University Katrina F Cooper Rowan University Randy Strich Rowan University Follow this and additional works at: https://rdw.rowan.edu/som_facpub Part of the Animal Experimentation and Research Commons, Genetic Phenomena Commons, Laboratory and Basic Science Research Commons, Medical Cell Biology Commons, and the Medical Genetics Commons Recommended Citation Stieg DC, Chang KT, Cooper KF, Strich R. Cyclin C regulated oxidative stress responsive transcriptome in mus musculus embryonic fibroblasts. G3 Genes - Genomes - Genetics. 2019 Jun 5;9(6):1901-1908. doi: 10.1534/g3.119.400077. PMID: 31036676. PMCID: PMC6553531. This Article is brought to you for free and open access by the School of Osteopathic Medicine at Rowan Digital Works. It has been accepted for inclusion in School of Osteopathic Medicine Faculty Scholarship by an authorized administrator of Rowan Digital Works. INVESTIGATION Cyclin C Regulated Oxidative Stress Responsive Transcriptome in Mus musculus Embryonic Fibroblasts David C. Stieg, Kai-Ti Chang, Katrina F. Cooper, and Randy Strich1 Department of Molecular Biology, Graduate School of Biological Sciences, Rowan University, Stratford, NJ 08084 ORCID IDs: 0000-0003-2746-3315 (D.C.S.); 0000-0003-3346-8972 (R.S.) ABSTRACT The transcriptional changes that occur in response to oxidative stress help direct the decision KEYWORDS to maintain cell viability or enter a cell death pathway. Cyclin C-Cdk8 is a conserved kinase that associates Cyclin C with the RNA polymerase II Mediator complex that stimulates or represses transcription depending on Oxidative Stress the locus. In response to oxidative stress, cyclin C, but not Cdk8, displays partial translocation into the RNA-seq cytoplasm. These findings open the possibility that cyclin C relocalization is a regulatory mechanism Cdk8 governing oxidative stress-induced transcriptional changes. In the present study, the cyclin C-dependent Mediator transcriptome was determined and compared to transcriptional changes occurring in oxidatively stressed Mus musculus embryonic fibroblasts. We observed a similar number (2000) of genes up or downregulated in oxidatively stressed cells. Induced genes include cellular repair/survival factors while repressed loci were generally involved in proliferation or differentiation. Depleting cyclin C in unstressed cells produced an approximately equal number of genes (2400) that were repressed by, or whose transcription required, cyclin C. Consistent with the possibility that cyclin C nuclear release contributes to tran- scriptional remodeling in response to oxidative stress, we found that 37% cyclin C-dependent genes were downregulated following stress. Moreover, 20% of cyclin C- repressed genes were induced in response to stress. These findings are consistent with a model that cyclin C relocalization to the cytoplasm, and corresponding inactivation of Cdk8, represents a regulatory mechanism to repress and stimulate transcription of stress-responsive genes. Reactive oxygen is a common cytotoxic compound that targets many be altered to repress growth-promoting genes, while inducing loci cellular compartments and macromolecules (Avery 2011). In response encoding damage control proteins such as chaperones or detoxify- to oxidative stress, the cell must decide whether to undergo a transient ing enzymes (Ma 2010). In either case, the activity of transcriptional growth arrest to repair the damage or enter a cell death pathway inducers and repressors of these genes must themselves be modulated. (Giannattasio, Guaragnella, Arbini, & Moro 2013; Han et al., 2008). Cyclin C, along with its cognate cyclin-dependent kinase 8 (Cdk8), are To accomplish either cell fate, the transcriptional program must components of the highly conserved Cdk8-Kinase Module (CKM). The CKM, also containing Med12 and Med13, interacts transiently with the mediator complex (Bourbon 2008) and plays both a positive and Copyright © 2019 Stieg et al. negative role in transcription (Nemet, Jelicic, Rubelj, & Sopta 2014). doi: https://doi.org/10.1534/g3.119.400077 For example, the CKM activates the b-catenin signaling pathway Manuscript received February 11, 2019; accepted for publication April 8, 2019; in colorectal cancer cells (Firestein et al., 2008) and functions as a published Early Online April 29, 2019. This is an open-access article distributed under the terms of the Creative coactivator with p53 to induce the Cdk inhibitor p21 (Donner, Szostek, Commons Attribution 4.0 International License (http://creativecommons.org/ Hoover, & Espinosa 2007). Conversely, the CKM represses transcription licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction by modifying cyclin H of TFIIH (Akoulitchev, Chuikov, & Reinberg in any medium, provided the original work is properly cited. 2000) and inducing the degradation of the intracellular domain (ICD) Supplemental material available at FigShare: https://doi.org/10.25387/ g3.8048756. involved in Notch signaling (Fryer, White, & Jones 2004). In support 1Corresponding author: Department of Molecular Biology, Two Medical Center of the importance of the CKM in ICD degradation, cyclin C has Drive, Stratford, NJ 08084 E-mail: [email protected] recently been identified as a tumor suppressor due to this activity Volume 9 | June 2019 | 1901 (Li et al., 2014). In mammalian cells, paralogs of Med12 (Med12L) were assessed using a Nanodrop UV Spectrophotometer (ThermoFisher). and Med13 (Med13L) also associate with cyclin C and Cdk8 but no Total RNA from each sample underwent further QC analysis including specific function has been described for these different CKM compo- NanoDrop2000, Qubit analysis and Agilent RNA ScreenTape System/ sitions (Allen & Taatjes 2015). In the budding yeast S. cerevisiae, the TapeStation (GENEWIZ). The RNA integrity numbers (RIN) for all CKM components play very similar roles primarily as transcriptional samples ranged from 8.5-10. The RNA was enriched for mRNA using repressors (van de Peppel et al., 2005). However, this may not be the polyA+ selection and a barcoded cDNA library was prepared for case during development in higher organisms (Li et al., 2014; Loncle each sample. The barcoded cDNA libraries were sequenced using an et al., 2007; Westerling, Kuuluvainen, & Makela 2007). Illumina HISEQ 2500 instrument to produce 25̴ million paired-end Although its role is more varied, the budding yeast CKM was initially reads for each sample. The raw data from the sequencing were identified as a repressor of stress responsive (Cooper, Mallory, Smith, & converted to FASTQ files for further analysis. Strich 1997; Holstege et al., 1998) and meiotic (Cooper et al., 1997; Surosky, Strich, & Esposito 1994) genes. To relieve this repression in Mapping RNA-seq reads response to oxidative stress, cyclin C translocates from the nucleus to The quality of each FASTQ file was assessed using FastQC then the cytoplasm where it is destroyed (Cooper et al., 2012). Preventing uploaded into the Galaxy environment (Afgan et al., 2018) for all cyclin C relocalization and destruction decreases, but does not elimi- subsequent analyses. The forward and reverse FASTQ files for each nate, gene induction (Cooper et al., 1997; Cooper et al., 2012). Prior to sample were paired and grouped into the following collections: WT, its proteolysis, the yeast cyclin C relocalizes to the mitochondria, where WT Stress, Ccnc2/2. The paired-end reads were mapped to the Ensembl it is necessary and sufficient for oxidative stress-induced fragmentation Mus musculus (GRCm38) cDNA FASTA file downloaded from of this organelle (Cooper, Khakhina, Kim, & Strich 2014; Cooper et al., UCSC (Casper et al.,2018).MappingwasperformedwithBowtie2 2012) and programmed cell death (PCD) (Cooper et al., 2014). This (Langmead & Salzberg 2012) using default settings. The Bowtie2 cytoplasmicroleforcyclinCinbothmitochondrialfission and mapping statistics for the indicated samples are listed in Table S1. PCD is conserved in mammalian cells (Ganesan et al., 2018; Wang, To generate count tables from the reads uniquely mapped to anno- Yan, Cooper, & Strich 2015). However, rather than all of cyclin C tated genes, featureCounts was performed on each sample using the translocating to the cytoplasm as observedinstressedyeastcells, GRCm38 annotation gtf file. The featureCounts data were filtered for only about 20% of total cyclin C is released following oxidative stress protein coding genes and aggregated for gene level analysis. in mammalian cells (Wang et al., 2015). Cyclin C nuclear release in yeast requires destruction of CKM component Med13 via the SCF DGE analysis ubiquitin ligase mediated by the F-box protein Grr1 (Stieg et al., The gene level data produced by featureCounts were used to de- 2018). Interestingly, the normal turnover of mammalian Med13 is termine differential gene expression (DGE) between samples. DGE dependent on the F-box protein Fbw7 (Davis et al., 2013), an ortho- analysis identifies genes that display statistically significant changes log of Grr1. Therefore, Med13 destruction in mammalian systems in transcript abundance between experimental conditions. DGE may be more selective allowing the precise removal of cyclin C, and analyses were completed using DESeq2 (Anders & Huber 2010) on thereby inhibiting
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