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Cyclin C Regulated Oxidative Stress Responsive Transcriptome in Mus 2 Musculus Embryonic Fibroblasts

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Cyclin C Regulated Oxidative Stress Responsive Transcriptome in Mus 2 Musculus Embryonic Fibroblasts G3: Genes|Genomes|Genetics Early Online, published on April 29, 2019 as doi:10.1534/g3.119.400077 1 Cyclin C Regulated Oxidative Stress Responsive Transcriptome in Mus 2 musculus Embryonic Fibroblasts 3 4 David C. Stieg*, Kai-Ti Chang*, Katrina F. Cooper*, Randy Strich*† 5 *Department of Molecular Biology, 6 Graduate School of Biological Sciences, 7 Rowan University, Stratford, NJ 08084. © The Author(s) 2013. Published by the Genetics Society of America. 8 Running title: Cyclin C oxidative stress transcriptome 9 10 †corresponding author 11 Department of Molecular Biology 12 Two Medical Center Drive 13 Stratford, NJ 08084 USA 14 Voice +1 856 566-6043 15 email: [email protected] 16 17 18 Key Words: 19 Cyclin C; Oxidative Stress; RNA-seq; Cdk8; Mediator 2 20 ABSTRACT 21 The transcriptional changes that occur in response to oxidative stress help direct the decision to 22 maintain cell viability or enter a cell death pathway. Cyclin C-Cdk8 is a conserved kinase that 23 associates with the RNA polymerase II Mediator complex that stimulates or represses 24 transcription depending on the locus. In response to oxidative stress, cyclin C, but not Cdk8, 25 displays partial translocation into the cytoplasm. These findings open the possibility that cyclin 26 C relocalization is a regulatory mechanism governing oxidative stress-induced transcriptional 27 changes. In the present study, the cyclin C-dependent transcriptome was determined and 28 compared to transcriptional changes occurring in oxidatively stressed Mus musculus embryonic 29 fibroblasts. We observed a similar number (~2000) of genes up or downregulated in oxidatively 30 stressed cells. Induced genes include cellular repair/survival factors while repressed loci were 31 generally involved in proliferation or differentiation. Depleting cyclin C in unstressed cells 32 produced an approximately equal number of genes (~2400) that were repressed by, or whose 33 transcription required, cyclin C. Consistent with the possibility that cyclin C nuclear release 34 contributes to transcriptional remodeling in response to oxidative stress, we found that 37% 35 cyclin C-dependent genes were downregulated following stress. Moreover, 20% of cyclin C- 36 repressed genes were induced in response to stress. These findings are consistent with a model 37 that cyclin C relocalization to the cytoplasm, and corresponding inactivation of Cdk8, represents 38 a regulatory mechanism to repress and stimulate transcription of stress-responsive genes. 39 40 41 3 42 INTRODUCTION 43 44 Reactive oxygen is a common cytotoxic compound that targets many cellular compartments 45 and macromolecules (Avery, 2011). In response to oxidative stress, the cell must decide 46 whether to undergo a transient growth arrest to repair the damage or enter a cell death 47 pathway (Giannattasio, Guaragnella, Arbini, & Moro, 2013; Han et al., 2008). To accomplish 48 either cell fate, the transcriptional program must be altered to repress growth-promoting 49 genes, while inducing loci encoding damage control proteins such as chaperones or detoxifying 50 enzymes (Ma, 2010). In either case, the activity of transcriptional inducers and repressors of 51 these genes must themselves be modulated. Cyclin C, along with its cognate cyclin-dependent 52 kinase 8 (Cdk8), are components of the highly conserved Cdk8-Kinase Module (CKM). The CKM, 53 also containing Med12 and Med13, interacts transiently with the mediator complex (Bourbon, 54 2008) and plays both a positive and negative role in transcription (Nemet, Jelicic, Rubelj, & 55 Sopta, 2014). For example, the CKM activates the β-catenin signaling pathway in colorectal 56 cancer cells (Firestein et al., 2008) and functions as a coactivator with p53 to induce the Cdk 57 inhibitor p21 (Donner, Szostek, Hoover, & Espinosa, 2007). Conversely, the CKM represses 58 transcription by modifying cyclin H of TFIIH (Akoulitchev, Chuikov, & Reinberg, 2000) and 59 inducing the degradation of the intracellular domain (ICD) involved in Notch signaling (Fryer, 60 White, & Jones, 2004). In support of the importance of the CKM in ICD degradation, cyclin C has 61 recently been identified as a tumor suppressor due to this activity (Li et al., 2014). In 62 mammalian cells, paralogs of Med12 (Med12L) and Med13 (Med13L) also associate with cyclin 63 C and Cdk8 but no specific function has been described for these different CKM compositions 4 64 (Allen & Taatjes, 2015). In the budding yeast S. cerevisiae, the CKM components play very 65 similar roles primarily as transcriptional repressors (van de Peppel et al., 2005). However, this 66 may not be the case during development in higher organisms (Li et al., 2014; Loncle et al., 2007; 67 Westerling, Kuuluvainen, & Makela, 2007). 68 Although its role is more varied, the budding yeast CKM was initially identified as a 69 repressor of stress responsive (Cooper, Mallory, Smith, & Strich, 1997; Holstege et al., 1998) 70 and meiotic (Cooper et al., 1997; Surosky, Strich, & Esposito, 1994) genes. To relieve this 71 repression in response to oxidative stress, cyclin C translocates from the nucleus to the 72 cytoplasm where it is destroyed (Cooper et al., 2012). Preventing cyclin C relocalization and 73 destruction decreases, but does not eliminate, gene induction (Cooper et al., 1997; Cooper et 74 al., 2012). Prior to its proteolysis, the yeast cyclin C relocalizes to the mitochondria, where it is 75 necessary and sufficient for oxidative stress-induced fragmentation of this organelle (Cooper, 76 Khakhina, Kim, & Strich, 2014; Cooper et al., 2012) and programmed cell death (PCD) (Cooper 77 et al., 2014). This cytoplasmic role for cyclin C in both mitochondrial fission and PCD is 78 conserved in mammalian cells (Ganesan et al., 2018; Wang, Yan, Cooper, & Strich, 2015). 79 However, rather than all of cyclin C translocating to the cytoplasm as observed in stressed yeast 80 cells, only about 20% of total cyclin C is released following oxidative stress in mammalian cells 81 (Wang et al., 2015). Cyclin C nuclear release in yeast requires destruction of CKM component 82 Med13 via the SCF ubiquitin ligase mediated by the F-box protein Grr1 (Stieg et al., 2018). 83 Interestingly, the normal turnover of mammalian Med13 is dependent on the F-box protein 84 Fbw7 (Davis et al., 2013), an ortholog of Grr1. Therefore, Med13 destruction in mammalian 5 85 systems may be more selective allowing the precise removal of cyclin C, and thereby inhibiting 86 Cdk8 activity, from specific promoters in response to stressors such as reactive oxygen. 87 In this study, we combined a bioinformatics approach with Ccnc+/+ and Ccnc-/- 88 immortalized mouse embryonic fibroblasts (MEFs) to identify genes repressed or induced in 89 response to oxidative stress exposure, and the role of cyclin C in the transcriptional regulation 90 of those genes. These studies revealed that 20-30 percent of stress-repressed or induced genes 91 is also controlled by cyclin C. In addition, the results point to a potential role for cyclin C nuclear 92 to cytoplasmic shuttling in mediating the correct cellular response to oxidative stress exposure. 93 94 MATERIALS AND METHODS 95 96 Cell culture 97 Ccnc+/+ (WT) and Ccnc−/− (Wang et al., 2015) immortalized MEF cells were cultured in Dulbecco’s 98 Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% -/- 99 penicillin/streptomycin at 37°C and 5% CO2. Replicate WT and Ccnc MEF cultures were grown 100 to 80% confluence and treated as follows. Oxidative stressed cells were exposed to 0.4 mM 101 H2O2 in DMEM without FBS or antibiotics for 4 h. Control unstressed WT MEFs were treated 102 with DMSO in DMEM without FBS and antibiotics for 4 h. Following treatments, cells were 103 harvested using trypsin and RNA was extracted as described below. All experiments were 104 conducted in triplicate for each condition. 105 106 107 Total RNA extraction and RNA-seq 6 108 Total RNA was prepared from the cell samples using mechanical disruption and the QIAGEN® 109 RNeasy kit. RNA quality and quantity were assessed using a Nanodrop UV Spectrophotometer 110 (ThermoFisher). Total RNA from each sample underwent further QC analysis including 111 NanoDrop™2000, Qubit analysis and Agilent RNA ScreenTape System™/TapeStation 112 (GENEWIZ®). The RNA integrity numbers (RIN) for all samples ranged from 8.5-10. The RNA was 113 enriched for mRNA using polyA+ selection and a barcoded cDNA library was prepared for each 114 sample. The barcoded cDNA libraries were sequenced using an Illumina® HISEQ 2500 115 instrument to produce ̴25 million paired-end reads for each sample. The raw data from the 116 sequencing were converted to FASTQ files for further analysis. 117 Mapping RNA-seq reads 118 119 The quality of each FASTQ file was assessed using FastQC then uploaded into the Galaxy 120 environment (Afgan et al., 2018) for all subsequent analyses. The forward and reverse FASTQ 121 files for each sample were paired and grouped into the following collections: WT, WT Stress, 122 Ccnc-/-. The paired-end reads were mapped to the Ensembl Mus musculus (GRCm38) cDNA 123 FASTA file downloaded from UCSC (Casper et al., 2018). Mapping was performed with Bowtie2 124 (Langmead & Salzberg, 2012) using default settings. The Bowtie2 mapping statistics for the 125 indicated samples are listed in Table S1. To generate count tables from the reads uniquely 126 mapped to annotated genes, featureCounts was performed on each sample using the GRCm38 127 annotation gtf file. The featureCounts data were filtered for protein coding genes and 128 aggregated for gene level analysis. 129 DGE analysis 7 130 The gene level data produced by featureCounts were used to determine differential gene 131 expression (DGE) between samples. DGE analysis identifies genes that display statistically 132 significant changes in transcript abundance between experimental conditions. DGE analyses 133 were completed using DESeq2 (Anders & Huber, 2010) on the following group comparisons: 134 WT:WT Stress; WT:Ccnc-/-.
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