Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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MYC is a major determinant of mitotic cell fate

ABSTRACT: Purpose: To determine global gene expression changes following siRNA knockdown of Myc, Kcnk1, and Snta1 compared to non-targeting siRNAs or mock-transfected cells Methods: Total RNA was processed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit according to manufacturer’s protocol. Generated cDNA libraries were sequenced using an Illumina HiSeq 2000 sequencer with four biological replicates sequenced per condition using single read, 50 cycle runs. Quality of sequencing reads were assessed using FastQC (Babraham Bioinformatics) and then aligned to a reference genome (hg19, UCSC Genome Browser) using TopHat. Sequencing yielded, on average, 23.7 million unique reads per sample with a 60.7 - 65.7% mapping rate. Cufflinks was used to generate transcript abundance for each annotated protein-coding gene as Fragments Per Kilobase of transcript per Million mapped reads (FPKM), and statistical analysis and comparison of FPKM values was calculated using R (Bioconductor). Results: Fold changes comparing mock and a non-targeting siRNA were highly congruent. Myc RNAi induced numerous changes, with 955 downregulated genes and 1214 upregulated genes. The effect on Myc itself was relatively modest, possibly reflecting its ability to negatively auto-regulate its own expression. Gene ontology analysis highlighted ribosome biogenesis, metabolism, gene expression, cell cycle, and apoptosis pathways, consistent with known Myc functions. The Kcnk1 siRNA affected 424 genes, with KCNK1 itself one of the most repressed. While gene ontology analysis also highlighted metabolism and biosynthesis pathways, the p-values and fold enrichment scores were substantially lower, indicating that DiM can be suppressed without major effects on metabolism and biosynthesis pathways. The Snta1 siRNA deregulated 575 genes, with SNTA1 itself the most repressed gene. Cell cycle and mitosis-related gene ontology terms feature heavily, consistent with this siRNA accelerating mitotic exit. Interestingly, FoxM1, which drives G2/M gene expression was reduced 1.75-fold, indicating that this siRNA may disrupt mitotic controls by deregulating FoxM1. Conclusions: Global gene expression profiling identifies Egr1 as regulator of mitotic cell fate. Total RNA was extracted from at least four replicates of siRNA-transfected cells and libraries for sequencing was prepared for each replicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Ying Sun

PROVIDER: E-GEOD-68219 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

MYC Is a Major Determinant of Mitotic Cell Fate.

Topham Caroline C Tighe Anthony A Ly Peter P Bennett Ailsa A Sloss Olivia O Nelson Louisa L Ridgway Rachel A RA Huels David D Littler Samantha S Schandl Claudia C Sun Ying Y Bechi Beatrice B Procter David J DJ Sansom Owen J OJ Cleveland Don W DW Taylor Stephen S SS

Cancer cell 20150701 1

Taxol and other antimitotic agents are frontline chemotherapy agents but the mechanisms responsible for patient benefit remain unclear. Following a genome-wide siRNA screen, we identified the oncogenic transcription factor Myc as a taxol sensitizer. Using time-lapse imaging to correlate mitotic behavior with cell fate, we show that Myc sensitizes cells to mitotic blockers and agents that accelerate mitotic progression. Myc achieves this by upregulating a cluster of redundant pro-apoptotic BH3-on ...[more]

PMID: 26175417

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Project description:Purpose: To determine global gene expression changes following siRNA knockdown of Myc, Kcnk1, and Snta1 compared to non-targeting siRNAs or mock-transfected cells Methods: Total RNA was processed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit according to manufacturer’s protocol. Generated cDNA libraries were sequenced using an Illumina HiSeq 2000 sequencer with four biological replicates sequenced per condition using single read, 50 cycle runs. Quality of sequencing reads were assessed using FastQC (Babraham Bioinformatics) and then aligned to a reference genome (hg19, UCSC Genome Browser) using TopHat. Sequencing yielded, on average, 23.7 million unique reads per sample with a 60.7 - 65.7% mapping rate. Cufflinks was used to generate transcript abundance for each annotated protein-coding gene as Fragments Per Kilobase of transcript per Million mapped reads (FPKM), and statistical analysis and comparison of FPKM values was calculated using R (Bioconductor). Results: Fold changes comparing mock and a non-targeting siRNA were highly congruent. Myc RNAi induced numerous changes, with 955 downregulated genes and 1214 upregulated genes. The effect on Myc itself was relatively modest, possibly reflecting its ability to negatively auto-regulate its own expression. Gene ontology analysis highlighted ribosome biogenesis, metabolism, gene expression, cell cycle, and apoptosis pathways, consistent with known Myc functions. The Kcnk1 siRNA affected 424 genes, with KCNK1 itself one of the most repressed. While gene ontology analysis also highlighted metabolism and biosynthesis pathways, the p-values and fold enrichment scores were substantially lower, indicating that DiM can be suppressed without major effects on metabolism and biosynthesis pathways. The Snta1 siRNA deregulated 575 genes, with SNTA1 itself the most repressed gene. Cell cycle and mitosis-related gene ontology terms feature heavily, consistent with this siRNA accelerating mitotic exit. Interestingly, FoxM1, which drives G2/M gene expression was reduced 1.75-fold, indicating that this siRNA may disrupt mitotic controls by deregulating FoxM1. Conclusions: Global gene expression profiling identifies Egr1 as regulator of mitotic cell fate.

Project description:FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS. Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knock-in mice expressing mislocalized cytoplasmic FUS and complete FUS knock-out mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knock-in mice, but not FUS knock-out mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons. Total RNA from brains of E18.5 knock-in (FusÎNLS/ÎNLS), and knock-out (Fus-/-) mice and their control littermates were extracted with Trizol and libraries were prepared for RNA sequencing and RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Generation sequencing (RASL-seq) (Li et al. 2012; Zhou et al. 2012). For the RNA-seq experiment, biological replicates were used with n=4-5 knock-in animals per group (4 wild-type and 5 homozygous FusÎNLS) and n=5 knock-out animals per group (5 wild-type and 5 homozygous Fus-/-). For the RASL-seq analysis, biological replicates were used with n=4 knock-in animals per group (4 wild-type, 4 heterozygous and 4 homozygous FusÎNLS) and n=5 animals per group (5 wild-type and 5 homozygous Fus-/- knock-out).

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MYC is a major determinant of mitotic cell fate

Publications

MYC Is a Major Determinant of Mitotic Cell Fate.

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