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Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors [Expression array]

ABSTRACT: We identified the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma derived U2OS cell line. This resulted in 2,140 transcripts mapping to 1,871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identified genomic loci bound by the G2/M transcription factor FOXM1 by Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associated these with cell cycle-regulated genes. FOXM1 was bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. ChIP-seq genomic loci were shown to be responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle-transcription factors (E2F1, E2F4, E2F6, and GABPA) from ENCODE and ChIP-seq data for the DREAM complex, found that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle regulated genes in a second cancer derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle.

ORGANISM(S): Homo sapiens

PROVIDER: GSE50988 | GEO | 2013/10/11

SECONDARY ACCESSION(S): PRJNA219724

REPOSITORIES: GEO

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Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors

Project description:We identified the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma derived U2OS cell line. This resulted in 2,140 transcripts mapping to 1,871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identified genomic loci bound by the G2/M transcription factor FOXM1 by Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associated these with cell cycle-regulated genes. FOXM1 was bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. ChIP-seq genomic loci were shown to be responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle-transcription factors (E2F1, E2F4, E2F6, and GABPA) from ENCODE and ChIP-seq data for the DREAM complex, found that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle regulated genes in a second cancer derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle. Cell cycle-regulated gene expression identified from three double thymidine time courses and one thymidine nocodazole time course.

2013-10-11 | E-GEOD-50988 | biostudies-arrayexpress

Genome-wide mapping of FOXM1 binding sites

Project description:There are nearly fifty forkhead (FOX) transcription factors encoded in the human genome and due to sharing a common DNA binding domain, they are all thought to bind to similar DNA sequences. It is therefore unclear how these transcription factors are targeted to specific chromatin regions to elicit specific biological effects. Here we used ChIP-seq to investigate the genome-wide chromatin binding mechanisms used by the forkhead transcription factor FOXM1. In keeping with its previous association with cell cycle control, we demonstrate that FOXM1 binds and regulates a group of genes which are mainly involved in controlling late cell cycle events in the G2 and M phases. However, rather than being recruited through canonical RYAAAYA forkhead binding motifs, FOXM1 binding is directed via CHR elements. FOXM1 binds these elements through protein-protein interactions with the MMB transcriptional activator complex. Thus, we have uncovered a novel and unexpected mode of chromatin binding of a FOX transcription factor which allows it to specifically control cell cycle-dependent gene expression. Examination of FOXM1 binding sites in G2/M U2OS cells

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The Forkhead box transcription factor FOXM1 is required for the maintenance of cell proliferation and protection against oxidative stress in human embryonic stem cells

Project description:Human embryonic stem cells (hESCs) exhibit unique cell cycle structure, self-renewal and pluripotency. The Forkhead box transcription factor M1 (FOXM1) is critically required for the maintenance of pluripotency in mouse embryonic stem cells and mouse embryonal carcinoma cells, but its role in hESCs remains unclear. Here, we show that FOXM1 expression was enriched in undifferentiated hESCs and was regulated in a cell cycle-dependent manner with peak levels detected at the G2/M phase. Expression of FOXM1 did not correlate with OCT4 and NANOG during in vitro differentiation of hESCs. Importantly, knockdown of FOXM1 expression led to aberrant cell cycle distribution with impairment in mitotic progression but showed no profound effect on the undifferentiated state. Interestingly, FOXM1 depletion sensitized hESCs to oxidative stress. Moreover, genome-wide analysis of FOXM1 targets by ChIP-seq identified genes important for M phase including CCNB1 and CDK1, which were subsequently confirmed by ChIP and RNA interference analyses. Further peak set comparison against a differentiating hESC line and a cancer cell line revealed substantial difference in the genomic binding profile of FOXM1 in hESCs. Taken together, our findings provide the first evidence to support FOXM1 as important regulator of cell cycle progression and defense against oxidative stress in hESCs.

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poly A+ RNA sequencing of cell cycle-synchronized RNA from U2OS cells

Project description:We performed paired-end poly A+ RNA sequencing using total RNA isolated from U2OS cells that are synchronised into G1, G1/S, S, G2 and M phase of cell cycle.

2020-10-27 | GSE143275 | GEO

Cell cycle expression profiles in HeLa cells

Project description:Periodic expression of cell cycle genes highlights the importance of precise temporal control of transcription in regulating cell cycle events. We used HeLa cells enriched for different phases of the cell cycle to identify genes that are expressed in a periodic fashion in specific cell cycle phases. These data were generated for comparison with ChIP-Sequencing data obtained for two subunits of the B-Myb-MuvB complex, B-Myb and LIN9, in HeLa cells. HeLa cells were synchronized at the beginning of S phase by double thymidine block and then released into the cell cycle by washing out the thymidine resulting in tight synchrony at S, G2 and M phases of the cell cycle. Most cells entered mitosis at 8 hours after release as determined by visual inspection. Three independent replicas of double thymidine block and release experiments were performed. RNA was isolated at specific times after release from the thymidine block (0, 2, 4, 6, 8 and 12 hours) and used for generating expression profiles.

2012-01-25 | E-GEOD-26922 | biostudies-arrayexpress

Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors [ChIP-seq]

Project description:We report the genome wide DNA binding patterns of wild type FOXM1 in asynchronous HeLa cells using chromatin precipitation followed by high-throughput sequencing (ChIP-seq). We find that FOXM1 is bound to the promoter of a number of cell cycle genes including PLK, AURKB, and CCNB1. FOXM1 ChIP-seq in asynchronous HeLa cells

2013-11-06 | E-GEOD-52098 | biostudies-arrayexpress

Transcription profiling of human cells expressing FoxM1-ER fusion protein

Project description:Experiment description To identify FoxM1-regulated genes, we carried out gene expression profile analysis after inducible activation of a FoxM1-ER fusion protein using high-density human cDNA microarrays. We constructed a stable line of U2OS cells expressing full-length HA-FoxM1 fused at its C-terminus to the ligand-binding domain of the estrogen receptor (ER), mutated such that it specifically responds to 4-hydroxytamoxifen (4-OHT) but not to estrogen. In the absence of 4-OHT the activity of the ER fusion protein is turned off, but can be rapidly induced when cells are exposed to 4-OHT. We isolated total RNA from untreated cultures and cultures treated with 4-OHT for 6 or 16 hr. As activation of FoxM1 results in accumulation of cells in G2/M, we additionally analyzed the induction of gene expression by FoxM1 in G1/S-synchronized cells by a thymidine block. Total RNA derived from untreated or stimulated FoxM1-ER cells was amplified, reverse transcribed, labeled and hybridized to cDNA microarrays containing 18,000 cDNAs and expressed sequence tags (ESTs). Bound cDNA was detected using Cy3 or Cy5 dyes. In each independent experiment we included reverted color controls (Cy3 (4-OHT treated); Cy5 (untreated) or Cy3 (untreated); Cy5 (treated)) and the expression profile was expressed as a Cy5:Cy3 ratio.

2005-01-01 | E-MEXP-230 | biostudies-arrayexpress

Hypoxia-induced alterations of G2 checkpoint regulators

Project description:Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, in addition to DNA repair, genome stability is also controlled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can alter the DNA damage cell cycle checkpoints. Here, we show that hypoxia (24h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, flow cytometric bar-coding analysis of individual cells showed that the levels of several G2 checkpoint regulators were reduced in G2 phase cells after hypoxic exposure, in particular cyclin B1. These effects were accompanied by decreased Cyclin dependent kinase (CDK) activity in G2 phase cells after hypoxia. Furthermore, cells pre-exposed to hypoxia showed a longer G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (~0.03 % O2 20h and 0.2% O2 72h). These results demonstrate that the DNA damage G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors. We measured gene expression changes in U2OS cells after treatment with 0.2% hypoxia for 24 hours. The data were used in the exploration of hyopxia induced alterations in the G2 checkpoint.

2016-01-04 | E-GEOD-70530 | biostudies-arrayexpress

ChIP-seq of H2A.Z and its histone chaperones during the cell cycle

Project description:The aim of this study was to uncover cell cycle dependent chromatin binding of H2A.Z and its histone chaperones. U2OS cells were stably transfected with Twin-Strep-tagged H2A.Z, ANP32e, or YL1 constructs, and WT cells were used as a negative/background control. U2OS cells were synchronised in G1 or G2-M phase using hydroxyurea or nocodazole, respectively, and protein-DNA complexes were isolated by affinity purification.

2023-06-29 | E-MTAB-11617 | biostudies-arrayexpress

Transcriptome of plants cells in the G2/M phase of the cell cycle

Project description:Gene expression must be reconfigured rapidly during the subsequent phases of the cell cycle to execute the cellular functions specific of each phase. Post-transcriptional regulation has a predominant role in modulating gene expression during the mitotic cell cycle, including among other mechanisms, protein phosphorylation and ubiquitination, differential protein stability and mRNA localization and translatability. Regulation at the transcriptional level is also important, as studies conducted in synchronized plant cell suspension cultures have identified hundreds of genes with periodic patterns of genes expression across the phases of the cell cycle. We describe here an alternative strategy to cell suspension cultures to profile the transcriptome of Arabidopsis root cells in the G2/M phase of the cell cycle. Through fluorescence activated cell sorting we first isolated cells in G2/M using CYCB1;1-GFP, a reporter of a mitotic cyclin. The analysis of the transcriptome of these cells allowed us to identify hundreds of genes whose expression is depleted or enriched in G2/M cells.

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