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Cited by 181Genomics and Chromatin DynamicsCancer-related Molecular PathwaysGene Regulatory Network Analysis

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

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Grant, Gavin D., Brooks, Lionel, Zhang, Xiaoyang, Mahoney, J. Matthew, Martyanov, Viktor, Wood, Tammara A., Sherlock, Gavin, Cheng, Chao, Whitfield, Michael L.·2013·Molecular Biology of the Cell
DOI 10.1091/mbc.e13-05-0264PMC PMC3842991
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Abstract

We identify the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma-derived U2OS cell line. This results in 2140 transcripts mapping to 1871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identify genomic loci bound by the G2/M transcription factor FOXM1 by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associate these with cell cycle-regulated genes. FOXM1 is bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. We show that ChIP-seq genomic loci are 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 the Encyclopedia of DNA Elements and ChIP-seq data for the DREAM complex finds 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.

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FIGURE 1: Periodic expression of well-characterized cell cycle genes. (A) Expression profiles of known periodically expressed genes for each of the four time courses. The purple bars indicate S phase, and black arrows indicate mitosis as estimated by flow cytometry or Western blots. (B) The idealized vector for each phase is the average expression of the known genes shown in A.
FIGURE 1: Periodic expression of well-characterized cell cycle genes. (A) Expression profiles of known periodically expressed genes for each of the four time courses. The purple bars indicate S phase, and black arrows indicate mitosis as estimated by flow cytometry or Western blots. (B) The idealized vector for each phase is the average expression of the known genes shown in A.CC BY-NC-SA · PubMed Central
FIGURE 2: Periodically expressed genes in the U2OS cell cycle. (A) The 2830 probes that show periodic expression in U2OS cells, ordered by the point of their peak expression as calculated from their sine and cosine values in the Fourier transform. Phase assignments were performed using the correlation coefficients with the ideal vectors defined in Figure 1. The color bar on the right indicates the phase of peak expression (G1/S, green; S, blue; G2, red; G2/M, yellow; and M/G1, black); blended colors indicate interspersed phase assignments. (B) The 2830 periodically expressed probes from U2OS cells were ordered by average linkage hierarchical clustering. The enriched biological processes of select clusters is indicated.
FIGURE 2: Periodically expressed genes in the U2OS cell cycle. (A) The 2830 probes that show periodic expression in U2OS cells, ordered by the point of their peak expression as calculated from their sine and cosine values in the Fourier transform. Phase assignments were performed using the correlation coefficients with the ideal vectors defined in Figure 1. The color bar on the right indicates the phase of peak expression (G1/S, green; S, blue; G2, red; G2/M, yellow; and M/G1, black); blended colors indicate interspersed phase assignments. (B) The 2830 periodically expressed probes from U2OS cells were ordered by average linkage hierarchical clustering. The enriched biological processes of select clusters is indicated.CC BY-NC-SA · PubMed Central
FIGURE 3: FOXM1 ChIP-seq indicates a role for FOXM1 in the cell cycle and translation. (A) CEAS analysis of the overlapping FOXM1 ChIP-seq analysis shows enrichment in promoters, downstream regions, 5′UTRs, and coding exons relative to background. (B) Venn diagram showing the overlapping gene targets of FOXM1, B-Myb, and LIN9 (b-Myb and LIN9 results from Sadasivam et al., 2012). There is enrichment in cell cycle Gene Ontology (GO) terms for biological process (BP) and cellular component (CC) in the overlapping gene lists for FOXM1/B-Myb, FOXM1/B-Myb/LIN9, and FOXM1/LIN9. FOXM1 genes that do not overlap with B-Myb or LIN9 show an enrichment in the GO term translation. (C) Bar graph showing the percentage of FOXM1 targets per cell cycle phase. (D) Bar graph showing the percentage of genes in each phase that are bound by FOXM1.
FIGURE 3: FOXM1 ChIP-seq indicates a role for FOXM1 in the cell cycle and translation. (A) CEAS analysis of the overlapping FOXM1 ChIP-seq analysis shows enrichment in promoters, downstream regions, 5′UTRs, and coding exons relative to background. (B) Venn diagram showing the overlapping gene targets of FOXM1, B-Myb, and LIN9 (b-Myb and LIN9 results from Sadasivam et al., 2012). There is enrichment in cell cycle Gene Ontology (GO) terms for biological process (BP) and cellular component (CC) in the overlapping gene lists for FOXM1/B-Myb, FOXM1/B-Myb/LIN9, and FOXM1/LIN9. FOXM1 genes that do not overlap with B-Myb or LIN9 show an enrichment in the GO term translation. (C) Bar graph showing the percentage of FOXM1 targets per cell cycle phase. (D) Bar graph showing the percentage of genes in each phase that are bound by FOXM1.CC BY-NC-SA · PubMed Central
FIGURE 4: The expression profiles of genes bound by FOXM1. The clustered U2OS and HeLa cell expression profiles of the 501 genes bound by FOXM1 that are cell cycle regulated in U2OS cells. FOXM1 transcription factor binding is shown as percentage coverage of the UCSC genome browser gene model as defined by GCA for each gene (see Supplemental Figure S6 for more detail).
FIGURE 4: The expression profiles of genes bound by FOXM1. The clustered U2OS and HeLa cell expression profiles of the 501 genes bound by FOXM1 that are cell cycle regulated in U2OS cells. FOXM1 transcription factor binding is shown as percentage coverage of the UCSC genome browser gene model as defined by GCA for each gene (see Supplemental Figure S6 for more detail).CC BY-NC-SA · PubMed Central
FIGURE 5: FOXM1 overexpression activates the expression of a number of target genes found via ChIP-seq. (A) Neither FOXM1 (light line) nor the empty vector control (dark line) activates the ACAP3 promoter. ACAP3 is a known FOXK1 target (Grant et al., 2012) and is used as a negative control. (B–D) FOXM1 activates the promoters of PLK1, CENPE, and TOP2A, known FOXM1 targets (Figure 4 and Supplemental Figure S6). (E) FOXM1 activates the MCM8 promoter. (F) FOXM1 activates the promoter of RPS6KB1, which phosphorylates the 6S ribosomal protein. (G) The previously unknown FOXM1 target RMI1 promoter is activated by FOXM1 overexpression. Error bars, SEM. Time points were binned and averaged for every 24 h for a one-way analysis of variance. *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.005.
FIGURE 5: FOXM1 overexpression activates the expression of a number of target genes found via ChIP-seq. (A) Neither FOXM1 (light line) nor the empty vector control (dark line) activates the ACAP3 promoter. ACAP3 is a known FOXK1 target (Grant et al., 2012) and is used as a negative control. (B–D) FOXM1 activates the promoters of PLK1, CENPE, and TOP2A, known FOXM1 targets (Figure 4 and Supplemental Figure S6). (E) FOXM1 activates the MCM8 promoter. (F) FOXM1 activates the promoter of RPS6KB1, which phosphorylates the 6S ribosomal protein. (G) The previously unknown FOXM1 target RMI1 promoter is activated by FOXM1 overexpression. Error bars, SEM. Time points were binned and averaged for every 24 h for a one-way analysis of variance. *p ≤ 0.05; **p ≤ 0.01, ***p ≤ 0.005.CC BY-NC-SA · PubMed Central
FIGURE 6: Biological process–specific gene clusters have coordinated transcription factor binding. (A) After the inclusion of FOXM1 ChIP-seq data as well as ENCODE data, hierarchical clustering shows that the genes in the U2OS S phase cluster are predominantly bound by E2F1 and E2F4. (B) The histone cluster shows E2F4 bound to more gene models than E2F1 or FOXM1. (C) Genes in the mitosis gene cluster are primarily bound by FOXM1 or E2F4. Transcription factor binding is shown as percentage of coverage of each portion of the gene model as defined by GCA (see Supplemental Figure S6 for more details).
FIGURE 6: Biological process–specific gene clusters have coordinated transcription factor binding. (A) After the inclusion of FOXM1 ChIP-seq data as well as ENCODE data, hierarchical clustering shows that the genes in the U2OS S phase cluster are predominantly bound by E2F1 and E2F4. (B) The histone cluster shows E2F4 bound to more gene models than E2F1 or FOXM1. (C) Genes in the mitosis gene cluster are primarily bound by FOXM1 or E2F4. Transcription factor binding is shown as percentage of coverage of each portion of the gene model as defined by GCA (see Supplemental Figure S6 for more details).CC BY-NC-SA · PubMed Central
FIGURE 7: Transcription factor binding as a function of cell cycle phase. (A) Enrichment of transcription factor targets using a sliding window across the cell cycle indicates enrichment of E2F1, E2F4, and FOXM1 binding to genes that show peak expression at specific times during the cell cycle. Phase angles were calculated from the arc tangent of the Fourier analysis. Transcription factor binding enrichment was calculated using Fischer's exact test over a sliding window of 30° with a 10° overlap between neighboring windows. Cell cycle phase is indicated (B) Gene density in each sliding window was calculated for the expression of all the cell cycle–regulated genes in U2OS cells. (C) Enrichment of transcription factor targets in each cell cycle phase was calculated using estimated phase boundaries rather than a sliding window. The significance of transcription factor binding enrichment was calculated using Fischer's exact test. Transcription factor binding data were from HeLa cells, except for E2F6, which was from K562 cells. The p values for each transcription factor at each phase are given in Table 2.
FIGURE 7: Transcription factor binding as a function of cell cycle phase. (A) Enrichment of transcription factor targets using a sliding window across the cell cycle indicates enrichment of E2F1, E2F4, and FOXM1 binding to genes that show peak expression at specific times during the cell cycle. Phase angles were calculated from the arc tangent of the Fourier analysis. Transcription factor binding enrichment was calculated using Fischer's exact test over a sliding window of 30° with a 10° overlap between neighboring windows. Cell cycle phase is indicated (B) Gene density in each sliding window was calculated for the expression of all the cell cycle–regulated genes in U2OS cells. (C) Enrichment of transcription factor targets in each cell cycle phase was calculated using estimated phase boundaries rather than a sliding window. The significance of transcription factor binding enrichment was calculated using Fischer's exact test. Transcription factor binding data were from HeLa cells, except for E2F6, which was from K562 cells. The p values for each transcription factor at each phase are given in Table 2.CC BY-NC-SA · PubMed Central
FIGURE 8: Core cell cycle–regulated genes between HeLa cells and U2OS cells are bound by FOXM1 or E2F1. The 253 genes that show periodic regulation in both HeLa cells and U2OS cells have been clustered after the inclusion of the FOXM1 and FOXK1 ChIP-seq data and the ENCODE data for selected transcription factors. Transcription factor binding is shown as percentage of coverage of each portion of the gene model as defined by GCA (see Supplemental Figure S6 for more details).
FIGURE 8: Core cell cycle–regulated genes between HeLa cells and U2OS cells are bound by FOXM1 or E2F1. The 253 genes that show periodic regulation in both HeLa cells and U2OS cells have been clustered after the inclusion of the FOXM1 and FOXK1 ChIP-seq data and the ENCODE data for selected transcription factors. Transcription factor binding is shown as percentage of coverage of each portion of the gene model as defined by GCA (see Supplemental Figure S6 for more details).CC BY-NC-SA · PubMed Central

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Research works printed but not linked: 6

  1. Chen W, 2011, Asian Pac J Cancer Prev, 12, 1785
  2. Cho RJ, 2001, Nat Genet, 27, 48
  3. Rozen S, 2000, Methods Mol Biol, 132, 365
  4. Slansky JE, 1996, Curr Top Microbiol Immunol, 208, 1
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Metadata

Authors
Grant, G. D., Brooks, L., Zhang, X., Mahoney, J. M., Martyanov, V., Wood, T. A., Sherlock, G., Cheng, C., & Whitfield, M. L.
Affiliations
  • Dartmouth College (US)
  • Stanford Medicine (US)
Corresponding author
Whitfield, Michael L.
ORCID
Venue
Molecular Biology of the Cell
Publisher
American Society for Cell Biology (ASCB)
Published
2013-12
Volume
24
Issue
23
Pages
3634-3650
ISSN
  • 1059-1524 (ISSN-L)
  • 1939-4586
DOI
10.1091/mbc.e13-05-0264
PMC
PMC3842991
OpenAlex
W2102123859
Cited by
179
References
92
Citations by year
2026: 5 · 2025: 11 · 2024: 13 · 2023: 6 · 2022: 10 · 2021: 21 · 2020: 19 · 2019: 17 · 2018: 14 · 2017: 20 · 2016: 17 · 2015: 14 · 2014: 12
FWCI
6.42
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • Molecular Biology of the Cell (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • Foundation for the National Institutes of Health
  • V Foundation for Cancer Research
  • National Human Genome Research Institute (R01HG004499)
  • National Cancer Institute (R25CA134286, R01CA130795)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
Keywords
cell cycle-regulated genes, periodic gene expression, FOXM1, E2F transcription factors, G2/M phase, S phase, osteosarcoma, U2OS cells, transcriptional regulation, DREAM complex, GABPA, E2F1, E2F4, E2F6, promoter activation
MeSH terms
Forkhead Box Protein M1, Cell Cycle, Multigene Family, HeLa Cells, Humans, Protein Binding, RNA, Messenger, Time Factors, Transcription, Genetic, Protein Biosynthesis, Gene Expression Regulation, Neoplastic, Genes, cdc, Gene Expression Profiling, Chromatin Immunoprecipitation, E2F Transcription Factors, Forkhead Transcription Factors

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