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

Grant GD, Brooks L, Zhang X, Mahoney JM, Martyanov V, Wood TA, Sherlock G, Cheng C, Whitfield ML - Mol. Biol. Cell (2013)

Bottom Line: This results in 2140 transcripts mapping to 1871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305.

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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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).
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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).

Mentions: To understand the network of transcription factors controlling cell cycle–regulated gene expression and better understand the role of FOXM1 in the transcriptional program that regulates mitosis, we performed ChIP-seq for endogenous FOXM1 in asynchronous HeLa cells. IPs were performed in duplicate using an antibody against the endogenous FOXM1 (C20; Santa Cruz Biotechnology) and sequenced independently. As a conservative estimate of FOXM1 binding, we focused the analysis on those ChIP-seq regions found in both ChIP-seq assays. The first sequencing analysis (run A) gave 17.1 million sequence reads, with 8.4 million reads mapped (Hg 19), and the second sequencing analysis (run B) gave 17.0 million sequence reads, with 8.4 million mapped. Using the MACS module in Cistrome (Zhang et al., 2008; Liu et al., 2011; www.cistrome.com), we found 5727 peaks in A and 2849 peaks in B, with 2215 regions identified in both; these regions could be associated with 2367 unique genes by the Gene Centered Annotation (GCA) module in Cistrome (Shin et al., 2009; Figure 4 and Supplemental Figure S4). We refer to these intersecting regions as FOXM1 genomic loci. Of the genomic loci, 36.8% were in promoter regions within 3000 base pairs upstream from the transcription start site, and 1.9% of the genomic loci were within 3000 base pairs downstream of the transcription start site. Of the genomic loci, 20.4% were in the 5′-untranslated region (5′UTR) of target genes and 1.1% were in the 3′UTR. Genomic loci in exonic regions accounted for 5.3%, and 17.8% of the loci were in intronic regions. Distal intergenic regions accounted for 16.6% of the ChIP-seq genomic loci (Figure 3A).


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

Grant GD, Brooks L, Zhang X, Mahoney JM, Martyanov V, Wood TA, Sherlock G, Cheng C, Whitfield ML - Mol. Biol. Cell (2013)

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3842991&req=5

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).
Mentions: To understand the network of transcription factors controlling cell cycle–regulated gene expression and better understand the role of FOXM1 in the transcriptional program that regulates mitosis, we performed ChIP-seq for endogenous FOXM1 in asynchronous HeLa cells. IPs were performed in duplicate using an antibody against the endogenous FOXM1 (C20; Santa Cruz Biotechnology) and sequenced independently. As a conservative estimate of FOXM1 binding, we focused the analysis on those ChIP-seq regions found in both ChIP-seq assays. The first sequencing analysis (run A) gave 17.1 million sequence reads, with 8.4 million reads mapped (Hg 19), and the second sequencing analysis (run B) gave 17.0 million sequence reads, with 8.4 million mapped. Using the MACS module in Cistrome (Zhang et al., 2008; Liu et al., 2011; www.cistrome.com), we found 5727 peaks in A and 2849 peaks in B, with 2215 regions identified in both; these regions could be associated with 2367 unique genes by the Gene Centered Annotation (GCA) module in Cistrome (Shin et al., 2009; Figure 4 and Supplemental Figure S4). We refer to these intersecting regions as FOXM1 genomic loci. Of the genomic loci, 36.8% were in promoter regions within 3000 base pairs upstream from the transcription start site, and 1.9% of the genomic loci were within 3000 base pairs downstream of the transcription start site. Of the genomic loci, 20.4% were in the 5′-untranslated region (5′UTR) of target genes and 1.1% were in the 3′UTR. Genomic loci in exonic regions accounted for 5.3%, and 17.8% of the loci were in intronic regions. Distal intergenic regions accounted for 16.6% of the ChIP-seq genomic loci (Figure 3A).

Bottom Line: This results in 2140 transcripts mapping to 1871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305.

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.

Show MeSH