Targeting an E2F site in the mouse genome prevents promoter silencing in quiescent and post-mitotic cells

Tavner, Fiona; Frampton, Jon; Watson, Roger J.

doi:10.1038/sj.onc.1210087

Cited by 35 publications

(33 citation statements)

References 22 publications

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“…Western blotting was carried out with primary antibodies as follows: Flag M2 (F1804 or F3165; Sigma), FOXM1 (C-20; Santa Cruz), LIN9 (ab62329; Abcam), LIN37 (kindly provided by J. DeCaprio [11]), B-MYB (BMyb LX015.1; kindly provided by R. Watson [15]), p130 (C-20; Santa Cruz), E2F4 (C-20; Santa Cruz), and NFYA (G2; Santa Cruz). Coimmunoprecipitation was carried out using the Miltenyi green fluorescent protein (GFP) kit (Miltenyi Biotech) for GFP-tagged FOXM1 according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

The Forkhead Transcription Factor FOXM1 Controls Cell Cycle-Dependent Gene Expression through an Atypical Chromatin Binding Mechanism

Chen

Müller

Quaas

et al. 2013

Molecular and Cellular Biology

205

239

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bThere are nearly 50 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 chromatin immunoprecipitation followed by sequencing (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 G 2 and M phases. However, rather than being recruited through canonical RYAAAYA forkhead binding motifs, FOXM1 binding is directed via CHR (cell cycle genes homology region) 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 that allows it to specifically control cell cycle-dependent gene expression. There are nearly 50 different forkhead transcription factors encoded in mammalian genomes, and these proteins all contain the conserved forkhead DNA binding domain (reviewed in references 1 and 2). Forkhead transcription factors are involved in controlling a wide range of biological processes and are aberrantly expressed or regulated in disease states, including cancer (reviewed in reference 2). However, due to sharing a common DNA binding domain, forkhead transcription factors are generally believed to bind to variations of the RYAAAYA motif. Hence, it is unclear how individual forkhead proteins are specifically recruited to the regulatory regions of different cohorts of target genes to control defined biological responses. One key process which is controlled by forkhead transcription factors is the cell cycle and, in particular, the G 2 -M transition. The initial links to G 2 -M control were made with the Saccharomyces cerevisiae forkhead protein Fkh2, which controls the temporal expression of a cluster of genes at this phase of the cell cycle (reviewed in reference 3). More recently, members of the FOXO and FOXM classes of forkhead transcription factors have been linked with controlling the same process in mammalian cells (4-6). In both cases, forkhead transcription factors coordinate the integration of signals from the cell cycle regulatory machinery to transcriptional outputs. This is exemplified by the links to the cell cycle regulated Polo-like kinase PLK1, which is recruited to cell cycle-regulated promoters through promoter elements bound by the forkhead transcription factors FOXM1 and Fkh2, albeit indirectly in the case of Fkh2 (7,8).In mammalian cells, the transcriptional control of a cluster of genes at the G 2 -M transition, is coordinated through promoter elements which typically contain CHR (cell cycle genes homology region) and...

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Section: Methodsmentioning

confidence: 99%

The Forkhead Transcription Factor FOXM1 Controls Cell Cycle-Dependent Gene Expression through an Atypical Chromatin Binding Mechanism

Chen

Müller

Quaas

et al. 2013

Molecular and Cellular Biology

205

239

View full text Add to dashboard Cite

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“…40), CDC25A (1:200; F-6, sc7389, Santa Cruz), FLAG (1:5,000; M2, 61415, Sigma), GFP (1:1,000; 11814460001, Roche), HA (1:2,000; MMS-101P, Covance), Histone 3 (1:10,000; ab1791, Abcam), p53 (1:100; DO-1), PLK1 (1:1,000; H152, sc5585, Santa Cruz), phospho-PLK1-T210 (1:200; ADI-KAP-CC107-D, Enzo Life Science), Vinculin (1:50,000; V9131, Sigma) and Actin (1:20,000; mAB1501, Chemicon Int.). The B-Myb LX015.1 monoclonal antibody (1:10) was a kind gift from Roger Watson 41 . B-Myb levels in Fig.…”

Section: Methodsmentioning

confidence: 99%

Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control

et al. 2015

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Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.

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“…The B-Myb LX015.1 monoclonal antibody was described previously (Tavner et al, 2007). Histone H3K4(trimethyl) (ab8580), polyclonal RbAp48 (ab50307) and monoclonal RbAp48 (ab488) antibodies were from Abcam; focal adhesion kinase (FAK) (sc-903), B-Myb (sc-724), p107 (sc-318), p130 (sc-317) and Cyclin B1 (sc-245) antibodies were from Santa Cruz (Santa Cruz, CA, USA); b-actin (A-2066) and b-tubulin (C-4585) antibodies were from Sigma (Poole, UK).…”

Section: Antibodiesmentioning

confidence: 99%

“…For luciferase assays, 10 5 F9 cells plated in 24-well dishes were transfected with 0.33 mg of pGL2-basic luciferase reporter, 0.4 mg of pSuper shRNA and 0.1 mg of pSV-bgal transfection control using 2 ml lipofectamine 2000 and OptiMEM. After incubating for 24-48 hours, the luciferase activity was measured as described previously (Tavner et al, 2007).…”

Section: Luciferase Reporter Assaysmentioning

confidence: 99%

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A Lin-9 complex is recruited by B-Myb to activate transcription of G2/M genes in undifferentiated embryonal carcinoma cells

2009

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It has recently been discovered that cell-cycle gene transcription is regulated by a core complex named LINC that switches from a transcriptionally repressive complex in G 0 -G 1 with the p130 or p107 pocket proteins and E2F4 to a transcriptionally active complex in S-G 2 containing B-Myb. We have studied the function of LINC in F9 embryonal carcinoma cells, which are distinguished by a rapid cell cycle resulting from an extremely short G 1 phase. We show that suppressing expression of the LINC component, Lin-9, in F9 cells causes arrest in mitosis, and we have used this system to screen for transcriptional targets. In these cells, B-Myb was found in complexes with Lin-9 and several other LINC constituents, however, the pocket proteins did not associate with LINC unless F9 cells were differentiated. Lin-9 and B-Myb were both required for transcription of G 2 /M genes such as Cyclin B1 and Survivin. Moreover, B-Myb was demonstrated to recruit Lin-9 to the Survivin promoter through multiple Myb-binding sites. The demonstration that a B-Myb/ LINC complex is vital for progression through mitosis in cells lacking a G 1 /S checkpoint has implications for both undifferentiated embryonal cells and for cancers in which pocket protein function is compromised.

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Targeting an E2F site in the mouse genome prevents promoter silencing in quiescent and post-mitotic cells

Cited by 35 publications

References 22 publications

The Forkhead Transcription Factor FOXM1 Controls Cell Cycle-Dependent Gene Expression through an Atypical Chromatin Binding Mechanism

The Forkhead Transcription Factor FOXM1 Controls Cell Cycle-Dependent Gene Expression through an Atypical Chromatin Binding Mechanism

Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control

A Lin-9 complex is recruited by B-Myb to activate transcription of G2/M genes in undifferentiated embryonal carcinoma cells

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