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Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control

We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell...

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Autores principales: Grant, Gavin D., Gamsby, Joshua, Martyanov, Viktor, Brooks, Lionel, George, Lacy K., Mahoney, J. Matthew, Loros, Jennifer J., Dunlap, Jay C., Whitfield, Michael L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418304/
https://www.ncbi.nlm.nih.gov/pubmed/22740631
http://dx.doi.org/10.1091/mbc.E11-02-0170
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author Grant, Gavin D.
Gamsby, Joshua
Martyanov, Viktor
Brooks, Lionel
George, Lacy K.
Mahoney, J. Matthew
Loros, Jennifer J.
Dunlap, Jay C.
Whitfield, Michael L.
author_facet Grant, Gavin D.
Gamsby, Joshua
Martyanov, Viktor
Brooks, Lionel
George, Lacy K.
Mahoney, J. Matthew
Loros, Jennifer J.
Dunlap, Jay C.
Whitfield, Michael L.
author_sort Grant, Gavin D.
collection PubMed
description We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.
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spelling pubmed-34183042012-10-30 Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control Grant, Gavin D. Gamsby, Joshua Martyanov, Viktor Brooks, Lionel George, Lacy K. Mahoney, J. Matthew Loros, Jennifer J. Dunlap, Jay C. Whitfield, Michael L. Mol Biol Cell Articles We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant. The American Society for Cell Biology 2012-08-15 /pmc/articles/PMC3418304/ /pubmed/22740631 http://dx.doi.org/10.1091/mbc.E11-02-0170 Text en © 2012 Grant et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell BD; are registered trademarks of The American Society of Cell Biology.
spellingShingle Articles
Grant, Gavin D.
Gamsby, Joshua
Martyanov, Viktor
Brooks, Lionel
George, Lacy K.
Mahoney, J. Matthew
Loros, Jennifer J.
Dunlap, Jay C.
Whitfield, Michael L.
Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control
title Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control
title_full Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control
title_fullStr Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control
title_full_unstemmed Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control
title_short Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control
title_sort live-cell monitoring of periodic gene expression in synchronous human cells identifies forkhead genes involved in cell cycle control
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418304/
https://www.ncbi.nlm.nih.gov/pubmed/22740631
http://dx.doi.org/10.1091/mbc.E11-02-0170
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