Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.
Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.
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DOI：
10.1091/mbc.e11-02-0170
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发表时间：
2012-08
影响因子：
3.3
通讯作者：
Whitfield ML
中科院分区：
文献类型：
--
作者：
Grant GD;Gamsby J;Martyanov V;Brooks L 3rd;George LK;Mahoney JM;Loros JJ;Dunlap JC;Whitfield ML
来自同步U2OS细胞周期调控启动子的周期性荧光素酶报告系统可测量活细胞中周期性的、细胞周期调控的基因表达。该试验用于鉴定控制周期性基因表达的叉头转录因子，并鉴定FOXK1是关键细胞周期基因的激活因子。我们开发了一个系统来监测同步细胞中细胞周期调节启动子的周期性荧光素酶活性。报告子由一个极小的人E2F1启动子驱动，该启动子在G1/S时达到峰值表达，或者由一个具有6个叉头dna结合位点的基础启动子驱动，该启动子在G2/M时达到峰值表达。在细胞周期同步后，在三到四个同步细胞周期中每隔10分钟在活细胞中测量荧光素酶活性，从而实现细胞周期调节基因表达的前所未有的分辨率。我们用这个实验筛选叉头转录因子来控制周期性基因表达。我们确认了FOXM1的作用，并鉴定了两个新的细胞周期调节因子FOXJ3和FOXK1。FOXJ3和FOXK1的敲除消除了细胞周期依赖性振荡，导致细胞增殖率下降。FOXJ3和FOXK1调控的基因分析表明，FOXJ3可能调控锌指蛋白网络，FOXK1与启动子结合，调控DHFR、TYMS、GSDMD和E2F结合伙伴TFDP1。染色质免疫沉淀和高通量测序分析鉴定出4329个FOXK1结合的基因组位点，其中83%含有FOXK1结合基序。我们证实了这些基因座的一个子集被野生型FOXK1激活，而不是被FOXK1 (H355A) dna结合突变体激活。
A periodic luciferase reporter system from cell cycle–regulated promoters in synchronous U2OS cells measures periodic, cell cycle–regulated gene expression in live cells. This assay is used to identify Forkhead transcription factors that control periodic gene expression, and it identifies FOXK1 as an activator of key cell cycle genes. 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.