PDF(Pubmed)
Abstract：
The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.
摘要：
丝裂原激活的蛋白激酶信号通路的功能是激活立即早期基因（IEGs）所必需的，包括EGR1和FOS，细胞生长和增殖。最近的研究已经确定拓扑异构酶II（TOP2）是IEGs转录激活的重要调节因子之一。然而,IEG激活中涉及TOP2的转录调控机制尚不清楚.这里,我们证明了ERK2而不是ERK1对IEG转录激活很重要，并报告了在EGR1基因上ERK2功能的关键ELK1结合序列。我们的数据表明，ERK1和ERK2均在相互和独特的残基上广泛磷酸化TOP2B的C末端结构域。尽管ERK1和ERK2都增强了松弛正DNA超螺旋所需的TOP2B的催化速率，ERK2延迟了负DNA超螺旋的TOP2B催化。此外,ERK1本身可以放松DNA超螺旋。ERK2催化抑制或敲低干扰转录并去调节IEGs中的TOP2B。此外，我们展示了人类细胞纯化的TOP2B和依托泊苷的第一个cryo-EM结构，以及对-423至332中的TOP2B具有最强亲和力的EGR1转录起始位点（-30至20）。该结构显示TOP2B介导的DNA断裂和剧烈弯曲。转录被依托泊苷激活，虽然它受到EGR1和FOS的ICRF193的抑制，表明TOP2B介导的DNA断裂有利于转录激活。一起来看,这项研究表明，激活的ERK2磷酸化TOP2B以调节TOP2-DNA相互作用并有利于IEGs的转录激活。我们建议TOP2B协会，催化作用,和在其底物DNA上的解离是调节转录的重要过程，而ERK2介导的TOP2B磷酸化可能是催化和解离步骤的关键。
