Mechanisms of Plk1 at the mitotic centromere

有丝分裂着丝粒 Plk1 的机制

• 批准号: 10179607
• 负责人: Mark E Burkard
• 金额: $ 30.54万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10179607
• 关键词: Affect; Binding; Biochemical; Biological Assay; Biological Process; Bloom Syndrome; Bloom syndrome protein; Case Study; Cells; Centromere; Chemicals; Chromatin; Chromosome Arm; Chromosome Segregation; Chromosomes; Complex; DNA-Directed RNA Polymerase; Disease; Dislocations; Environment; Enzymes; Event; Genetic Transcription; Genome; Genomic Instability; Goals; Human; Kinetochores; Learning; Location; Mammalian Cell; Maps; Mediating; Microscopic; Microtubules; Mitosis; Mitotic; Mitotic spindle; PLK1 gene; Phenotype; Phosphorylation; Phosphorylation Site; Production; RNA; Regulation; Resolution; Role; Signal Transduction; Structure; System; Techniques; Testing; Transcript; Transcriptional Regulation; Work; arm; base; centromere protein C; chemical genetics; experimental study; genome integrity; helicase; human disease; innovation; insight; recruit

SUMMARY Our goal is to identify how Polo-like kinase 1 (Plk1) signals at the human centromere to maintain genome integrity. In mitosis, the kinetochore (KT) is assembled at the centromere and this complex is a major focus of Plk1 activity. The KT is a 100 nm-structure assembled on the centromere that attaches chromosomes to the mitotic spindle. Using super-resolution techniques, we have discovered that the bulk of Plk1 localizes to the centromere, directly on chromatin >50 nm from the outer KT. Our long-term goal is to delineate Plk1 partners, substrates, and timing of its activities to maintain faithful chromosome segregation. We are currently focused on activities at the centromere, building on our key findings: Bloom Syndrome RecQ Helicase (BLM) directly or indirectly mediates chromosome arm dislocation and centromere unwinding that occur with loss of Plk1 activity; Plk1 regulates nascent transcripts on chromatin in mitosis and phosphorylates the N-terminus of Centromere Protein C (CENP-C), both known to maintain KT integrity. Our central hypothesis is that a discrete centromere pool of Plk1 stabilizes the mitotic centromere and operates through inactivating helicases, by supporting transcription, and by maintaining CENP-C function. In Aim 1, we will map the spatial environments of Plk1 that contribute to its functions along the KT-centromere axis. We will test the idea that a chromatin-localized pool of Plk1 targets substrates and mediates activities separately from a KT-localized pool, to regulate faithful chromosome segregation. Aim 2 will test how Plk1 dampens BLM helicase function to maintain centromere integrity against mitotic pulling forces. To do this, we will identify Plk1 phosphorylation sites on the BLM protein, determine the role of Plk1 on BLM localization, and evaluate how phosphorylation modulates its helicase activities in biochemical assays and cells. Aim 3 will identify the role of Plk1 on CENP-C stabilization via direct phosphorylation and transcriptional regulation. We have already mapped Plk1 phosphorylation sites on CENP- C and have discovered that Plk1 regulates mitotic transcription. Towards this end, we will functionally analyze the CENP-C phosphorylation events and identify the functional effect of Plk1 on RNA polymerases and mitotic RNAs. Together, this work will reveal how Plk1 operates regionally in the KT to maintain genomic integrity.

概括 我们的目标是确定人类丝粒中的类似polo样激酶1（PLK1）信号如何维持基因组 正直。在有丝分裂中，动力学（KT）在丝粒处组装，该复合物是 PLK1活性。 KT是在Centromere上组装的100 nm结构，将染色体连接到 有丝分裂主轴。使用超分辨率技术，我们发现大部分PLK1本地化 Centromere，直接在距离外KT> 50 nm的染色质上。我们的长期目标是描述PLK1合作伙伴， 底物及其维持忠实染色体隔离的活动的时机。我们目前专注于 CentRomere的活动，基于我们的主要发现：Bloom综合征RECQ解旋酶（BLM）或直接 间接介导PLK1活性丧失而发生的染色体臂脱位和丝粒脱落； PLK1调节有丝分裂中染色质的新生转录本，并磷酸化着丝粒的N末端 蛋白C（CENP-C），都已知可以维持KT完整性。我们的中心假设是一个离散的中心粒 PLK1池稳定有丝分裂的丝粒，并通过灭活的解旋酶进行操作 转录，并通过维护CENP-C功能。在AIM 1中，我们将绘制PLK1的空间环境 沿着KT-中心轴轴的功能有助于其功能。我们将测试一个染色质定位池的想法 PLK1靶向基材并分别与KT位置的池分开进行调节活动，以规范忠实 染色体分离。 AIM 2将测试PLK1如何抑制BLM解旋酶的功能以保持中心粒 对有丝分裂拉力的完整性。为此，我们将确定BLM蛋白上的PLK1磷酸化位点， 确定PLK1在BLM定位中的作用，并评估磷酸化如何调节其解旋酶 生化测定和细胞中的活性。 AIM 3将通过直接确定PLK1对CENP-C稳定的作用 磷酸化和转录调节。我们已经在CENP-上绘制了PLK1磷酸化位点 C并发现PLK1调节有丝分裂转录。为此，我们将在功能上分析 CENP-C磷酸化事件并确定PLK1对RNA聚合酶和有丝分裂的功能效应 RNA。这项工作将共同揭示PLK1在KT中的区域运行以维持基因组完整性。