Mechanisms of Plk1 at the mitotic centromere

有丝分裂着丝粒 Plk1 的机制

• 批准号: 10598560
• 负责人: Mark E Burkard
• 金额: $ 30.57万
• 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/10598560
• 关键词: Affect; Binding; Biochemical; Biological Assay; Biological Process; Bloom Syndrome; Case Study; Cells; Centromere; Chemicals; Chromatin; Chromosome Arm; Chromosome Segregation; Chromosomes; Complex; DNA-Directed RNA Polymerase; Disease; Dislocations; Environment; Enzymes; Event; Genetic Transcription; Genome Stability; Genomic Instability; Goals; Human; Kinetochores; Learning; Location; Mammalian Cell; Maps; Mediating; Microscopic; Microtubules; Mitosis; Mitotic; Mitotic spindle; PLK1 gene; Phenotype; Phosphorylation; Phosphorylation Site; Production; Proteins; RNA; Regulation; Role; Signal Transduction; Structure; System; Techniques; Testing; Transcript; Transcriptional Regulation; Work; arm; centromere protein C; chemical genetics; experimental study; genome integrity; helicase; human disease; innovation; insight; recruit; ultra high resolution

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 Polo 样激酶 1 (Plk1) 如何在人类着丝粒上发出信号以维持基因组 正直。在有丝分裂中，着丝粒 (KT) 在着丝粒处组装，该复合体是有丝分裂的主要焦点 Plk1 活动。 KT 是组装在着丝粒上的 100 nm 结构，将染色体附着在着丝粒上 有丝分裂纺锤体。使用超分辨率技术，我们发现 Plk1 的大部分定位于 着丝粒，直接位于距离外部 KT >50 nm 的染色质上。我们的长期目标是圈定Plk1合作伙伴， 底物及其活动的时间以维持忠实的染色体分离。我们目前专注于 着丝粒的活动，以我们的主要发现为基础：直接或直接布卢姆综合征 RecQ 解旋酶 (BLM) 间接介导因 Plk1 活性丧失而发生的染色体臂错位和着丝粒解旋； Plk1 调节有丝分裂中染色质上的新生转录本并磷酸化着丝粒的 N 末端 蛋白 C (CENP-C)，两者均已知可维持 KT 完整性。我们的中心假设是离散的着丝粒 Plk1 池可稳定有丝分裂着丝粒，并通过失活解旋酶发挥作用，通过支持 转录，并维持 CENP-C 功能。在目标 1 中，我们将绘制 Plk1 的空间环境， 有助于其沿 KT 着丝粒轴的功能。我们将测试染色质定位池的想法 Plk1 靶向底物并介导与 KT 局部池分开的活动，以调节忠实的 染色体分离。目标 2 将测试 Plk1 如何抑制 BLM 解旋酶功能以维持着丝粒 对抗有丝分裂拉力的完整性。为此，我们将识别 BLM 蛋白上的 Plk1 磷酸化位点， 确定 Plk1 对 BLM 定位的作用，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。目标 3 将通过直接确定 Plk1 对 CENP-C 稳定的作用 磷酸化和转录调控。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，我们将从功能上进行分析 CENP-C 磷酸化事件并确定 Plk1 对 RNA 聚合酶和有丝分裂的功能影响 RNA。这项工作将共同揭示 Plk1 如何在 KT 中进行区域性运作以维持基因组完整性。