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Regulation of Ku70 methylation and functions by SETD4

SETD4 对 Ku70 甲基化和功能的调节

基本信息

批准号：
10546482
负责人：
Zhiyuan Shen
金额：
$ 35.2万
依托单位：
RUTGERS BIOMEDICAL AND HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10546482
关键词：
Acetylation Acinus organ component Actins Affect Alleles Animal Model Antibodies Apoptosis Apoptotic BAX gene Bax protein Binding Biochemistry C-terminal Cell Nucleus Cells Cellular biology Chromatin Cytoplasm DNA DNA Damage DNA Double Strand Break DNA Repair Development Dimerization Double Strand Break Repair Elements Ensure Enzymes Epitopes Event Excision G22P1 gene Genome Goals Health Histidine Histones Human Human Genome Induction of Apoptosis Ionizing radiation Knock-in Mouse Ku70 protein LoxP-flanked allele Lysine Malignant Neoplasms Measures Mediating Methylation Methyltransferase Mitochondria Modeling Molecular Molecular Biology Molecular Conformation Mus Nonhomologous DNA End Joining Nuclear Oxygenases Plants Play Positioning Attribute Proteins Reagent Regulation Ribulose-Bisphosphate Carboxylase Role S-Adenosylhomocysteine S-Adenosylmethionine SET Domain Side Signal Transduction Site Tertiary Protein Structure Testing Time XRCC5 gene antibody detection dimer in vivo irradiation methyl group monomer mouse genetics non-histone protein novel overexpression recruit response restraint ribulose-1,5-bisphosphate success therapy outcome tumor tumorigenesis

项目摘要

Regulation of Ku70 Methylation and Functions by SETD4 (Abstract) The objective of this study is to elucidate a novel regulatory mechanism of Ku70 functions that are controlled by lysine methylation. Ku70 is a critical protein in DNA damage repair, especially during the initiation of non- homologous end-joining after irradiation. This function is carried out by its dimerization with Ku80 and encircling of DNA at the break sites. The free form of Ku70 is known for its anti-apoptosis activity in the cytoplasm, due to its binding with the pro-apoptosis protein BAX. Our preliminary studies suggest that SETD4, a putative non- histone methyl-transferase, methylated Ku70 to cause Ku70 relocation to the cytoplasm. Over-expression of SETD4 suppressed apoptosis, while SETD4 depletion sensitized it. SETD4’s chromatin-binding was dependent on Ku70, but not vice versa. SETD4 can be recruited to DNA damage sites, but only at a relatively mid-late time point after DNA damage. Based on these novel findings, we hypothesize that Ku70 methylation by SETD4 plays a critical role for the functional translocation of Ku70 from DNA double strand breaks (DSB) to the cytoplasm. We have generated highly specific antibodies against methylated Ku70 and SETD4, and several Ku70 and SETD4 knock-in mouse lines. We strive to use a combined approach that integrates biochemistry, cell and molecular biology, and mouse genetics to test our hypothesis. In Aim 1, we will focus on Ku70-methylation and its anti-apoptotic and DNA repair activities. First, the consequence of Ku70 methylation on Ku70/Ku80 dimer stability and its binding to DNA will be determined. Second, the cytoplasmic activity of methylated Ku70 in apoptosis will be verified with non-DNA damaging agents. Third, the direct effect of Ku70 methylation on Ku70 recruitment and retention at DNA damage sites, DSB repair efficiency, and cellular sensitivity to ionizing radiation will be measured. Lastly, we will use in-house developed Ku70 knock-in mice to characterize the functions of the lysine-containing SAP domain of Ku70 and its methylation in vivo. In Aim 2, we will focus on how SETD4 regulates apoptosis and DNA damage response through Ku70. First, we will identify the structural elements that are critical for SETD4 to methylate Ku70. Second, the consequence of SETD4 modulation on apoptosis will be measured in cells incapable of Ku70 methylation. Third, we predict that, while Ku70 is required for SETD4 recruitment to DNA damage sites, the SETD4’s enzymatic activity may be required for Ku70 disassociation from DNA damage sites. Thus, the mutual roles of SETD4 and Ku70 on their recruitment and/or retention at DNA damage sites will be determined, and their effects on DNA repair will be assessed. Lastly, we have tagged the floxed mouse Setd4 allele with V5 and Flag (V5F) epitopes. We will use this mouse line to systematically analyze SETD4’s role in Ku70 methylation, and its subsequent contributions in development and tumorigenesis. These studies are expected to elucidate a previous unknown mechanism that coordinates Ku70 functions in the nucleus and cytoplasm. The success of this project is ensured by our unique reagents and animal models as part of a rigorous approach.

SETD4 对 Ku70 甲基化和功能的调节（抽象的）本研究的目的是阐明 Ku70 功能的一种新的调控机制，该机制由赖氨酸甲基化。 Ku70 是 DNA 损伤修复中的关键蛋白，尤其是在非损伤修复过程中辐射后的同源末端连接。该功能是通过其与 Ku80 的二聚化并环化来实现的 DNA 断裂位点。 Ku70 的游离形式以其在细胞质中的抗凋亡活性而闻名，这是由于它与促凋亡蛋白 BAX 结合。我们的初步研究表明 SETD4，一个假定的非组蛋白甲基转移酶将 Ku70 甲基化，导致 Ku70 迁移到细胞质。过度表达 SETD4 抑制细胞凋亡，而 SETD4 缺失则使其敏感。 SETD4 的染色质结合具有依赖性在 Ku70 上，但反之则不然。 SETD4可以被招募到DNA损伤位点，但只能在相对中后期的时间 DNA 损伤后的点。基于这些新发现，我们假设 SETD4 的 Ku70 甲基化作用 Ku70 从 DNA 双链断裂 (DSB) 到细胞质的功能易位发挥着关键作用。我们已经生成了针对甲基化 Ku70 和 SETD4 的高度特异性抗体，以及几种 Ku70 和 SETD4 敲入小鼠系。我们努力采用综合方法，将生物化学、细胞和分子生物学和小鼠遗传学来检验我们的假设。在目标 1 中，我们将重点关注 Ku70 甲基化和其抗凋亡和 DNA 修复活性。一、Ku70甲基化对Ku70/Ku80二聚体的影响稳定性及其与 DNA 的结合将被确定。二、甲基化Ku70的细胞质活性细胞凋亡将用非DNA损伤剂来验证。三、Ku70甲基化对Ku70的直接影响 DNA 损伤位点的募集和保留、DSB 修复效率以及细胞对电离辐射的敏感性将被测量。最后，我们将使用内部开发的 Ku70 敲入小鼠来表征 Ku70 含有赖氨酸的 SAP 结构域及其体内甲基化。在目标 2 中，我们将重点关注 SETD4 如何通过 Ku70 调节细胞凋亡和 DNA 损伤反应。首先，我们将确定结构要素对于 SETD4 甲基化 Ku70 至关重要。其次，SETD4 调节对细胞凋亡的影响将是在无法进行 Ku70 甲基化的细胞中进行测量。第三，我们预测，虽然 SETD4 需要 Ku70 募集到 DNA 损伤位点后，SETD4 的酶活性可能是 Ku70 从 DNA 解离所必需的。 DNA 损伤位点。因此，SETD4 和 Ku70 在 DNA 招募和/或保留方面的相互作用将确定损伤位点，并评估其对 DNA 修复的影响。最后，我们已经标记了 floxed 小鼠 Setd4 等位基因与 V5 和 Flag (V5F) 表位。我们将利用这条小鼠线来系统地分析 SETD4 在 Ku70 甲基化中的作用及其随后在发育和肿瘤发生中的贡献。这些研究有望阐明先前未知的协调细胞核中 Ku70 功能的机制和细胞质。我们独特的试剂和动物模型确保了该项目的成功，作为该项目的一部分严格的方法。