Regulation of Ku70 methylation and functions by SETD4

SETD4 对 Ku70 甲基化和功能的调节

• 批准号: 10330477
• 负责人: Zhiyuan Shen
• 金额: $ 35.2万
• 依托单位: RBHS -CANCER INSTITUTE OF NEW JERSEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10330477
• 关键词: Acetylation; Acinus organ component; 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; 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-Adenosylmethionine; SET Domain; Side; Signal Transduction; Site; Tertiary Protein Structure; Testing; Time; Transferase; XRCC5 gene; antibody detection; base; dimer; in vivo; irradiation; methyl group; mouse genetics; novel; overexpression; recruit; response; ribulose-1,5-bisphosphate; success; therapy outcome; tumor; tumorigenesis

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.

调节Ku70甲基化和setD4的功能 （抽象的） 这项研究的目的是阐明由KU70功能的新型调节机制控制的。 赖氨酸甲基化。 Ku70是DNA损伤修复中的关键蛋白质，尤其是在非 - 照射后同源末端连接。此功能是通过用ku80二聚和包围来执行的 在休息地点的DNA。 Ku70的自由形式以其在细胞质中的抗凋亡活性而闻名 它与凋亡蛋白Bax的结合。我们的初步研究表明，SetD4是一种推定的非 - 组蛋白甲基转移酶，甲基化的Ku70导致KU70迁移到细胞质。过表达 SETD4抑制了凋亡，而SETD4部署对IT的敏感。 SetD4的染色质结合是依赖的 在Ku70上，但反之亦然。 SETD4可以招募到DNA损伤位点，但仅在相对较短的时间 DNA损伤后的点。基于这些新颖的发现，我们假设setd4播放的ku70甲基化 KU70从DNA双链断裂（DSB）到细胞质的功能转移的关键作用。 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敲入鼠标线。我们努力使用整合生物化学，细胞和整合的组合方法 分子生物学和小鼠遗传学以检验我们的假设。在AIM 1中，我们将专注于Ku70-甲基化和 它的抗凋亡和DNA修复活性。首先，KU70甲基化在KU70/KU80二聚体上的后果 将确定稳定性及其与DNA的结合。其次，甲基化ku70在中的细胞质活性 凋亡将通过非DNA损伤药物进行验证。第三，KU70甲基化对KU70的直接影响 DNA损伤位点的募集和保留，DSB修复效率以及对电离辐射的细胞敏感性 将测量。最后，我们将使用内部开发的Ku70敲入小鼠来表征 ku70的含赖氨酸的SAP结构域及其在体内的甲基化。在AIM 2中，我们将重点介绍SETD4的方式 通过KU70调节细胞凋亡和DNA损伤反应。首先，我们将确定结构元素 对于setD4至关重要的甲基化ku70。其次，SetD4调制对凋亡的后果将是 在无法ku70甲基化的细胞中测量。第三，我们预测，虽然SETD4需要KU70 招募DNA损伤部位，ku70与SetD4的酶活性可能需要 DNA损伤位点。那就是setd4和ku70在募集和/或在DNA上保留的相互作用 将确定损伤部位，并评估它们对DNA修复的影响。最后，我们已经标记了 带有V5和标志（V5F）表位的Floxed Mouse SetD4等位基因。我们将使用此鼠标线进行系统分析 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 预计研究将阐明以前的未知机制，该机制在核中协调KU70的功能 和细胞质。我们的独特试剂和动物模型作为一个的一部分，确保了该项目的成功 严格的方法。