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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.

SETD 4对Ku 70甲基化及功能的调控（摘要）本研究的目的是阐明Ku 70功能的一种新的调节机制，该机制由赖氨酸甲基化Ku 70是DNA损伤修复中的关键蛋白，尤其是在非损伤启动过程中。辐照后同源末端连接。该功能是通过其与Ku 80的二聚化和环断裂处的DNA已知游离形式的Ku 70在细胞质中具有抗凋亡活性，这是由于其与促凋亡蛋白BAX结合。我们的初步研究表明，SETD 4，一个假定的非- 组蛋白甲基转移酶，甲基化Ku 70以引起Ku 70重新定位到细胞质。过表达 SETD 4抑制细胞凋亡，而SETD 4缺失使其敏感。 Ku 70上，但反之亦然。SETD 4可以被募集到DNA损伤位点，但仅在相对中后期 DNA损伤后。基于这些新的发现，我们假设由SETD 4引起的Ku 70甲基化在 Ku 70从DNA双链断裂（DSB）到细胞质的功能性易位的关键作用。我们已经产生了针对甲基化Ku 70和SETD 4的高度特异性抗体，以及几种Ku 70和SETD 4。 SETD 4敲入小鼠系。我们努力使用一种综合方法，将生物化学、细胞和分子生物学和小鼠遗传学来验证我们的假设。在目标1中，我们将关注Ku 70-甲基化和其抗凋亡和DNA修复活性。首先，Ku 70甲基化对Ku 70/Ku 80二聚体的影响将测定其稳定性及其与DNA的结合。第二，甲基化的Ku 70在细胞内的活性，将用非DNA损伤剂验证细胞凋亡。三、Ku 70甲基化对Ku 70的直接影响 DNA损伤位点的募集和保留、DSB修复效率和细胞对电离辐射的敏感性将被衡量。最后，我们将使用内部开发的Ku 70基因敲入小鼠来表征 Ku 70的含赖氨酸SAP结构域及其体内甲基化。在目标2中，我们将重点关注SET 4如何通过Ku 70调控细胞凋亡和DNA损伤反应。首先，我们将确定结构要素，是SETD 4甲基化Ku 70的关键。第二，SETD 4对细胞凋亡的调节的结果将是：在不能Ku 70甲基化的细胞中测量。第三，我们预测，虽然Ku 70是需要为SETD 4 由于Ku 70被募集到DNA损伤位点，因此可能需要SETD 4的酶活性来使Ku 70从DNA损伤位点解离。 DNA损伤部位。因此，SETD 4和Ku 70在它们的DNA募集和/或保留中的相互作用是相互的。将确定损伤部位，并评估其对DNA修复的影响。最后，我们标记了具有V5和Flag（V5 F）表位的floxed小鼠Setd 4等位基因。我们将用这条鼠标线来系统地分析 SETD 4在Ku 70甲基化中的作用及其在发育和肿瘤发生中的后续贡献。这些这项研究有望阐明一种以前未知的机制，这种机制协调了Ku 70在核中的功能和细胞质。我们独特的试剂和动物模型确保了该项目的成功，严格的方法。