Regulation of Ku70 methylation and functions by SETD4

Project Details

Description

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.
StatusActive
Effective start/end date2/1/211/31/22

ASJC

  • Genetics
  • Molecular Biology

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