Related Experiment Video
Updated: Mar 26, 2026
Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique
Published on: January 14, 2016
iTRAQ-based chromatin proteomic screen reveals CHD4-dependent recruitment of MBD2 to sites of DNA damage
Yazhou Sun1, Yeran Yang1, Hongyan Shen2
1Key Laboratory of Genomic and Precision Medicine, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing, 100049, China.
Abstract:
Many DNA repair proteins can be recruited to DNA damage sites upon genotoxic stress. In order to search potential DNA repair proteins involved in cellular response to mitomycin C treatment, we utilized a quantitative proteome to uncover proteins that manifest differentially enrichment in the chromatin fraction after DNA damage. 397 proteins were identified, among which many factors were shown to be involved in chromatin modification and DNA repair by GO analysis. Specifically, methyl-CpG-binding domain protein 2 (MBD2) is revealed to be recruited to DNA damage sites after laser microirradiation, which was mediated through MBD domain and MBD2 C-terminus. Additionally, the recruitment of MBD2 is dependent on poly (ADP-ribose) and chromodomain helicase DNA-binding protein 4 (CHD4). Moreover, knockdown of MBD2 by CRISPR-Cas9 technique results in MMC sensitivity in mammalian cells.
Insights
Methyl-CpG-binding domain protein 2 (MBD2) is recruited to DNA damage sites, aiding cellular repair. Its knockdown increases sensitivity to genotoxic agents like mitomycin C.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA repair proteins are crucial for cellular response to genotoxic stress.
- Understanding these proteins aids in developing strategies against DNA-damaging agents.
Purpose of the Study:
- To identify DNA repair proteins involved in the cellular response to mitomycin C (MMC).
- To investigate the role of methyl-CpG-binding domain protein 2 (MBD2) in DNA damage response.
Main Methods:
- Quantitative proteomic analysis of chromatin fractions after DNA damage.
- Gene Ontology (GO) analysis to identify protein functions.
- Laser microirradiation and CRISPR-Cas9 mediated knockdown for functional studies.
Main Results:
- Identified 397 differentially enriched proteins, including chromatin modifiers and DNA repair factors.
- MBD2 recruitment to DNA damage sites was observed, dependent on its MBD domain, C-terminus, poly(ADP-ribose), and CHD4.
- Knockdown of MBD2 resulted in increased sensitivity to MMC in mammalian cells.
Conclusions:
- MBD2 plays a significant role in the DNA damage response pathway.
- MBD2's recruitment mechanism involves specific protein interactions and domains.
- MBD2 is a potential therapeutic target for enhancing sensitivity to genotoxic cancer treatments.
