Capturing Common Fragile Site Breaks by Native γH2A.X ChIP

Summary

We present a rapid and efficient method to detect common fragile site breaks through native γH2A.X chromatin immunoprecipitation (ChIP). This approach significantly reduces both the time and labor associated with traditional γH2A.X ChIP assays while maintaining high reproducibility and reliability of results.

Abstract

Replication stress induced by exposure to extrinsic agents can lead to DNA breaks at common fragile sites, which are regions in the genome known to be prone to structural instability. The γH2A.X chromatin immunoprecipitation (ChIP) assay serves as a powerful tool in genotoxicity studies, as γH2A.X phosphorylation is a well-established marker for DNA double-strand breaks. Traditional γH2A.X ChIP assays, however, are often labor-intensive and involve multiple, time-consuming steps. In this study, we present a simplified yet effective method that combines subcellular fractionation with native ChIP to isolate γH2A.X-associated complexes. This approach is particularly suitable for analyzing γH2A.X-chromatin interactions with enhanced specificity and efficiency. Using subcellular fractionation, chromatin-unbound materials are effectively removed, resulting in a purified chromatin fraction. Subsequent micrococcal nuclease (MNase) digestion under mild conditions allows chromatin fragmentation while preserving physiological interactions between γH2A.X and its associated protein complexes. This preservation is essential for studying native interaction partners involved in DNA damage response pathways. This optimized native ChIP protocol substantially reduces the time and labor associated with conventional γH2A.X ChIP assays. The streamlined procedure not only simplifies the workflow but also yields highly reproducible results, making it particularly advantageous in settings where high-throughput processing of multiple samples is required. This method has broad applicability in studies focused on genome stability, DNA repair, and chromatin biology, where accurate and efficient detection of DNA damage sites is critical. By employing optimized protocols and streamlined steps, this method enables the detection of DNA damage at fragile sites with improved sensitivity and minimal sample handling, making it a valuable tool for studies on genome stability and DNA damage response.

Introduction

Common fragile sites (CFSs) are large chromosomal regions found on every human chromosome prone to breaking during metaphase. Under replication stress, replication at these regions is significantly delayed, preventing their complete duplication before mitotic entry¹, which ultimately results in site-specific gaps and breaks. CFSs are hotspots for chromosomal instability and are a major cause of chromosomal rearrangements during early cancer development. Replication stress, which is often present under tumorigenic conditions, can lead to the loss of tumor suppressor genes and amplification of oncogenes-collectively referred to as copy number var....

Protocol

1. Cell harvesting

1. Seed about 5 x 10⁵ HEK 293T cells into each of the four 6 cm dishes, each containing 4 mL of complete DMEM medium.
2. After 24 h, treat one dish with 2 µL of 1 mM Aphidicolin (refer to Table of Material) stock solution (final concentration of 0.5 µM) and another dish with 20 µL of 1 M hydroxyurea (refer to Table of Material) stock solution (final concentration of 5 mM) to induce replication stress. Add DMSO .......

Discussion

Environmental pollution is a significant contributor to human cancers. Many pollutants are carcinogenic, meaning they can cause genetic damage that leads to the development of cancer^40,41. However, determining whether a particular substance is tumorigenic is a challenging task. A fast, reliable, and cost-efficient method for identifying carcinogenic potential would empower scientists to efficiently screen environmental pollutants and assess their impact on genomi.......

Materials

Name	Company	Catalog Number	Comments
0.2 µm nitrocellulose membrane	Amersham	10600011
Actin B	proteintech	20536-1-AP
Aphidicolin	MedChemExpress	HY-N6733
ChIP-grade magnetic Protein A/G beads	ThermoFisher	26162
Clarity Western ECL Substrate	Bio-Rad	#1705061
Glycogen, molecular biology grade	ThermoFisher	Cat. No.  R0561
HRP-conjugated secondary antibody	proteintech	SA00001-2
hydroxyurea	MedChemExpress	HY-B0313
Micrococcal Nuclease	NEB	M0247S
normal IgG	Santa Cruz	sc-2025
Taq Universal SYBR Green Supermix	BioRad	1725120
γH2A.X antibody  (for ChIP)	Sigma-Aldrich	05-636
γH2A.X antibody (for WB)	Cell Signaling	#25955