H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy

Abstract

DNA double-strand breaks (DSBs) caused by cellular exposure to genotoxic agents or produced by inherent metabolic processes initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and repair. Phosphorylation of histone H2AX to form ␥-H2AX is one of the earliest of these events and is important for coordination of signaling and repair activities. An intriguing aspect of H2AX phosphorylation is that ␥-H2AX spreads a limited distance up to 1-2 Mbp from the site of a DNA break in mammalian cells. However, neither the distribution of H2AX throughout the genome nor the mechanism that defines the boundary of ␥-H2AX spreading have yet been described. Here, we report the identification of previously undescribed H2AX chromatin structures by successfully applying 4Pi microscopy to visualize endogenous nuclear proteins. Our observations suggest that H2AX is not distributed randomly throughout bulk chromatin, rather it exists in distinct clusters that themselves are uniformly distributed within the nuclear volume. These data support a model in which the size and distribution of H2AX clusters define the boundaries of ␥-H2AX spreading and also may provide a platform for the immediate and robust response observed after DNA damage.␥-H2AX ͉ H2AX chromatin clusters ͉ super-resolution 4Pi microscopy ͉ chromatin response to DNA damage ͉ 3D quantification of chromatin structures M aintenance of genome integrity is critical for organism development and survival, and higher organisms have evolved sophisticated mechanisms for detection and repair of chromosome breaks. DNA damage results in the rapid and coordinated action of various pathways, including activation of cell cycle checkpoints (1, 2), histone modification near the site of the break (3, 4), and recruitment of chromatin remodeling enzymes (4-6), cohesins (7-9), and DNA repair proteins (1, 2, 10). The significance of these molecular processes is highlighted by the fact that defects in many are associated with an increased risk of cancer and developmental and immunologic abnormalities (1). Important insights into the positioning of nuclear signaling and repair proteins and their response to various types and levels of genomic insults have been achieved by using immunofluorescence methods (10-12). However, it has been impossible to distinguish f luorescent signals in a threedimensional (3D) environment that are closer together than 500-800 nm in distance given the depth resolution limits of current light microscopes. With Ϸ100-nm resolution along the optic axis (z axis), 4Pi microscopy (13, 14) provides a significant increase in resolution and has allowed more defined images of cellular structures such as microtubules, mitochondria, or the Golgi apparatus (15). However, until now, imaging of endogenous nuclear proteins had not been achieved (a comparison of 4Pi vs. confocal is described in Materials and Methods). In this study, we describe the successful use of 4Pi microscopy to visualize endogenous nuclear proteins. By applying previo...

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“…S7). This is likely a result of the endogenous cellular amplification as the phosphorylation of H2AX spreads extensively around a site of DNA breakage while TUNEL directly labels a single DNA break (32,33). High-content immunofluorescence quantitation of H2AXP levels showed clear enrichment after 12 h of doxycycline treatment over the vehicle control (Fig.…”

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“…The (53) provides an explanation for the gaps between individual gH2AX subfoci and the H2AX clusters observed with 4Pi microscopy (48).…”