[Cell Cycle 5:17, 1910-1912, 1 September 2006]; ©2006 Landes Bioscience
Spatio-Temporal Dynamics of Chromatin Containing DNA Breaks Extra View
ABSTRACT The cellular response to DNA breaks consists of a complex signaling network that coordinates the initial recognition of the lesion with the induction of cell cycle checkpoints and DNA repair. With DNA wrapped around histone proteins and packaged into higher order levels of chromatin structure, the detection of a single DNA break (DSB) in the genome is the molecular equivalent of finding a needle in a haystack. A recent study from our laboratory used high-resolution electron microscropy and live cell imaging to demonstrate that chromatin undergoes a marked reorganization in response to a DSB. In an energy dependent manner, chromatin rapidly decondenses to a more open configuration in the regions surrounding the lesion. We propose that this ATP dependent chromatinremodeling event facilitates the subsequent recognition and processing of damaged DNA. While the chromatin surrounding the lesion remodels to a more open configuration, the DNA break itself remains relatively immobile over time, consistent with the idea that DNA damage response proteins migrate to positionally stable sites of damaged DNA.1 The lack of significant movement of chromatin regions containing DSBs has implications for the process by which chromosomal translocations form.
Original manuscript submitted: 07/05/06 Manuscript accepted: 07/13/06
More than 20 years ago, it was recognized that DNA damage induces global changes in chromatin structure.2,3 A major rearrangement in the packaging of nucleosomes was deduced by finding changes in accessibility of DNA binding dyes and alterations in the susceptibility to nuclease digestion in response to DNA damaging agents. Other studies documented a correlation between the radiation sensitivity of certain cell lines (e.g., AT fibroblasts) with anamolies in their chromatin structure.4 More recently, it was proposed that the alteration in the chromatin state, rather than the break itself, might be what initially triggers the damage stress alarm.5 Nevertheless, the actual structural change in chromatin that occurs upon introduction of DSBs has remained unclear. Our laboratory used a correlative fluorescence and electron microscopy method to characterize the biophysical properties of damaged chromatin.6 This method allowed us to monitor chromatin changes at high resolution immediately after the introduction of DSBs either by laser microirradiation or by less directed ionizing radiation. To track chromatin containing DSBs in living cells, we utilized photoactivatable-GFP (PAGFP) tagged to core histone H2B, which allowed us to mark specific regions of chromatin while simultaneously introducing DSBs in the same regions. Chromatin was found to decondense immediately after the introduction of DSBs, as evidenced both by the expansion of the photoactivated GFP-H2B signal beyond the boundary of the damaged area and by 35% reduction in density of chromatin fibers in the vicinity of DSBs visualized by electron microscropy. The chromatin opening occurred immediately (within seconds) after the DSBs were introduced, corresponding to the time period when damage sensors, such as Nbs1 and 53BP1, are initially recruited to DSBs. Interestingly, when DSBs were introduced under ATP-depleting conditions, the chromatin did not decondense and the DNA damage sensor proteins failed to be recruited to damaged sites. However, the damage response proteins were able to survey the area of the nucleus containing DSBs (capable of diffusing throughout the nucleus), but did not stably associate with the break sites and did not establish a steady-state accumulation at the damaged DNA. Thus, both chromatin remodeling and recruitment of sensors are energy dependent. This is consistent with a series of recent reports demonstrating a role for ATP dependent chromatin remodeling complexes in the early stages of the DNA damage response. For example, the yeast SWI/SNF is recruited to DSBs prior to DNA repair enzymes, and this complex might remodel chromatin in a way that facilitates the loading of recombinational repair proteins.7 In mammalian cells, the formation of Rad51 foci appears to depend on
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chromatin, histones, DNA repair, doublestrand breaks, ATP, GFP, PAGFP, electron microscopy, H2AX
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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3169
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*Correspondence to: Andre Nussenzweig; Experimental Immunology Branch; National Institutes of Health, NCI; 10 Center Drive; Building 10, Room 4B04; Bethesda, Maryland 20892-1360 USA; Tel.: 301.435.6425; Fax: 301.496.0887; Email:
[email protected]
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Experimental Immunology Branch; National Cancer Institute; National Institutes of Health; Bethesda, Maryland USA
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Michael J. Kruhlak Arkady Celeste André Nussenzweig*
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Spatio-Temporal Dynamics of Chromatin Containing DNA Breaks
Figure 1. Changes in chromatin structure in the presence of a DNA double-strand break. Chromatin is dynamically remodeled by ATP-dependent activities in wildtype and H2AX-/- cells after the introduction of DSBs. This decondensation may increase the accessibility of the lesion or expose preexisting methylated residues in core histones, which in turn may serve as docking sites for certain DNA damage sensors. We speculate that the open chromatin configuration is maintained in the wildtype cells but not in the H2AX-/- cells.
chromatin unwinding by the IP60-TRRAP complex, which has both acetylation and ATPase activities.8 In addition to chromatin relaxation, other studies indicate that histones immediately proximal to the break site need to be removed in order to load Rad51 onto the lesion.9 Thus, the unraveling of nucleosomes in the immediate vicinity of the break may make the lesion more accessible to DNA repair proteins. Several chromatin remodelers, including INO80 and NuA4 complexes are recruited to DNA breaks in phospho-H2AX dependent manner.10-12 This indicates that the phosphorylation of H2AX may somehow regulate the modulation of chromatin architecture during the DNA damage response. Although it is rapidly phosphorylated in response to DSBs,13 H2AX is neither essential for the initial expansion in chromatin at the break site,6 nor is it required for the initial recruitment of damage sensors such as Nbs1, 53BP1 and Brca1.14 Nevertheless, whereas DNA damage response proteins are retained in a steady-state accumulation at DSBs, they appear to “fall off ” of chromatin in the absence H2AX before lesions are repaired.14 Moreover, the dephosphorylation of H2AX appears to be critical for turning off the checkpoint signal.15 Herein may lay the importance of the H2AX-dependent chromatin remodeling activities. These factors may be required during subsequent stages of the DNA damage response to maintain a stable open chromatin environment (Fig. 1). Without maintaining such an environment a resultant gradual loss of DNA repair factors and checkpoint signaling ensues, regardless of whether the DSB itself persists. The prediction that the chromatin eventually reverts back to its pre-DSB state in WT cells, www.landesbioscience.com
but that it does so prematurely in the absence of H2AX (Fig. 1) could in principle be tested by electron microscopy. We propose a model in which chromatin alterations are necessary during different stages of the DNA damage response (Fig. 1). When a DSB is first introduced, chromatin is deconsensed by ATP dependent chromatin remodeling proteins. This facilitates the subsequent recruitment of DNA damage checkpoint and repair proteins, including the kinases (ATM, ATR, DNA-PKcs) that phosphorylate H2AX. In addition to increasing DNA accessibility, chromatin decondensation may also expose methylated residues in core histones, which serve as binding sites for certain DNA damage sensors.16,17 The phosphorylation of H2AX establishes large sub-nuclear domains surrounding the DSBs that support the steady-state accumulation of DNA repair proteins and subsequent chromatin remodelers, which in turn maintain the chromatin in an open configuration. In the H2AX knockout cells, however, this environment is lost, due to the scattering of DNA repair proteins and chromatin remodeling complexes. Without the stable accumulation of remodeling complexes the chromatin state may simply revert back to the pre-DSB configuration, with one detrimental difference, the DSB lesion may remain unprotected. The presence of multiple DSBs creates the physiologically heightened need to resolve the individual breaks successfully and to prevent adverse chromosomal rearrangements that have the potential to initiate tumorigenesis. DNA lesions resulting from exogenous sources, such as ionizing radiation, or spontaneous lesions during replication occur in an apparently random fashion scattered
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throughout the cell nucleus. If multiple DSBs interact during the DNA repair process then the potential for aberrant chromosomal rearrangements increases concomitantly. If cellular mechanisms exist that purposefully bring multiple lesions together, then the potential for intermingling of chromosomes further increases. A previous study in mammalian cells suggested that chromatin domains containing DSBs tend to cluster together, particularly in the G1 phase of the cell cycle, with the clustering being mediated by the Mre11 complex.18 These observations support a model in which DSBs can move over a large distance before interacting. However, when we examined the distribution and mobility of DSBs introduced in living cells either by laser microirradiation or by γ-radiation we found that in contrast to the previous study, DSBs exhibit limited mobility and cohesiveness independently of the cell cycle stage. Rather the limited mobility was similar to the random movement of chromatin not containing DSBs,19 and the potential for individual separated DSBs to interact was very low over a number of hours.6 Although it remains unclear how to account for the disparity in these results, it is notable that the earlier study produced DSBs with alpha particles (charged ions of helium), which induce a higher local density of lesions in DNA and more complex chromosomal aberrations than gamma-irradiation.20 The more complex lesions generated by alpha particles are rejoined with slower kinetics than gamma irradiation,21,22 and the longerterm gradual shifting of the chromosomes may inadvertently bring multiple DSBs within close proximity. Thus, the increased potential for movement of chromatin regions (and chromosomal translocations) would depend on the nature of the DSB and the kinetics of repair. Likewise, one would expect that in repair deficient cells, DNA lesions persist for a longer time and therefore multiple DSBs may eventually encounter each other stochastically, increasing the probability of a translocation. In summary, chromatin-containing DSBs is reorganized but undergoes limited migration. Future studies will aim at identifying specific proteins that bring about and determine the extent and timing of local chromatin decondensation at DSBs.
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