Chromatin stiffening underlies enhanced locus mobility afterDNAdamage in budding yeast

Abstract

DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome-wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility.

Chromatin Dynamics upon DNA Damage

“…Chromatin dynamics therefore appears to be scale-dependent: in response to DNA damage, chromatin is more mobile at large time scales but, surprisingly, its mobility is reduced at short time scales, this effect being stronger at the damaged site. Such a pattern of dynamics is consistent with a global chromatin stiffness that has been proposed to arise in response to DNA damage [65,89,90]. These results underline the importance of performing multiscale tracking to fully understand the complex dynamics of chromatin at each scale.…”

DNA Repair: The Search for Homology

“…Two regions on the same DNA molecule are linked such that their relative motion is still random, but not independent of each other. Their relative motion is “subdiffusive.” Yeast chromosomes exhibit this subdiffusive behavior [63-66]. Both simulations and direct observations of fluorescently tagged immunoglobulin loci in mouse B cells lead to the same conclusion [67]: for sites that are within a radius of 1 micron (the size of the yeast nucleus), the mean free passage time (MFPT) is about 2100 sec (i.e.…”

“…One modification is the phosphorylation of histone H2A (called γ-H2AX) that spreads for a distance of ~50 kb on either side of the DSB [74]. In cells lacking ATRMec1 or carrying a non-phosphorylatable histone H2A allele, Rc for a region near a DSB or the distance between two fluorescently marked sites around the DSB does not show the increases seen in wildtype cells [63, 68]. …”

“…One kinetochore protein, Cep3, is phosphorylated by checkpoint kinases after DNA damage, but other observers have found that introducing a non-phosphorylatable Cep3 mutant does not seem to affect an increase in the distance between interchromosomal sites or to change in Rc of a broken chromosome [63, 78, 79]. …”

“…The loss is not simply around the site of damage but global, and appears to cause a general decondensation of yeast chromatin (though yeast don’t have much higher-order chromatin beyond an 11 nm fiber) [86, 87]. Damage-provoked histone loss (or artificial histone depletion) was suggested to create increased fiber flexibility, which seems to be the opposite of the conclusion reached by Fabre and Zimmer [63] that damage leads to chromatin stiffening. However, another study of nucleosome depletion, but without DNA damage, also concluded that chromatin fibers became more stiff, with a reduced R c [79].…”