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Chromatin, Nuclear Organization, and Genome Stability in Mammals
Abstract
The genomes of mammalian cells exist as chromatin—a complex and dynamic structure that both serves as the background and actively participates in all fundamental nuclear processes such as transcription, replication, and DNA repair. Chromatin is characterized by multiple levels of organization: at the primary level, DNA is wrapped around a set of proteins called histones; interactions between histones promote further folding of the nucleoprotein fiber into a 30-nm structure with an unknown mechanical composition. The 30-nm fibers are then additionally folded into higher-order domains with differing structural and functional properties, which are then arranged in the nucleus in a probabilistic manner, with gene-poor regions preferring the periphery and gene-rich regions accumulating in the interior. Every level of chromatin organization has relevance for genome stability. At the 30-nm fiber level, the chromatin response to DNA damage is driven by the “access, repair, restore model,” while higher levels of organization determine the frequency and nature of chromosomal translocations. A modern view of genome stability aims to integrate the influence of fundamental cellular processes such as transcription and replication with chromatin context to give a better understanding of the processes that shaped our genomes.
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The focal accumulation of DNA repair factors, including the MRE11/Rad50/NBS1 (MRN) complex and the phosphohistone variant gamma-H2A.X, is a key cytological feature of the DNA damage response (DDR). Although these foci have been extensively studied by light microscopy, there is comparatively little known regarding their ultrastructure. Using correlative light microscopy and electron spectroscopic imaging (LM/ESI) we have characterised the ultrastructure of chromatin and DNA repair foci within the nuclei of normal human fibroblasts in response to DNA double-strand breaks (DSBs). The induction of DNA DSBs by etoposide leads to a global decrease in chromatin density, which is accompanied by the formation of invaginations of the nuclear envelope as revealed by live-cell microscopy. Using LM/ESI and the immunogold localisation of gamma-H2A.X and MRE11 within repair foci, we also observed decondensed 10 nm chromatin fibres within repair foci and the accumulation of large non-chromosomal protein complexes over three hours recovery from etoposide. At 18 h after etoposide treatment, we observed a close juxtapositioning of PML nuclear bodies and late repair foci of gamma-H2A.X, which exhibited a highly organised chromatin arrangement distinct from earlier repair foci. Finally, the dual immunogold labelling of MRE11 with either gamma-H2A.X or NBS1 revealed that gamma-H2A.X and the MRN complex are sub-compartmentalised within repair foci at the sub-micron scale. Together these data provide the first ultrastructural comparison of gamma-H2A.X and MRN DNA repair foci, which are structurally dynamic over time and strikingly similar in organisation.
It is well established that modification of lysines in histone molecules correlates with gene expression and chromatin structure. It is not known whether this operates entirely at a local level, e.g. through the recruitment of specific proteins, or whether histone modifications might impact on more long-range aspects of chromatin organization. There is a distinctive organization of chromatin within the nucleus and the chromatin at the nuclear periphery of mammalian cells appears to be hypoacetylated. Previously it had been suggested that inhibition of histone deacetylases by TSA causes a gross remodeling of nuclear structure, specifically the recruitment of centromeric heterochromatin to the nuclear periphery. Here, we have quantified the nuclear organization of histone modifications and the localization of centromeric domains in human cells before and after TSA treatment. TSA alters the nuclear distribution of histone acetylation, but not that of histone methylation. TSA elevates levels of histone acetylation at the nuclear periphery but we see no alteration in the position of centromeric domains in the nuclei of treated cells. We conclude that the distinctive nuclear localization of centromeric domains is independent of histone acetylation.
In eukaryotic cells, the inheritance of both the DNA sequence and its organization into chromatin is critical to maintain genome stability. This maintenance is challenged by DNA damage. To fully understand how the cell can tolerate genotoxic stress, it is necessary to integrate knowledge of the nature of DNA damage, its detection and its repair within the chromatin environment of a eukaryotic nucleus. The multiplicity of the DNA damage and repair processes, as well as the complex nature of chromatin, have made this issue difficult to tackle. Recent progress in each of these areas enables us to address, both at a molecular and a cellular level, the importance of inter-relationships between them. In this review we revisit the 'access, repair, restore' model, which was proposed to explain how the conserved process of nucleotide excision repair operates within chromatin. Recent studies have identified factors potentially involved in this process and permit refinement of the basic model. Drawing on this model, the chromatin alterations likely to be required during other processes of DNA damage repair, particularly double-strand break repair, are discussed and recently identified candidates that might perform such alterations are highlighted.