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In the premature ageing disease Hutchinson-Gilford progeria syndrome (HGPS), the underlying genetic defect in the lamin A gene leads to accumulation at the nuclear lamina of progerin-a mutant form of lamin A that cannot be correctly processed. This has been reported to result in defects in the DNA damage response and in DNA repair, leading to the hypothesis that, as in normal ageing and in other progeroid syndromes caused by mutation of genes of the DNA repair and DNA damage response pathways, increased DNA damage may be responsible for the premature ageing phenotypes in HGPS patients. However, this hypothesis is based upon the study of markers of the DNA damage response, rather than measurement of DNA damage per se or the consequences of unrepaired DNA damage-mutation. Here, using a mutation reporter cell line, we directly compared the inherent and induced mutation rates in cells expressing wild-type lamin A or progerin. We find no evidence for an elevated mutation rate in progerin-expressing cells. We conclude that the cellular defect in HGPS cells does not lie in the repair of DNA damage per se.

The ability to visualize specific DNA sequences, on chromosomes and in nuclei, by fluorescence in situ hybridization (FISH) is fundamental to many aspects of genetics, genomics and cell biology. Probe selection is currently limited by the availability of DNA clones or the appropriate pool of DNA sequences for PCR amplification. Here, we show that liquid-phase probe pools from sequence capture technology can be adapted to generate fluorescently labelled pools of oligonucleotides that are very effective as repeat-free FISH probes in mammalian cells. As well as detection of small (15 kb) and larger (100 kb) specific loci in both cultured cells and tissue sections, we show that complex oligonucleotide pools can be used as probes to visualize features of nuclear organization. Using this approach, we dramatically reveal the disposition of exons around the outside of a chromosome territory core and away from the nuclear periphery.

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections.

It has been suggested that chromatin at the nuclear periphery could act to shield DNA sequences in the nuclear interior from damage. To test this hypothesis, we have examined the nuclear distribution of sites of DNA repair induced by oxidation or UV-C. We do not detect more damage (repair) at the nuclear periphery than in the nuclear interior. In fact, contrary to the body guard hypothesis, there is an excess of damage detected in the nuclear interior. This is further supported by sequence comparison between genes on human chromosomes 18 or 19, and their counterparts in the chimpanzee. The synonymous substitution rate for genes on chromosome 19, which is located towards the centre of the human nucleus, was higher than that for genes on chromosome 18, which is located at the nuclear periphery. We conclude that chromatin at the periphery of the human nucleus is not able to protect more internally located sequences from damage and mutation. We suggest that features of the chromatin structure, or base composition, of sequences in the nuclear centre make them more susceptible to damage.

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.

Translocations are the most frequent chromosome structural aberration in the human population, yet little is known about their aetiology. Here, factors that might influence the occurrence of constitutional translocations in the population are examined. By analysing >10000 translocations from two large databases of cytogenetic abnormalities, chromosome size is identified as the major determinant of translocation frequency. This probably reflects the large target size for double-strand breakage and repair presented by the largest chromosomes. There is also evidence for selection against translocations that involve breakage through the most gene-dense chromosomes. Lastly, it is suggested that nuclear organization of chromosomes impinges on the frequency of translocations amongst the smallest autosomes.