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The KRAB-zinc finger proteins (KRAB-ZFPs) represent a very large, but poorly understood, family of transcriptional regulators in mammals. They are thought to repress transcription via their interaction with KRAB-associated protein 1 (KAP1), which then assembles a complex of chromatin modifiers to lay down histone marks that are associated with inactive chromatin. Studies of KRAB-ZFP/KAP1-mediated gene silencing, using reporter constructs and ectopically expressed proteins, have shown colocalisation of both KAP1 and repressed reporter target genes to domains of constitutive heterochromatin in the nucleus. However, we show here that although KAP1 does indeed become recruited to pericentric heterochromatin during differentiation of mouse embryonic stem (ES) cells, endogenous KRAB-ZFPs do not. Rather, KRAB-ZFPs and KAP1 relocalise to novel nucleoplasmic foci that we have termed KRAB- and KAP1-associated (KAKA) foci. HP1s can also concentrate in these foci and there is a close spatial relationship between KAKA nuclear foci and PML nuclear bodies. Finally, we reveal differential requirements for the recruitment of KAP1 to pericentric heterochromatin and KAKA foci, and suggest that KAKA foci may contain sumoylated KAP1 - the form of the protein that is active in transcriptional repression.

The extent to which the nuclear organisation of a gene impacts on its ability to be expressed, or whether nuclear organisation merely reflects gene expression states, remains an important but unresolved issue. A model system that has been instrumental in investigating this question utilises the murine Hox gene clusters encoding homeobox-containing proteins. Nuclear reorganisation and chromatin decondensation, initiated towards the 3' end of the clusters, accompanies activation of Hox genes in both differentiation and development, and might be linked to mechanisms underlying colinearity. To investigate this, and to delineate the cis-acting elements involved, here we analyse the nuclear behaviour of a 3' Hoxb1 transgene transposed to the 5' end of the Hoxd cluster. We show that this transgene contains the cis-acting elements sufficient to initiate ectopic local nuclear reorganisation and chromatin decondensation and to break Hoxd colinearity in the primitive streak region of the early embryo. Significantly, in rhombomere 4, the transgene is able to induce attenuated nuclear reorganisation and decondensation of Hoxd even though there is no detectable expression of the transgene at this site. This shows that reorganisation of chromosome territories and chromatin decondensation can be uncoupled from transcription itself and suggests that they can therefore operate upstream of gene expression.

Nuclear organisation is thought to be important in regulating gene expression. Here we investigate whether human embryonic stem cells (hES) have a particular nuclear organisation, which could be important for maintaining their pluripotent state. We found that whereas the nuclei of hES cells have a general gene-density-related radial organisation of chromosomes, as is seen in differentiated cells, there are also distinctive localisations for chromosome regions and gene loci with a role in pluripotency. Chromosome 12p, a region of the human genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p we find no overall change in nuclear chromosome position, but instead we detect a relocalisation of the OCT4 locus, to a position outside its chromosome territory. There is also a smaller proportion of centromeres located close to the nuclear periphery in hES cells compared to differentiated cells. We conclude that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency. Understanding this level of hES cell biology provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.

The burgeoning wealth of gene sequences contrasts with our ignorance of gene function. One route to assigning function is by determining the sub-cellular location of proteins. We describe the identification of mouse genes encoding proteins that are confined to nuclear compartments by splicing endogeneous gene sequences to a promoterless betageo reporter, using a gene trap approach. Mouse ES (embryonic stem) cell lines were identified that express betageo fusions located within sub-nuclear compartments, including chromosomes, the nucleolus and foci containing splicing factors. The sequences of 11 trapped genes were ascertained, and characterisation of endogenous protein distribution in two cases confirmed the validity of the approach. Three novel proteins concentrated within distinct chromosomal domains were identified, one of which appears to be a serine/threonine kinase. The sequence of a gene whose product co-localises with splicesome components suggests that this protein may be an E3 ubiquitin-protein ligase. The majority of the other genes isolated represent novel genes. This approach is shown to be a powerful tool for identifying genes encoding novel proteins with specific sub-nuclear localisations and exposes our ignorance of the protein composition of the nucleus. Motifs in two of the isolated genes suggest new links between cellular regulatory mechanisms (ubiquitination and phosphorylation) and mRNA splicing and chromosome structure/function.

It is generally agreed that, above the level of the 30 nm fibre, eukaryotic chromatin is constrained into loops, but there is disagreement about the nature of the substructure that serves to anchor loops and the DNA sequences that act as the attachment sites. This problem may stem from the very different methods that all purport to separate loop and attached DNAs. We have tested ideas about how the genome is arranged into loops by analysing the average loop size over different cytologically resolvable regions of human chromosomes using fluorescence in situ hybridisation with loop and attached DNA fractions. Variations in average loop size, along and between chromosomes, measurable at this level of resolution were small but significant and were dependent on the extraction method. This emphasises the fundamental differences between the nuclear substructure probed by different protocols. DNA attached to the nuclear 'scaffold' or 'matrix' hybridises preferentially to gene-poor regions of the genome (G-bands). Conversely, fractions attached to the nuclear 'skeleton' hybridise preferentially to gene-rich R-bands and sites of high levels of transcription. The inactive X chromosome has a deficit of associations with the nuclear skeleton but not with the matrix or scaffold. A large excess of attached sequences is found at some sites of constitutive heterochromatin, but not at centromeres.

Fluorescence in situ hybridisation has been used to follow replication of the short arm of human chromosome 11 using chromosome anomalies to distinguish the maternally-and paternally-derived homologues. The temporal difference in replication timing within and between chromosomes has been estimated by combining S phase detection with dual colour fluorescence in situ hybridisation. Proximal regions of 11p, including the WT1 gene, tend to replicate earlier on the maternally-derived chromosome than on the paternally-derived homologue. More distal parts of 11p (including the IGF2 gene) have the opposite imprint. The average difference in replication timing between homologous loci in the population of cells is small compared to the differences between loci along a single chromosome. The imprint is not strictly adhered to since many nuclei have hybridisation patterns opposite to the trend within the population. The nature of the imprinting signal has been investigated. Absolute replication time, but not the imprint, was affected by azacytidine, an inhibitor of DNA methylation. The replication imprint was modified by treatments that inhibit histone deacetylation. We suggest that replication imprinting reflects differences in chromatin structure between homologues.

Chromosomes from the fission yeast Schizosaccharomyces pombe have been introduced into mouse cells by protoplast fusion. In most cell lines the yeast DNA integrates into a single site within a mouse chromosome and results in striking chromosome morphology at metaphase. Both light and electron microscopy show that the yeast chromosome region is narrower than the flanking mouse DNA. Regions of the yeast insert stain less intensely with propidium iodide than surrounding DNA and bear a morphological resemblance to fragile sites. We investigate the composition of the yeast transgenomes and the modification and chromatin structure of this yeast DNA in mouse cells. We suggest that the underlying basis for the structure we see lies above the level of DNA modification and nucleosome assembly, and may reflect the attachment of the yeast DNA to the rodent cell nucleoskeleton. The yeast integrant replicates late in S phase at a time when G bands of the mouse chromosomes are being replicated, and participates in sister chromatid exchanges at a high frequency. We discuss the implications of these studies to the understanding of how chromatin folding relates to metaphase chromosome morphology and how large stretches of foreign DNA behave when introduced into mammalian cells.