Attention has been focused towards mapping the position of whole porcine chromosome territories in interphase nuclei in a quest to understand further how and why the genome is organised in the way that it is, within the nuclear environment. In this study, the position of porcine chromosomes within interphase nuclei from a number of cell types was mapped and correlated to both gene density and size theory of chromosome positioning.
This study has determined that, like many other genomes studied, the porcine genome is highly organised into territories that are non-randomly positioned within the nucleus. Indeed, this is the largest study so far to investigate porcine genome organisation and the nuclear positioning of porcine whole chromosomes. This study has mapped the position of the majority of porcine whole chromosome territories in a number of different cell types, surpassing that of any other species. It remains unclear however how chromosomes assume these nuclear positions.
Existing evidence shows that there are correlations between the nuclear positioning of a chromosome and its gene density [5
] or size [8
]. We have, for the purposes of this study, attempted to calculate the gene density of each chromosome via the distribution of porcine CpG islands [39
], H3 isochores [40
], using synteny to the human genome with respect to CpG island distribution, early and late replicating DNA and the known nuclear positioning of syntenic chromosomes (Figure and Table ) [5
] and via the current gene assignments mapped to porcine chromosomes on e!Ensembl (http://www.ensembl.org/Sus_scrofa/Location/Genome
- last accessed 26.08.10) (Table ), it should be noted that the genome has not yet been fully annotated. Table displays our predicted porcine chromosome territory interphase positioning according to these various estimates of gene density of each porcine chromosome or known positioning of human chromosomes in proliferating fibroblasts or lymphoblasts. With these correlations, less than half of the porcine chromosomes actual positions fit with the predicted positions. Although the porcine genome has been sequenced, the assignment of a number of genes still needs to be determined.
Figure 6 Schematic diagram showing the porcine karyotype. Each chromosome has its size represented (Mbp) . Porcine CpG island  and H3-rich isochore distribution  is shown on the right-hand side of each chromosome. Synteny to human CpG island distribution (more ...)
A comparison between the chromosome territory positioning of actual porcine chromosomes from 2D FISH studies with respect to synteny with human chromosomes, porcine epigenetic markers,in silicogene data and chromosome size
Using the latest porcine genome information we have calculated a gene-density value for each chromosome by dividing the possible genes on each chromosome by the Mbp size for that chromosome. These values range from 3.9 for porcine chromosome 11 to 12.9 for chromosome 7 (Table ). In Table we display the chromosomes and their categorised position in SOB adult fibroblasts in descending order of gene density and the correlation between gene density and position is weak with the poorest gene dense chromosome being in the nuclear interior and the richest at the nuclear periphery. Given that the genome sequencing is only near completion there may still be a gene density distribution that presently remains elusive. However, the correlation appears much more convincing for size with the three smallest chromosomes being found in the nuclear interior and the two largest chromosomes at the nuclear periphery. It should also be noted that these are proliferating cells but they are all in S-phase. For humans no difference in chromosome positioning for human chromosomes 18 and 19 was revealed in S-phase [6
Our studies allowed comparison in the positioning of homologous chromosomes in kidney fibroblasts derived from both adult and embryonic tissue. It was revealed that chromosomes had comparable interphase nuclear locations. Since cells from the embryonic lineage had already differentiated to kidney fibroblasts and followed the same differentiation pathway as the adult kidney fibroblasts, then similar chromosome positioning was perhaps likely.
The more interesting comparison perhaps is the comparison between the ex vivo
lymphocytes and the tissue culture cells, since lymphocytes are derived from haemopoietic lineage and therefore may also exert distinct tissue specific control of genome organisation. Despite over 95% of cultured lymphocytes being deficient in A-type lamins and the difference in stem cell lineage, the majority of chromosomes occupied similar nuclear locations. We did however note that we found less chromosome signal at the nuclear periphery in shell 1 for lymphocytes, which may be due to the role A-type lamins play in anchoring the genome to the nuclear periphery [44
]. However, the amount of total chromatin as stained by DAPI was not dissimilar to other cell types used in this study.
The use of pig tissues and development of a 3D FISH protocol has allowed us also to perform pig chromosome territory delineation and position analysis in cells in 3D preserved frozen tissue sections. So to determine whether the spatial positioning of chromosomes within nuclei is tissue specific, the nuclear position of four porcine chromosomes 5, 13, 17 and X were ascertained in three different tissues types including brain, kidney, and lung. Although each tissue type arose from different origins (endoderm, mesoderm or ectoderm) and contained specialised cells, analogous porcine chromosomes occupied equivalent interphase nuclear positions in each of the tissue types analysed. These data differed to a previous study that investigated genome organisation in nuclei derived from specific mouse tissues [11
]. Parada and colleagues found that although some chromosomes shared similar interphase nuclear positions, many exhibited a tissue specific organisation, particularly cells that followed divergent differentiation pathways [11
From this study it is apparent that chromosomes occupy similar interphase nuclear positions regardless of in vitro
or in vivo
conditions. Comparisons between chromosome positioning of chromosomes 5, 13, 17 and X in cultured nuclei (during S-phase) and nuclei within ex vivo
tissue sections, reveals that chromosomes 5, 13 and X occupy peripheral positions, whereas chromosome 17 is localised at more internal nuclear locations (Figure ). Nuclear positioning data for chromosomes 5, 13, 17 and X verify that chromosomes share analogous nuclear positions between in vitro
cultured nuclei and in vivo
nuclei. However, variations in culture conditions, such as serum starvation, can substantially affect relative interphase chromosome positioning as demonstrated with human chromosomes becoming repositioned from a peripheral nuclear location to an internal location upon quiescence [9
]. Considering the possible influence of altered culture conditions on genome organisation, serum was removed from adult porcine fibroblasts cultures for several days in an effort to quiesce cells. Unfortunately, we were unable to quiesce the cells via a decrease in serum levels to 0.5% or via contact inhibition since apoptosis was induced and many cells were lost, a phenomenon previously reported in cultured pig cells [45
]. Therefore we could not determine if chromosome territories were repositioned within the nuclei in quiescent porcine cells. Nevertheless, despite the proliferation status of in vivo
cells in tissue sections being undetermined, equivalent chromosome positions within nuclei were exhibited regardless of their proliferation status. The brain is composed of two main cell types neurons and glial cells, with glial cells representing 90% of brain cells. Brain cells are terminally differentiated and have ceased cell division [46
], however, despite their quiescent nature no alterations in the nuclear position of specific porcine chromosomes were ascertained. The conditions used to make in vitro
cells quiescent, such as serum starvation or contact inhibition do not typically mimic in vivo
conditions of quiescent cells. Thus, in vitro
quiescent cells may exhibit altered chromosome territory position due to specific signal transduction pathways not normally activated or silenced in in vivo
cells. Although the pig seems an excellent animal model it is also possible that species-specific differences may arise resulting in conflicting data. This is evident when using the mouse as a model for genome organisation. The mouse genome is uniform in size and gene density and chromosomes are positioned at different nuclear locations within mouse nuclei compared to the nuclear position of syntenic human chromosomes in human nuclei [47
In summary, the porcine genome is non-randomly organised with most chromosomes occupying similar nuclear positions despite developmental origin or lineage and regardless of being cultured in vitro or from in vivo tissue. From epigenetic data, projected gene assignments and chromosome size, it would appear presently that the porcine genome does not fit completely into either the size or gene-density model of chromosome positioning. We will assess gene-density correlated positioning again in the future when the porcine genome is well established. Thus it appears that other parameters also influence the positioning of whole chromosomes within the nucleus.