PML nuclear bodies and interphase nuclear organisation
Acute promyelocytic leukemia (APL) manifests as a block in the differentiation of promyelocytes, precursors of the granulocyte/neutrophil pathway, which leads to an accumulation of promyelocytes that infiltrate bone marrow (for review see Jensen et al., 2001). This block in differentiation results from a reciprocal translocation event, between promyelocytic leukaemia (PML) protein and retinoic acid receptor alpha (RARA) producing the fusion proteins PML-RARA and RARA-PML. At the molecular level, normal PML is a nuclear protein localising to discrete subnuclear domains as part of a multi-protein complex termed ND10, Kr bodies, PML oncogenic domains (PODs) or PML nuclear bodies (NBs), (for review see Negorev and Maul, 2001). In APL, PML NBs become disrupted into a microgranular appearance with numerous PML foci, which is due to the presence of the PML-RARA fusion protein. PML NBs are also the target for a variety of virus-derived proteins and the subsequent loss of organisation of these domains is an important phase of the viral infection cycle. Thus, it appears that PML NBs are functionally important multi-protein complexes which when disrupted or reorganised have profound cellular consequences.
Given the accumulating evidence that PML regulates the expression of specific target genes, we set out to determine the three-dimensional spatial relationship between PML NBs and gene dense/gene poor genomic regions in primary human fibroblast nuclei. We used immuno-FISH to visualise two gene-rich regions, namely the Major Histocompatibility Complex (MHC) on chromosome 6 and the Epidermal Differentiation Complex (EDC) on chromosome 1; and the gene-poor 6p24 region on chromosome 6 and PML NBs (Shiels et al., 2001). Using novel statistical methods, we found that there is a significantly higher association between PML NBs and the MHC region in comparison to the other genomic regions examined. These data showed for the first time that PML NBs have specific genomic associations that are independent of transcription and support a model for PML NBs as functional domains.
More recently we have focused upon the investigation of PML NBs and their spatial relationships with other nuclear compartments in the mammalian cell nucleus (for review see Batty et al., 2009). The mammalian nucleus shows functional compartmentalisation, with many other nuclear structures and compartments existing in addition to PML NBs. Using a systems biology approach including cell culture, confocal microscopy imaging, and statistical inference we are currently investigating novel quantitative methods for analysis of cell nucleus organisation. (A review of standard methods used in the field can be found in Shiels et al., 2007.) This includes the Stable Count Thresholding (SCT) algorithm which, in combination with statistical analysis, has been successfully applied to the quantification of the spatial configuration of PML NBs with respect to SC35 domains (also known as splicing speckles) (Russell et al., 2009). We showed that PML NBs and SC35 domains are significantly closer than expected under a null model for their spatial point distribution in both interphase and serum-starved MRC5 human primary fibroblast nuclei.