Identifying gene locus associations with promyelocytic leukemia nuclear bodies using immuno-TRAP

Genetics & Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
The Journal of Cell Biology (Impact Factor: 9.83). 04/2013; 201(2):325-35. DOI: 10.1083/jcb.201211097
Source: PubMed


Important insights into nuclear function would arise if gene loci physically interacting with particular subnuclear domains could be readily identified. Immunofluorescence microscopy combined with fluorescence in situ hybridization (immuno-FISH), the method that would typically be used in such a study, is limited by spatial resolution and requires prior assumptions for selecting genes to probe. Our new technique, immuno-TRAP, overcomes these limitations. Using promyelocytic leukemia nuclear bodies (PML NBs) as a model, we used immuno-TRAP to determine if specific genes localize within molecular dimensions with these bodies. Although we confirmed a TP53 gene-PML NB association, immuno-TRAP allowed us to uncover novel locus-PML NB associations, including the ABCA7 and TFF1 loci and, most surprisingly, the PML locus itself. These associations were cell type specific and reflected the cell's physiological state. Combined with microarrays or deep sequencing, immuno-TRAP provides powerful opportunities for identifying gene locus associations with potentially any nuclear subcompartment.

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Available from: David Bazett-Jones, Apr 04, 2014
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    • "The eukaryotic nucleus contains domains organized by master proteins, such as promyelocytic leukemia (PML), which drives the formation of PML nuclear bodies (NBs; Lallemand-Breitenbach and de Thé, 2010). PML NBs are stress-regulated, dynamic structures that concentrate hundreds of proteins and nely tune multiple pathways including senescence, stemness, stress response, and defense against viruses (Koken et al., 1995; Dellaire and Bazett-Jones, 2004; Bernardi and Pandol, 2007; Ching et al., 2013). Functionally, NB disruption through expression of the PML/RARA oncogene has been implicated in acute promyelocytic leukemia (APL) pathogenesis. "
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    ABSTRACT: The promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are stress-responsive domains where many partner proteins accumulate. Here, we clarify the basis for NB formation and identify stress-induced partner sumoylation as the primary NB function. NB nucleation does not rely primarily on intermolecular interactions between the PML SUMO-interacting motif (SIM) and SUMO, but instead results from oxidation-mediated PML multimerization. Oxidized PML spherical meshes recruit UBC9, which enhances PML sumoylation, allow partner recruitment through SIM interactions, and ultimately enhance partner sumoylation. Intermolecular SUMO-SIM interactions then enforce partner sequestration within the NB inner core. Accordingly, oxidative stress enhances NB formation and global sumoylation in vivo. Some NB-associated sumoylated partners also become polyubiquitinated by RNF4, precipitating their proteasomal degradation. As several partners are protein-modifying enzymes, NBs could act as sensors that facilitate and confer oxidative stress sensitivity not only to sumoylation but also to other post-translational modifications, thereby explaining alterations of stress response upon PML or NB loss.
    The Journal of Cell Biology 03/2014; 204(6):931-45. DOI:10.1083/jcb.201305148 · 9.83 Impact Factor
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    ABSTRACT: Researchers describe technique to identify genomic loci that interact with specific nuclear structures.
    The Journal of Cell Biology 04/2013; 201(2):169-169. DOI:10.1083/jcb.2012if · 9.83 Impact Factor
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