Identifying gene locus associations with promyelocytic leukemia nuclear bodies using immuno-TRAP.
ABSTRACT 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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ABSTRACT: Histone variant H3.3 is deposited in chromatin at active sites, telomeres and pericentric heterochromatin by distinct chaperone, but the mechanisms of regulation and coordination of chaperone-mediated H3.3 loading remain largely unknown. We show here that the chromatin-associated oncoprotein DEK regulates differential HIRA- and DAAX/ATRX-dependent distribution of H3.3 on chromosomes in somatic cells and in embryonic stem cells. Live cell imaging studies show that non-nucleosomal H3.3 normally destined to PML nuclear bodies is re-routed to chromatin after depletion of DEK. This results in HIRA-dependent wide-spread chromatin deposition of H3.3, and H3.3 incorporation in foci of heterochromatin, in process requiring the DAXX/ATRX complex. In embryonic stem cells, loss of DEK leads to displacement of PML bodies and ATRX from telomeres, redistribution of H3.3 from telomeres to chromosome arms and pericentric heterochromatin, induction of a fragile telomere phenotype and telomere dysfunction. Our results indicate that DEK is required for proper loading of ATRX and H3.3 on telomeres and for telomeric chromatin architecture. We propose that DEK acts as a 'gate-keeper' of chromatin, controlling chromatin integrity by restricting broad access to H3.3 by dedicated chaperones. Our results also suggest that telomere stability relies on mechanisms ensuring proper histone supply and routing.Genome Research 07/2014; 24(10). DOI:10.1101/gr.173831.114 · 13.85 Impact Factor
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ABSTRACT: The eukaryotic cell nucleus enclosed within the nuclear envelope harbors organized chromatin territories and various nuclear bodies as sub-nuclear compartments. This higher-order nuclear organization provides a unique environment to regulate the genome during replication, transcription, maintenance, and other processes. In this review, we focus on the plant four-dimensional nuclear organization, its dynamics and function in response to signals during development or stress.Frontiers in Plant Science 08/2014; 5:378. DOI:10.3389/fpls.2014.00378 · 3.64 Impact Factor
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ABSTRACT: The relative positioning of gene loci within a mammalian nucleus is non-random and plays a role in gene regulation. Some sub-nuclear structures may represent “hubs” that bring specific genetic loci into close proximity where co-regulatory mechanisms can operate. The identification of loci in proximity to a shared sub-nuclear structure can provide insights into the function of the associated structure, and reveal relationships between the loci sharing a common association. A technique is introduced based on the nano-dissection of DNA from thin sections of cells by high-precision nano-tools operated inside a scanning electron microscope. The ability to dissect and identify gene loci occupying a shared site at a single sub-nuclear structure is demonstrated here for the first time. The technique is applied to the nano-dissection of DNA in vicinity of a single promyelocytic leukemia nuclear body (PML NB), and reveals novel loci from several chromosomes that are confirmed to associate at PML NBs with statistical significance in a cell population. Furthermore, it is demonstrated that pairs of loci from different chromosomes congregate at the same nuclear body. It is proposed that this technique is the first that allows the de novo determination of gene loci associations with single nuclear sub-structures.Small 08/2014; 10(16). DOI:10.1002/smll.201400075 · 7.51 Impact Factor