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

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

Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR

The promyelocytic leukemia (PML) nuclear body (NB) is a dynamic subnuclear compartment that is implicated in tumor suppression, as well as in the transcription, replication, and repair of DNA. PML NB number can change during the cell cycle, increasing in S phase and in response to cellular stress, including DNA damage. Although topological changes in chromatin after DNA damage may affect the integrity of PML NBs, the molecular or structural basis for an increase in PML NB number has not been elucidated. We demonstrate that after DNA double-strand break induction, the increase in PML NB number is based on a biophysical process, as well as ongoing cell cycle progression and DNA repair. PML NBs increase in number by a supramolecular fission mechanism similar to that observed in S-phase cells, and which is delayed or inhibited by the loss of function of NBS1, ATM, Chk2, and ATR kinase. Therefore, an increase in PML NB number is an intrinsic element of the cellular response to DNA damage

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

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