KAP1 depletion increases PML nuclear body number in concert with ultrastructural changes in chromatin
Copyright policy )KAP1 depletion increases PML nuclear body number in concert with ultrastructural changes in chromatin
The promyelocytic leukemia (PML) protein is the main structural component of subnuclear domains termed PML nuclear bodies (PML NBs), which are implicated in tumor suppression by regulating apoptosis, cell senescence, and DNA repair. Previously, we demonstrated that ATM kinase can regulate changes in PML NB number in response to DNA double-strand breaks (DSBs). PML NBs make extensive contacts with chromatin and ATM mediates DNA damage-dependent changes in chromatin structure in part by the phosphorylation of the KRAB-associated protein 1 (KAP1) at S824. We now demonstrate that in the absence of DNA damage, reduced KAP1 expression results in a constitutive increase in PML NB number in both human U2-OS cells and normal human diploid fibroblasts. This increase in PML NB number correlated with decreased nuclear lamina-associated heterochromatin and a 30% reduction in chromatin density as observed by electron microscopy, which is reminiscent of DNA damaged chromatin. These changes in chromatin ultrastructure also correlated with increased histone H4 acetylation, and treatment with the HDAC inhibitor TSA failed to further increase PML NB number. Although PML NB number could be restored by complementation with wild-type KAP1, both the loss of KAP1 or complementation with phospho-mutants of KAP1 inhibited the early increase in PML NB number and reduced the fold induction of PML NBs by 25-30% in response to etoposide-induced DNA DSBs. Together these data implicate KAP1-dependent changes in chromatin structure as one possible mechanism by which ATM may regulate PML NB number in response to DNA damage.
- Dalhousie University Canada
Nuclear Proteins, Acetylation, Cell Cycle Proteins, Ataxia Telangiectasia Mutated Proteins, Promyelocytic Leukemia Protein, Protein Serine-Threonine Kinases, Tripartite Motif-Containing Protein 28, Cell Nucleus Structures, Chromatin, Histone Deacetylases, DNA-Binding Proteins, Histone Deacetylase Inhibitors, Histones, Repressor Proteins, Mutation, Humans, Phosphorylation, Cells, Cultured, DNA Damage, Transcription Factors
Nuclear Proteins, Acetylation, Cell Cycle Proteins, Ataxia Telangiectasia Mutated Proteins, Promyelocytic Leukemia Protein, Protein Serine-Threonine Kinases, Tripartite Motif-Containing Protein 28, Cell Nucleus Structures, Chromatin, Histone Deacetylases, DNA-Binding Proteins, Histone Deacetylase Inhibitors, Histones, Repressor Proteins, Mutation, Humans, Phosphorylation, Cells, Cultured, DNA Damage, Transcription Factors
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