Abstract
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 doublestrand 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.
| Original language | English |
|---|---|
| Pages (from-to) | 308-322 |
| Number of pages | 15 |
| Journal | Cell Cycle |
| Volume | 10 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jan 15 2011 |
Bibliographical note
Funding Information:matin using Image J v1.42 software (NIH). Pixel measurements G.D. is a Canadian Institutes of Health Research (CIHR) New (n = 50 measurements taken from 5 cells) were converted into Investigator and the Cameron Research Scientist of the Dalhousie microns (μm) and then averaged. Statistical significance between University Cancer Research Program. Y.S. is an Israel Cancer cell lines was generated using the Student’s t-test in Excel Research Fund Research Professor. R.K. was a recipient of a (Microsoft). Killam Predoctoral Fellowship and K.A. is supported by a trainee For the statistical analysis of chromatin density, chromatin award from The Beatrice Hunter Cancer Research Institute with was first segmented within composite ESI micrographs of Mock funds provided by The Terry Fox Foundation Strategic Health and ΔKAP1 5757-TERT NHDFs as previously described in Research Training Program in Cancer Research at the CIHR. reference 27. Briefly, the N and P ESI micrographs were first This work was funded by an operating grant awarded to G.D. false colored in green and red, respectively, and combined into a from the Canadian Institutes of Health Research (MOP-84260). single RGB image using Photoshop 11.0 (Adobe). In the merged Work in the laboratory of Y.S. is funded by the A-T Medical image chromatin becomes apparent as yellow 10 nm and higher-Research Foundation and the Israel Cancer Research Fund. order fibers. These images were then imported into ImageJ v1.42 (NIH) and the Color Threshold Plugin (Gabriel Landini) was Note
ASJC Scopus Subject Areas
- Molecular Biology
- Developmental Biology
- Cell Biology
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't