E2F-dependent repression of topoisomerase II regulates heterochromatin formation and apoptosis in cells with melanoma-prone mutation.
RB pathway mutations, especially at the CDK4 and INK4A loci, are hallmarks of melanomagenesis. It is presently unclear what advantages these alterations confer during melanoma progression and how they influence melanoma therapy. Topoisomerase II inhibitors are widely used to treat human malignancies, including melanoma, although their variable success is attributable to a poor understanding of their mechanism of action. Using mouse and human cells harboring the melanoma-prone p16Ink4a-insensitive CDK4R24C mutation, we show here that topoisomerase II proteins are direct targets of E2F-mediated repression. Drug-treated cells fail to load repressor E2Fs on topoisomerase II promoters leading to elevated topoisomerase II levels and an enhanced sensitivity of cells to apoptosis. This is associated with the increased formation of heterochromatin domains enriched in structural heterochromatin proteins, methylated histones H3/H4, and topoisomerase II. We refer to these preapoptotic heterochromatin domains as apoptosis-associated heterochromatic foci. We suggest that cellular apoptosis is preceded by an intermediary chromatin remodeling state that involves alterations of DNA topology by topoisomerase II enzymes and gene silencing via formation of heterochromatin. These observations provide novel insight into the mechanism of drug action that influence treatment outcome: drug sensitivity or drug resistance.
Jiao
Wan
W
Cell Cycle and Human Diseases Group, Laboratory of Cell Regulation and Carcinogenesis, NIH, Bethesda, MD 20892, USA.
Lin
Huei-Min
HM
Timmons
Jamie
J
Nagaich
Akhilesh K
AK
Ng
Shu-Wing
SW
Misteli
Tom
T
Rane
Sushil G
SG
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004317:Doxorubicin / Q000494:pharmacology; D050684:E2F Transcription Factors; D005047:Etoposide / Q000494:pharmacology; D006570:Heterochromatin / Q000378:metabolism*; D006801:Humans; D008297:Male; D008545:Melanoma / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009154:Mutation; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D006570:Heterochromatin; D011518:Proto-Oncogene Proteins; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors; D005047:Etoposide; D004317:Doxorubicin; C495900:CDK4 protein, human; C495902:Cdk4 protein, mouse; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases