Deacetylase Activity Associates with Topoisomerase II and Is Necessary for Etoposide-induced Apoptosis

Abstract

DNA topoisomerase II (topo II) is a ubiquitous nuclear enzyme that is involved in DNA replication, transcription, chromosome segregation, and apoptosis.Here we show by immunoprecipitation, pull down with glutathione S-transferase fusion proteins, and yeast two-hybrid analysis that both topo II␣ and -␤ physically interact with the histone deacetylase HDAC1. The in vitro DNA decatenation activity of recombinant topo II␣ and -␤ is inhibited by association with catalytically inactive, recombinant HDAC1. We provide evidence for the in vivo significance of the topo II-HDAC1 association, showing that inhibition of HDAC activity with trichostatin A suppresses apoptosis induced by the topo II poison etoposide, but not by the topoisomerase I inhibitor camptothecin. We suggest that chromatin remodeling by an HDAC-containing complex facilitates both topo II-catalyzed DNA rearrangement and etoposide-induced DNA damage in vivo.For completion of cell division, the DNA of replicated chromosomes must be disentangled to allow the segregation of sister chromatids. In humans, this is achieved by the unique decatenation activity of DNA topoisomerase II (topo 1 II). Topo II is essential for normal and neoplastic cellular proliferation, and several common anti-cancer drugs exert their cytotoxic effects through this enzyme (1, 2).Topoisomerase II activity in mammalian cells has been attributed to at least two isoforms. Topo II␣ (p170) associates with chromosomes during prophase and throughout mitosis and is thought to be a major component of the nuclear scaffold (3, 4). It has a peak of expression during G 2 /M of the cell cycle (5). In contrast, the closely related topo II␤ (p180) isoform is thought to have a more general role in DNA metabolism, with expression levels that remain relatively constant during cell and growth cycles (5). Both isoforms interact with the C-terminal region of the tumor suppressor protein, p53 (6). p53 is a component of a multiprotein complex that contains the histone deacetylase HDAC1 and the corepressor Sin3a (7-11).HDAC1, and the closely related HDAC2, are both components of two separate multiprotein complexes. The NuRD/Mi-2 repression complex contains both nucleosome remodeling and histone deacetylase activities (12), whereas the Sin3 complex contains only the latter (9). Both complexes contain the Rbassociated proteins RbAp46 and RbAp48 and associate with various, sometimes DNA-binding, transcriptional repressor and corepressor proteins (11). The Xenopus NuRD complex (which contains homologues of mammalian HDAC1, RbAp48, and the methyl-CpG-binding protein MBD3) copurifies with DNA topoisomerase II (13), raising the possibility that mammalian topo II isoforms and HDAC1 may interact in a multiprotein complex.Here we show that HDAC1 and DNA topoisomerase II isoforms physically interact both in vivo and in vitro. We also show that the HDAC inhibitor, TSA, suppresses apoptosis induced by the topo II poison etoposide, but not by the topo I inhibitor camptothecin. Our results raise the interesting possibility t...

Effects of Scaffold/Matrix Alteration on Centromeric Function and Gene Expression

“…One such protein is Topo II␣, which is a major component of the chromosomal scaffold/matrix (43) that has been shown to be enriched at the 10q25 neocentromere (5). A direct interaction between Topo II␣ and the histone deacetylases HDAC-1 and HDAC-2 has been described in mammalian cells, with protein complexes containing these proteins demonstrating both deacetylation and topoisomerase activities (44,45). Furthermore, TSA has been shown to disrupt the association of other scaffold/matrix proteins (46 -48).…”

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis

“…Indeed, the POK protein FAZF has been recently shown to interact with the Fanconi Anemia gene FANCC and to form nuclear dots at or nearby RF, suggesting that POK proteins may be implied in replication and DNA damage repair (Dai et al, 2002). We thus have subjected the induced UTA-L cells to DNA injuries, either upon treatment with topoisomerases poisons (etoposide or camptothecine; Johnson et al, 2001;Suzuki et al, 2001) or to UV irradiation (either UVA or UVC). However, we never found that these treatments are able to mimic or potentiate the effects of BrdU on RF localization with respect to BCL6 bodies (unpublished data).…”

Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis–renal dysfunction–cholestasis (ARC) syndrome

“…We incubated the cell suspension on ice for 30 min with gentle stirring, sonicated it on ice and clarified it by centrifuging at 13,000g for 5 min. We processed the supernatant for immunoprecipitation using 5 µg of purified mouse monoclonal antibody to hemagglutinin or antibody to syntaxin7 coupled to protein G-or protein A-sepharose beads (Amersham Biosciences) 30 . …”