Topoisomerase poisons such as the epipodophyllotoxin etoposide are widely used effective cytotoxic anticancer agents. However, they are associated with the development of therapy-related acute myeloid leukemias (t-AMLs), which display characteristic balanced chromosome translocations, most often involving the mixed lineage leukemia (MLL) locus at 11q23. MLL translocation breakpoints in t-AMLs cluster in a DNase I hypersensitive region, which possesses cryptic promoter activity, implicating transcription as well as topoisomerase II activity in the translocation mechanism. We find that 2-3% of MLL alleles undergoing transcription do so in close proximity to one of its recurrent translocation partner genes, AF9 or AF4, consistent with their sharing transcription factories. We show that most etoposide-induced chromosome breaks in the MLL locus and the overall genotoxicity of etoposide are dependent on topoisomerase IIβ, but that topoisomerase IIα and -β occupancy and etoposide-induced DNA cleavage data suggest factors other than local topoisomerase II concentration determine specific clustering of MLL translocation breakpoints in t-AML. We propose a model where DNA double-strand breaks (DSBs) introduced by topoisomerase IIβ into pairs of genes undergoing transcription within a common transcription factory become stabilized by antitopoisomerase II drugs such as etoposide, providing the opportunity for illegitimate end joining and translocation.TOP2 | acute leukemia | nuclear organization T herapy-related acute leukemia is an unfortunate side effect of chemotherapy used to treat primary cancers. Chromosomal translocations are a crucial early event in many leukemias, but the mechanism(s) by which these translocations occur are not fully understood (1, 2). Chromosome translocation requires double-strand breaks (DSBs) in each chromosome and close proximity of the breaks, allowing aberrant joining of the heterologous ends. Some debate has existed as to whether chromatin fibers need to be juxtaposed at the time of DNA damage or whether breaks formed at distal sites are able to come together to generate translocations. However, the former model is supported by the positional stability of breaks, the limited mobility of chromatin fibers (3-5), and by the relationship between translocation frequencies and average interphase proximity of the loci involved (6, 7). Several mechanisms of DSB formation contribute to events leading to leukemias and lymphomas (1, 2), but therapy-related acute myeloid leukemia (t-AML) with balanced reciprocal translocation is strongly associated with prior exposure to a class of widely used anticancer drugs known as topoisomerase poisons, which includes etoposide (8). Balanced chromosome translocations involving the mixed lineage leukemia locus (MLL) at 11q23 account for approximately one-third of t-AML translocations (8) and appear with a short latency period following chemotherapy with topoisomerase poisons (9).Estrogen exposure in utero has also been linked to MLL rearrangements (10).Topoisomerase...

We report the whole genome ChIP seq for human TOP2B from MCF7 cells. Using three different peak calling methods, regions of binding were identified in the presence or absence of the nuclear hormone estradiol, as TOP2B has been reported to play a role in ligand-induced transcription. TOP2B peaks were found across the whole genome, 50% of the peaks fell either within a gene or within 5 kb of a transcription start site. TOP2B peaks coincident with gene promoters were less frequently associated with epigenetic features marking active promoters in estradiol treated than in untreated cells. Significantly enriched transcription factor motifs within the DNA sequences underlying the peaks were identified. These included SP1, KLF4, TFAP2A, MYF, REST, CTCF, ESR1 and ESR2. Gene ontology analysis of genes associated with TOP2B peaks found neuronal development terms including axonogenesis and axon guidance were significantly enriched. In the absence of functional TOP2B there are errors in axon guidance in the zebrafish eye. Specific heparin sulphate structures are involved in retinal axon targeting. The glycosaminoglycan biosynthesis–heparin sulphate/heparin pathway is significantly enriched in the TOP2B gene ontology analysis, suggesting changes in this pathway in the absence of TOP2B may cause the axon guidance faults.

Rearrangements involving the RUNX1 gene account for approximately 15% of balanced translocations in therapy-related acute myeloid leukemia (t-AML) patients and are one of the most common genetic abnormalities observed in t-AML. Drugs targeting the topoisomerase II (TOP2) enzyme are implicated in t-AML; however, the mechanism is not well understood and to date a single RUNX1-RUNX1T1 t-AML breakpoint junction sequence has been published. Here we report an additional five breakpoint junction sequences from t-AML patients with the RUNX1-RUNX1T1 translocation. Using a leukemia cell line model, we show that TOP2 beta (TOP2B) is required for induction of RUNX1 chromosomal breaks by the TOP2 poison etoposide and that, while TOP2 alpha (TOP2A) and TOP2B proteins are both present on RUNX1 and RUNX1T1 chromatin, only the TOP2B enrichment reached significance following etoposide exposure at a region on RUNX1 where translocations occur. Furthermore, we demonstrate that TOP2B influences the separation between RUNX1 and two translocation partners (RUNX1T1 and EVI) in the nucleus of lymphoid cells. Specifically, we identified a TOP2B-dependent increase in the number of nuclei displaying juxtaposed RUNX1 and RUNX1T1 loci following etoposide treatment.

Chromatin immunoprecipitation is a method to isolate a protein of interest coupled to DNA following cross-linking with formaldehyde and to quantify the relative abundance or occupancy of the protein at specific genomic loci. After immunoprecipitation of protein-DNA complexes protein-DNA cross-links are reversed and the DNA is extracted. Various methods exist to identify binding sites and determine relative occupancy of the protein of interest; these include quantitative PCR, probing microarrays or sequencing the isolated DNA (ChIP-seq). This chapter details the method of chromatin immunoprecipitation of TOP2 to the point of DNA extraction from the precipitated protein-DNA complexes.