C-Terminal regions of topoisomerase IIα and IIβ determine isoform-specific functioning of the enzymes in vivo

Topoisomerase II removes supercoils and catenanes generated during DNA metabolic processes such as transcription and replication. Vertebrate cells express two genetically distinct isoforms (α and β) with similar structures and biochemical activities but different biological roles. Topoisomerase IIα is essential for cell proliferation, whereas topoisomerase IIβ is required only for aspects of nerve growth and brain development. To identify the structural features responsible for these differences, we exchanged the divergent C-terminal regions (CTRs) of the two human isoforms (α 1173-1531 and β 1186-1621) and tested the resulting hybrids for complementation of a conditional topoisomerase IIα knockout in human cells. Proliferation was fully supported by all enzymes bearing the α CTR. The α CTR also promoted chromosome binding of both enzyme cores, and was by itself chromosome-bound, suggesting a role in enzyme targeting during mitosis. In contrast, enzymes bearing the β CTR supported proliferation only rarely and when expressed at unusually high levels. A similar analysis of the divergent N-terminal regions (α 1-27 and β 1-43) revealed no role in isoform-specific functions. Our results show that it is the CTRs of human topoisomerase II that determine their isoform-specific functions in proliferating cells. They also indicate persistence of some functional redundancy between the two isoforms

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

<p>To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.</p

Infection Exposure Promotes ETV6-RUNX1 Precursor B-cell Leukemia via Impaired H3K4 Demethylases

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/peripheral cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss-of-function resulted in increased levels of H3K4me3, which co-precipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches