ARTICLES 1114 VOLUME 11 NUMBER 11 NOVEMBER 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY The PML protein is a target of the t(15;17) chromosomal translocation typical of promyelocytic variants of acute myeloid leukemia, which fuses PML reciprocally with retinoic acid receptor α (RARα) 1 . Overexpression of PML in cancer cell lines induces growth arrest and apoptosis 2 . PML-RARα fusion proteins that interfere with the func- tion of endogenous PML in a trans-dominant manner 1,3 cause leukemia in transgenic mice, whereas PML –/– mice develop a range of cancers including papillomas, carcinomas and lymphomas after expo- sure to carcinogens 4 ; this is paralleled by the loss of PML in human cancers from diverse tissues 5 . Thus, PML is a potent tumor suppressor in vitro and in vivo, in many cell types. PML accumulates in focal nuclear structures termed PML bodies 2 , with an average size of ∼1 μm, which vary in number from 10–30 per nucleus. PML bodies are not formed in PML –/– cells 4,6 , or in cells that express PML-RARα fusions 1 . Their disappearance is correlated with abnormalities in cell growth and death through effects on transcrip- tional regulation, as well as p53-dependent and independent apopto- sis 7 . Because PML bodies contain several different molecules 2 , including the proapoptotic factor DAXX and the tumor suppressors p53, Rb, Chk2 and BRCA1, it has been proposed that they may serve as repositories for protein storage or modification 8 . However, the vari- able movement of proteins to and from PML bodies predicted by this hypothesis has yet to be directly demonstrated. We investigated the intracellular trafficking of the checkpoint protein CHFR and report here its hitherto unrecognized interaction with PML bodies. CHFR, a RING- and FHA-domain-containing nuclear protein, is inactivated by epigenetic mechanisms in up to 30% of epithelial cancer cell lines 9 . CHFR inactivation abrogates the transient delay in passage through early mitotic prophase induced by microtubule-disrupting agents 9 . We find that the nuclear distribution and mobility of CHFR depend on its movement through PML bodies, revealing novel aspects of their putative role as depots for nuclear proteins. This regulation is per- turbed by a trans-dominant inhibitory mutant of CHFR or in PML –/– cells, and is accompanied by abnormalities in mitotic progression and the response to microtubule depolymerization. Our findings identify a novel interaction between two tumor suppressor proteins that provides insight into the biology of PML bodies, and has implications for cancer therapy. RESULTS Alterations in GFP-CHFR distribution during the cell cycle Green fluorescent protein (GFP) was fused in-frame to the N terminus of human CHFR (Fig. 1a) to yield a GFP-CHFR fusion protein with a predicted molecular mass of ∼110 kDa (Fig. 1b). A similar GFP-CHFR fusion protein has been used previously, and can reconstitute function in CHFR-deficient cells 10 . We used it to examine the intracellular dis- tribution of CHFR, because antibodies for fluorescent staining of the endogenous human protein have not yet been reported, nor have we been able to raise them. To provide evidence that GFP-CHFR behaves similarly to the endogenous molecule, we cloned the CHFR ortholog from Xenopus laevis (xCHFR), and raised an antibody against it that detects a single band of the appropriate size in cell extracts (Fig. 1c). Staining of endogenous xCHFR in the XR1 cell line during interphase (Fig. 1d) revealed a punctate pattern of nuclear distribution very simi- lar to GFP-CHFR in HeLa cells (Fig. 1e). Moreover, endogenous x-CHFR and transfected GFP-CHFR colocalized extensively in XR1 cells (Fig. 1d). Collectively, these findings provide evidence that GFP-CHFR accurately reflects the behavior of endogenous CHFR. In HeLa cells during interphase, GFP-CHFR accumulates, primarily in large and small focal structures that are excluded from the nucleolus, with a small amount distributed through the nucleoplasm. Coincident University of Cambridge, Cancer Research UK Department of Oncology and The Medical Research Council Cancer Cell Unit, Hills Road, Cambridge CB2 2XZ, UK. Correspondence should be addressed to A.R.V. (arv22@cam.ac.uk). Published online 3 October 2004; doi:10.1038/nsmb837 PML bodies control the nuclear dynamics and function of the CHFR mitotic checkpoint protein Matthew J Daniels, Alexander Marson & Ashok R Venkitaraman Nuclear foci containing the promyelocytic leukemia protein (PML bodies), which occur in most cells, play a role in tumor suppression. Here, we demonstrate that CHFR, a mitotic checkpoint protein frequently inactivated in human cancers, is a dynamic component of PML bodies. Intermolecular fluorescence resonance energy transfer analysis identified a distinct fraction of CHFR that interacts with PML in living cells. This interaction modulates the nuclear distribution and mobility of CHFR. A trans-dominant mutant of CHFR that inhibits checkpoint function also prevents colocalization and interaction with PML. Conversely, the distribution and mobility of CHFR are perturbed in PML –/– cells, accompanied by aberrations in mitotic entry and the response to spindle depolymerization. Thus, PML bodies control the distribution, dynamics and function of CHFR. Our findings implicate the interaction between these tumor suppressors in a checkpoint response to microtubule poisons, an important class of anticancer drugs. © 2004 Nature Publishing Group http://www.nature.com/nsmb