INTRODUCTION Despite the recognition that Polycomb-group (Pc-G) proteins play an important role in sustaining developmentally established expression boundaries of homeotic genes in Drosophila and Hox gene clusters in mammals, relatively little is known about the molecular principles of this maintenance function. In Drosophila, Pc-G dependent silencing is mediated via polycomb responsive elements (PREs) (Zink and Paro, 1989; Rastelli et al., 1993; Simon et al., 1993; Chan et al., 1994; Chiang et al., 1995; Müller, 1995; Strutt and Paro, 1997). Several lines of evidence predict an extensive interaction between known Pc-G members in both flies and mammals. Their colocalization on polytene chromosomes and in interphase nuclei of cultured insect cells as well as mammalian cells provides a visual indication of physical association (Franke et al., 1995; Alkema et al., 1997a; Gunster et al., 1997; Buchenau et al., 1998). Furthermore, copurification and coimmunoprecipitation of several Pc-G protein classes yields direct evidence for their biochemical interaction (Franke et al., 1992; Alkema et al., 1997a; Gunster et al., 1997; Kyba and Brock, 1998). Studies in Drosophila have shown that, when mutated, the Pc gene product seems to dislodge Pc-G complexes in embryonal cells; this correlates with a severely disturbed larval development (Franke et al., 1995). Taken together, these data have prompted the idea that multiple binding sites with varying binding affinities for Pc-G members may assist in recruitment of multiple factors into larger nucleation sites for Pc-G repression (Zink and Paro, 1995; Pirotta, 1997, 1998; Strutt and Paro, 1997; Van Lohuizen, 1998). How Pc-G proteins recognize and interact with target genes is still largely unclear. Most Pc-G proteins fail to recognize specific DNA sequences, although recently the Drosophila Pc-G gene pleiohomeotic was found to encode a protein with homology to a mammalian transcriptional regulator with sequence-specific DNA binding properties (Brown et al., 1998). Moreover, a conserved sequence motif was reported to occur in Polycomb-responsive elements (Mihaly et al., 1998) suggesting that at least some Pc-G group members may be in direct DNA contact. Polycomb function has been conserved in mammals. The Bmi1 proto-oncogene was the first functional mammalian Pc- G identified (Van Lohuizen et al., 1991b; Brunk et al., 1991). Subsequent gain- and loss-of-function mutational analysis in the mouse showed that morphological transformations of the vertebra along the antero-posterior axis of the skeleton are associated with Hox gene expression boundary shifts (Van der Lugt et al., 1994, 1996; Alkema et al., 1995). Several Pc-G 4627 Journal of Cell Science 112, 4627-4639 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS4692 The human proto-oncogene Bmi1 is a member of the mammalian Polycomb Group (Pc-G) genes. The subnuclear distribution of the BMI1 protein was studied in several primary human and tumor-derived cell lines using immunohistochemical and biochemical methods. In primary and tumor cells, nuclear BMI1 shows a fine-grain distribution over chromatin, usually dense in interphase nuclei and significantly weaker along mitotic chromosomes. In addition, BMI1 preferentially associates with several distinct heterochromatic domains in tumor cell lines. In both primary and tumor cell lines a marked cell cycle- regulation of Pc-G-chromatin interaction is observed: nuclear BMI1-staining dissipates in late S phase and is re- established early in G 1 -phase. Chromatin-association of BMI1 inversely correlates with its phosphorylation status in a cell cycle-dependent fashion: at G 1 /S, hypophosphorylated BMI1 is specifically retained in the chromatin-associated nuclear protein fraction, whereas during G 2 /M, phosphorylated BMI1 is not chromatin- bound. Our findings indicate a strict cell cycle-controlled regulation of Pc-G complex-chromatin association and provide molecular tools for improving our understanding of Pc-G complex regulation and function in mammalian cells. Key words: Polycomb, Chromatin, Phosphorylation SUMMARY Chromatin-association of the Polycomb group protein BMI1 is cell cycle- regulated and correlates with its phosphorylation status Jan Willem Voncken 1 , Dieter Schweizer 2 , Louise Aagaard 3 , Lydia Sattler 2 , Michael F. Jantsch 2 and Maarten van Lohuizen 1, * 1 The Netherlands Cancer Institute, Division of Molecular Carcinogenesis, NL-1066 CX Amsterdam, The Netherlands 2 Department of Cytology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria 3 Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Dr Bohrgasse 7, A-1030 Vienna, Austria *Author for correspondence (e-mail: lohuizen@nki.nl) Accepted 13 October; published on WWW 30 November 1999