Abstract

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

Highlights

  • Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation

  • We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin

  • We present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes

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Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—Eukaryotic expression vectors for untagged and tagged wild type or mutant 3pK have been described elsewhere [3, 22]. Human U2OS osteosarcoma cells and TIG3 primary human fibroblasts expressing the murine ecotropic receptor were provided by Dr D. Immunofluorescence—Immunofluorescence on methanol-fixed U2OS cells was carried out as described previously [26]. The anti-Bmi mAb F6 and anti-MPh 1 (HPH1) pAb 70092 were kindly provided by Dr M. van Lohuizen. Cells were extracted with ELB buffer [5, 26]; immune complexes were precipitated with protein A- or G-agarose. The pellet was carefully washed once in nuclear buffer and extracted in IP0.1 buffer (20 mM HEPES, pH 7.6, 10% glycerol, 25 mM MgCl2, 0.1 mM EDTA, 0.2% Nonidet P-40, 0.1 M Potassium acetate, 1 mM NaVO4, 50 mM NaF; chromatin-bound fraction) as described previously [34]. In vitro kinase assays with recombinant protein utilized 1 ␮g of kinase (3pK-EE) and 5 ␮g of substrate (His- or GST-tagged Bmi1)

RESULTS
DISCUSSION
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