Abstract
Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function.
Highlights
Post-translational modification of Polycomb protein Cbx7 remains poorly understood
Identification of a Novel Cbx7 Phosphorylation Site—To investigate the post-translational modification profile of Cbx7, we performed immunoprecipitation followed by LC-MS/MS using an established HEK 293 cell line that expresses C-terminal FLAG-tagged mouse Cbx7 (Cbx7-FLAG) [4] versus the parental cell line as a control
Expressing mouse Cbx7 in HEK 293 cells reconstitutes a bona fide Polycomb repressive complex 1 (PRC1), because we detected core PRC1 proteins interacting with Cbx7 by MS: RING1A, RING1B, BMI1, and MEL-18, as well as less abundant components such as PHC3 and SCML2 (Fig. 1A) [39, 40]
Summary
Post-translational modification of Polycomb protein Cbx remains poorly understood. Results: Here we identify and characterize a novel phosphorylation site at threonine 118 of mouse Cbx. We have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx and propose that mitogen signaling to the chromatin template regulates PRC1 function. Multiple PcG protein complexes exist, and two core complexes have been well characterized biochemically and functionally [5] These are the Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), which contain unique histone modifying activities. We report a novel phosphorylation site on Thr-118 of mouse Cbx, which lies in close proximity to the Pc box This site is highly conserved from Drosophila Pc to mammalian Cbx, suggesting an important regulatory function. We report a novel Cbx phosphorylation site and demonstrate that MAPK-mediated Cbx7T118ph regulates PRC1 function
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