REST Interacts with Cbx Proteins and Regulates Polycomb Repressive Complex 1 Occupancy at RE1 Elements

Abstract

Polycomb group (PcG) proteins control the epigenetic inheritance of transcription regulatory states during development. Progression from pluripotency to differentiation requires the concurrent activation and repression of different PcG target genes. We found that REST and nine REST-associated proteins copurified with Cbx family PcG proteins from mouse embryonic stem (ES) cells. REST interacted with Cbx proteins in live cells and coprecipitated with endogenous Ring1b. Endogenous PRC1 subunits occupied all sites tested that were bound by REST in ES cells. Antibodies directed against different PRC1 subunits precipitated proximal versus distal RE1 elements with opposite relative efficiencies, suggesting that PRC1 bound different sites in distinct configurations. Deletion of the amino-terminal region of REST (Rest^ΔN knockout) as well as short hairpin RNA depletion of REST (REST knockdown) in ES cells reduced PRC1 binding at distal RE1 elements and increased PRC1 binding at proximal RE1 elements. Rest^ΔN and PRC1 subunit knockout as well as REST and PRC1 subunit knockdown had similar relative effects on transcription of neuronal genes in ES cells, derepressing genes with distal, but not genes with proximal, RE1 elements. In differentiating neurons, Rest^ΔN knockout reduced PRC1 occupancy and derepressed transcription at distal RE1 elements but increased PRC1 occupancy and repressed transcription at proximal RE1 elements. The opposite effects of REST on PRC1 occupancy at different RE1 elements contributed to the gene-specific control of PRC1 functions during ES cell differentiation.

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Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.05088-11.

ACKNOWLEDGMENTS

We thank Haruhiko Koseki for providing Cbx2^−/−, Ring1b^fl/fl Ring1a^−/−, Ring1b^fl/fl, and Bmi1^−/− Mel18^−/− ES cells; Helle Jørgensen for providing Rest^ΔN ES cells; Gail Mandel for providing the REST cDNA; Steven Gygi and the Taplin mass spectrometry facility for LC-MS/MS analysis; Lingqun Liu for bioinformatic analysis; and members of the Kerppola laboratory, in particular Claudius Vincenz, for sharing reagents and for constructive criticisms.

This work was funded by the NIDA (grant R01 DA030339).