Polycomb Complexes and the Propagation of the Methylation Mark at the Drosophila Ubx Gene

Vincenzo Pirrotta,Gaetano I. Dellino,Yuri B. Schwartz,Tatyana G. Kahn

doi:10.1074/jbc.m605430200

Abstract

Polycomb group proteins are transcriptional repressors that control many developmental genes. The Polycomb group protein Enhancer of Zeste has been shown in vitro to methylate specifically lysine 27 and lysine 9 of histone H3 but the role of this modification in Polycomb silencing is unknown. We show that H3 trimethylated at lysine 27 is found on the entire Ubx gene silenced by Polycomb. However, Enhancer of Zeste and other Polycomb group proteins stay primarily localized at their response elements, which appear to be the least methylated parts of the silenced gene. Our results suggest that, contrary to the prevailing view, the Polycomb group proteins and methyltransferase complexes are recruited to the Polycomb response elements independently of histone methylation and then loop over to scan the entire region, methylating all accessible nucleosomes. We propose that the Polycomb chromodomain is required for the looping mechanism that spreads methylation over a broad domain, which in turn is required for the stability of the Polycomb group protein complex. Both the spread of methylation from the Polycomb response elements, and the silencing effect can be blocked by the gypsy insulator.

Highlights

In this work we examined the in vivo distribution of me3K27 and several Polycomb Group (PcG) proteins in the repressed Ubx gene using quantitative chromatin immunoprecipitation (ChIP)
While this article was in preparation, other reports have described the distribution of PcG proteins over part or all of the Ubx gene using nonquantitative or semiquantitative analyses of chromatin immunoprecipitation that have given different results from ours [12, 19, 20]
Our results suggest that trimethylation of H3 at K27 is unlikely to serve as a recruiter of PcG proteins to their response elements but may be important for stabilizing the complexes and may participate more directly in transcriptional repression

Summary

EXPERIMENTAL PROCEDURES

Fly Lines—The YGPhsW transposon construct in lines F22 and M7 is described in Ref. 21. Anti-E(Z) antibodies were raised against a peptide containing amino acids 8 –190 fused to GST and affinity-purified as described in Ref. 22. Anti-PC and anti-PSC antibodies were described in Ref. 22. Chromatin Cross-linking and Immunoprecipitation—Cells of a derivative of the Schneider L2 cultured cell line, Sg4, were grown and cross-linked as described [25], the embryos were collected overnight and cross-linked as described in Ref. 21. 5– 6 ␮g of anti-PC, PSC, me3K27, me2K27, H2B, 1–3 ␮g of anti-H3, E(Z), Trithorax (TRX), GAGA, 1 ␮l of anti-TBP, and 5 ␮l of anti-me3K9 and anti-RNA polymerase (8WG16) antibodies were used per one reaction. PCR reactions were assembled by mixing 5 ␮l (V2) of immunoprecipitated DNA solution with 10 ␮l of 2ϫSYBR Green PCR Master Mix, 0.1 ␮M of corresponding primers and pure water to 20 ␮l. The hybridization data are deposited to ArrayExpress data base with accession number: E-MEXP-535

RESULTS

Ubx promoter

DISCUSSION