Dominant Alleles Identify SET Domain Residues Required for Histone Methyltransferase of Polycomb Repressive Complex 2

Ellen L Miller,Elizabeth A Carrington,Liangjun Wang,Richard S Jones,Preeti Joshi,Carrie S Ketel,Jeffrey A Simon

doi:10.1074/jbc.m804442200

Ellen L Miller, Elizabeth A Carrington + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m804442200

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Abstract

Polycomb gene silencing requires histone methyltransferase activity of Polycomb repressive complex 2 (PRC2), which methylates lysine 27 of histone H3. Information on how PRC2 works is limited by lack of structural data on the catalytic subunit, Enhancer of zeste (E(Z)), and the paucity of E(z) mutant alleles that alter its SET domain. Here we analyze missense alleles of Drosophila E(z), selected for molecular study because of their dominant genetic effects. Four missense alleles identify key E(Z) SET domain residues, and a fifth is located in the adjacent CXC domain. Analysis of mutant PRC2 complexes in vitro, and H3-K27 methylation in vivo, shows that each SET domain mutation disrupts PRC2 histone methyltransferase. Based on known SET domain structures, the mutations likely affect either the lysine-substrate binding pocket, the binding site for the adenosylmethionine methyl donor, or a critical tyrosine predicted to interact with the substrate lysine epsilon-amino group. In contrast, the CXC mutant retains catalytic activity, Lys-27 specificity, and trimethylation capacity. Deletion analysis also reveals a functional requirement for a conserved E(Z) domain N-terminal to CXC and SET. These results identify critical SET domain residues needed for PRC2 enzyme function, and they also emphasize functional inputs from outside the SET domain.

Highlights

The Polycomb group (PcG)3 proteins are highly conserved chromatin components that work together to silence target genes during development
Deletion analysis reveals a functional requirement for a conserved E(Z) domain N-terminal to CXC and SET. These results identify critical SET domain residues needed for Polycomb repressive complex 2 (PRC2) enzyme function, and they emphasize functional inputs from outside the SET domain
Genetic studies show that individual components of each complex are required for Hox gene silencing in vivo, indicating that these complexes work together to maintain silent chromatin states

Summary

EXPERIMENTAL PROCEDURES

Sequence Determination of Mutant Alleles—Genomic DNA was extracted from larvae or adult flies that were hemizygous for the E(z)son, E(z)son, E(z)son, E(z), or E(z) alleles in trans to a deficiency of the E(z) chromosomal region. By these criteria, which include lethal lowing modifications: unmethylated (H3 peptide 27– 40 plus phase analysis and Hox patterning defects, these five mutants three propionyl groups), monomethylated (peptide 27– 40 plus were determined to exert dominant effects stronger than a refthree propionyl groups plus one methyl group), dimethylated erence E(z) null, E(z), in one or more phenotypic tests (Table (peptide 27– 40 plus two propionyl groups plus two methyl 1) These mutants likely produce functionally altered vergroups), and trimethylated (peptide 27– 40 plus two propionyl sions of E(Z), and if missense alleles, they could identify critical groups plus three methyl groups). Lated H3 peptide peak at 1601.9 m/z was confirmed by E(z) Missense Mutations Alter SET Domain Motifs Implimanual MS/MS sequencing

RESULTS

PRO to MESOc ϩϩ ϩ Ϫ Ϫ Ϫ

This critical tyrosine is analogous to

DISCUSSION