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J. Biol. Chem., Vol. 283, Issue 41, 27757-27766, October 10, 2008

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

Preeti Joshi, Elizabeth A. Carrington, Liangjun Wang, Carrie S. Ketel, Ellen L. Miller, Richard S. Jones¹, and Jeffrey A. Simon²

From the Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minneapolis 55455 and the Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275

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 -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.

Received for publication, June 10, 2008 , and in revised form, August 6, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health grants (to J. S. and R. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Dept. of Biological Sciences, Southern Methodist University, Dallas, TX 75275. Tel.: 214-768-3810; Fax: 214-768-3955; E-mail: rjones{at}mail.smu.edu. ² To whom correspondence should be addressed: Dept. of Genetics, Cell Biology and Development, 321 Church St. SE, Minneapolis, MN 55455. Tel.: 612-626-5097; Fax: 612-626-6140; E-mail: simon004{at}umn.edu.

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