Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (ESC) and Enhancer of Zeste [E(Z)] proteins are partners in an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(Z) suggests this complex might methylate histones. We purified an ESC-E(Z) complex from Drosophila embryos and found four major subunits: ESC, E(Z), NURF-55, and the PcG repressor, SU(Z)12. A recombinant complex reconstituted from these four subunits methylates lysine-27 of histone H3. Mutations in the E(Z) SET domain disrupt methyltransferase activity in vitro and HOX gene repression in vivo. These results identify E(Z) as a PcG protein with enzymatic activity and implicate histone methylation in PcG-mediated silencing.

The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is >1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.

Polycomb/Trithorax response elements (PRE/TREs) can switch their function reversibly between silencing and activation, by mechanisms that are poorly understood. Here we show that a switch in forward and reverse noncoding transcription from the Drosophila vestigial (vg) PRE/TRE switches the status of the element between silencing (induced by the forward strand) and activation (induced by the reverse strand). In vitro, both ncRNAs inhibit PRC2 histone methyltransferase activity, but in vivo only the reverse strand binds PRC2. Over-expression of the reverse strand evicts PRC2 from chromatin and inhibits its enzymatic activity. We propose that interactions of RNAs with PRC2 are differentially regulated in vivo, allowing regulated inhibition of local PRC2 activity. Genome-wide analysis shows that strand switching of ncRNAs occurs at several hundred PcG binding sites in fly and vertebrate genomes. This work identifies a novel and potentially widespread class of PRE/TREs that switch function by switching the direction of ncRNA transcription.

No abstract

The Extra sex combs (ESC) protein is a Polycomb group (PcG