Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants1–6. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive1,7,8. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes1–4. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1–4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB.

Sex comb on midleg (Scm) is a member of the Polycomb group of proteins involved in the maintenance of repression of Hox and other developmental control genes in Drosophila. The two malignant brain tumour (MBT) repeats of Scm form a domain that preferentially binds to monomethylated lysine residues either as a free amino acid or in the context of peptides, while unmodified or di-or trimethylated lysine residues are bound with significantly lower affinity. The crystal structure of a monomethyl-lysinecontaining histone tail peptide bound to the MBT repeat domain shows that the methyl-lysine side chain occupies a binding pocket in the second MBT repeat formed by three conserved aromatic residues and one aspartate. Insertion of the monomethylated side chain into this pocket seems to be the main contributor to the binding affinity. Functional analyses in Drosophila show that the MBT domain of Scm and its methyl-lysine-binding activity are required for repression of Hox genes.

Polycomb group (PcG) genes encode evolutionarily conserved transcriptional repressors that are required for the long-term silencing of particular developmental control genes in animals and plants. PcG genes were first identified in Drosophila as regulators that keep HOX genes inactive in cells where these genes must remain silent during development. Here, we report the results of a genetic screen aimed at isolating novel PcG mutants in Drosophila. In an EMS mutagenesis, we isolated 82 mutants that show Polycomb-like phenotypes in clones in the adult epidermis and misexpression of the HOX gene Ubx in clones in the imaginal wing disc. Analysis of these mutants revealed that we isolated multiple new alleles in most of the already-known PcG genes. In addition, we isolated multiple mutant alleles in each of ten different genes that previously had not been known to function in PcG repression. We show that the newly identified PcG gene calypso is required for the long-term repression of multiple HOX genes in embryos and larvae. In addition, our studies reveal that the Kto/Med12 and Skd/Med13 subunits of the Med12ÁMed13ÁCdk8ÁCycC repressor subcomplex of Mediator are needed for repression of the HOX gene Ubx. The results of the mutant screen reported here suggest that the majority of nonredundant Drosophila genes with strong classic PcG phenotypes have been identified.