Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt

Polycomb group (PcG) proteins repress transcription by modifying chromatin structure in target genes. dSfmbt is a subunit of the Drosophila melanogaster PcG protein complex PhoRC and contains four malignant brain tumour (MBT) repeats involved in the recognition of various mono- and dimethylated histone peptides. Here, we present the crystal structure of the four-MBT-repeat domain of dSfmbt in complex with a mono-methylated histone H4 peptide. Only a single histone peptide binds to the four-MBT-repeat domain. Mutational analyses show high-affinity binding with low peptide sequence selectivity through combinatorial interaction of the methyl-lysine with an aromatic cage and positively charged flanking residues with the surrounding negatively charged surface of the fourth MBT repeat. dSfmbt directly interacts with the PcG protein Scm, a related MBT-repeat protein with similar methyl-lysine binding activity. dSfmbt and Scm co-occupy Polycomb response elements of target genes in Drosophila and they strongly synergize in the repression of these target genes, suggesting that the combined action of these two MBT proteins is crucial for Polycomb silencing

Structural and functional analyses of methyl-lysine binding by the malignant brain tumour repeat protein Sex comb on midleg

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-lysine-containing 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

Chromatin-modifying Complex Component Nurf55/p55 Associates with Histones H3 and H4 and Polycomb Repressive Complex 2 Subunit Su(z)12 through Partially Overlapping Binding Sites

Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners

Histone methylation by PRC2 is inhibited by active chromatin marks

The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through deposition of histone H3 lysine 27 tri-methyl (H3-K27me3) modifications. Here, we analyzed the molecular mechanism by which PRC2 recognizes and discriminates between transcriptionally repressed and active chromatin. We show structurally and biochemically that the unmodified histone H3 tail comprising residues 1-14 is bound by the PRC2 Nurf55-Su(z)12 sub-complex. We find that H3-K4me3 modified tails are no longer retained by Nurf55-Su(z)12, and that H3-K4me3 triggers allosteric PRC2 inhibition in conjunction with the Su(z) VEFS domain and E(z). Inhibition requires H3-K4me3 to be on the same tail as H3-K27. The PRC2 HMTase activity is also inhibited by H3-K36me2/3, but not by the heterochromatin mark such as H3-K9me3. Inhibition by H3-K4me3 and H3-K36me2/3 marks of transcriptionally active chromatin is conserved in mammalian and fly PRC2. In the case of the diverse plant PRC2 complexes, the specific Su(z)12 subunit present determines whether the complex is inhibited by these active chromatin marks. PRC2 inhibition by active chromatin marks, coupled with PRC2 stimulation by transcriptionally repressive H3-K27me3, enables PRC2 to autonomously template repressive marks H3-K27me3 without overwriting active chromatin domains

Mitotic lamin disassembly is triggered by lipid-mediated signaling.

International audienceDisassembly of the nuclear lamina is a key step during open mitosis in higher eukaryotes. The activity of several kinases, including CDK1 (cyclin-dependent kinase 1) and protein kinase C (PKC), has been shown to trigger mitotic lamin disassembly, yet their precise contributions are unclear. In this study, we develop a quantitative imaging assay to study mitotic lamin B1 disassembly in living cells. We find that CDK1 and PKC act in concert to mediate phosphorylation-dependent lamin B1 disassembly during mitosis. Using ribonucleic acid interference (RNAi), we showed that diacylglycerol (DAG)-dependent PKCs triggered rate-limiting steps of lamin disassembly. RNAi-mediated depletion or chemical inhibition of lipins, enzymes that produce DAG, delayed lamin disassembly to a similar extent as does PKC inhibition/depletion. Furthermore, the delay of lamin B1 disassembly after lipin depletion could be rescued by the addition of DAG. These findings suggest that lipins activate a PKC-dependent pathway during mitotic lamin disassembly and provide evidence for a lipid-mediated mitotic signaling event

Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB

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 plants(1–6). 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 inactive(1,7,8). Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes(1–4). 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