Role of the polycomb protein EED in the propagation of repressive histone marks.

Polycomb group proteins have an essential role in the epigenetic maintenance of repressive chromatin states. The gene-silencing activity of the Polycomb repressive complex 2 (PRC2) depends on its ability to trimethylate lysine 27 of histone H3 (H3K27) by the catalytic SET domain of the EZH2 subunit, and at least two other subunits of the complex: SUZ12 and EED. Here we show that the carboxy-terminal domain of EED specifically binds to histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent it from recognizing repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and, in Drosophila, reduce global methylation and disrupt development. These findings suggest a model for the propagation of the H3K27me3 mark that accounts for the maintenance of repressive chromatin domains and for the transmission of a histone modification from mother to daughter cells.

Small Molecule Inhibitor Targeting CDT1/Geminin Protein Complex Promotes DNA Damage and Cell Death in Cancer Cells.

DNA replication initiation requires the loading of MCM2-7 complexes at the origins of replication during G1. Replication licensing renders chromatin competent for DNA replication and its tight regulation is essential to prevent aberrant DNA replication and genomic instability. CDT1 is a critical factor of licensing and its activity is controlled by redundant mechanisms, including Geminin, a protein inhibitor of CDT1. Aberrant CDT1 and Geminin expression have been shown to promote tumorigenesis in vivo and are also evident in multiple human tumors. In this study, we developed an in vitro AlphaScreen™ high-throughput screening (HTS) assay for the identification of small-molecule inhibitors targeting the CDT1/Geminin protein complex. Biochemical characterization of the most potent compound, AF615, provided evidence of specific, dose-dependent inhibition of Geminin binding to CDT1 both in-vitro and in cells. Moreover, compound AF615 induces DNA damage, inhibits DNA synthesis and reduces viability selectively in cancer cell lines, and this effect is CDT1-dependent. Taken together, our data suggest that AF615 may serve as a useful compound to elucidate the role of CDT1/Geminin protein complex in replication licensing and origin firing as well as a scaffold for further medicinal chemistry optimisation.

Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein.

Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an approximately 100 kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0 A resolution and allowed the collection of complete diffraction data to about 2.6 A resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed.

The Arabidopsis (ASHH2) CW domain binds monomethylated K4 of the histone H3 tail through conformational selection.

Chromatin post-translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N-terminal tail lysine residues constitutes one of many such modifications, executed by families of histone lysine methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain, CW, and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains has so far been emphasizing lock-key models, missing important aspects of histone-tail CW interactions. We here present an analysis of the ASHH2 CW-H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. ß-augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the η1, η3 and C-terminal coils, but also in the ß1 and ß2 strands and the C-terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affect the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct post-translational modification state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome. DATABASES: Structural data are available in the PDB database under the accession code 6QXZ. Resonance assignments for CW42 in its apo- and holo-forms are available in the BMRB database under the accession code 27251.

Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2.

Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification.

Bacteria co-transformed with recombinant proteins and chaperones cloned in independent plasmids are suitable for expression tuning.

The efficient over-expression of several recombinant proteins in the same bacterial cell is usually prevented due to metabolic limitations. Nevertheless, the possibility to co-produce high amounts of the sub-units of a complex or to express a wide set of chaperones and foldases could be technologically very useful. We developed a system based on three vectors. Two are under IPTG regulation and enable the recombinant expression of six chaperones, the third one is arabinose-inducible and harbours the sequence for the target protein. In such a way the independent induction and the level of expression of both chaperones and target protein is possible. The data show that the expression leakage from pET vectors was prevented by the introduction of further plasmids in the cell and that the recombinant proteins compete for their expression. In fact, the high rate induction of one of them could switch off the accumulation of the other recombinant proteins. The first information was used to maximise the expression of toxic proteins while the cross-inhibition among recombinant proteins was exploited to modulate and optimise the target protein expression and to induce the chaperone-assisted in vivo re-folding of aggregated target protein.

Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment.

The mechanisms by which the major Polycomb group (PcG) complexes PRC1 and PRC2 are recruited to target sites in vertebrate cells are not well understood. Building on recent studies that determined a reciprocal relationship between DNA methylation and Polycomb activity, we demonstrate that, in methylation-deficient embryonic stem cells (ESCs), CpG density combined with antagonistic effects of H3K9me3 and H3K36me3 redirects PcG complexes to pericentric heterochromatin and gene-rich domains. Surprisingly, we find that PRC1-linked H2A monoubiquitylation is sufficient to recruit PRC2 to chromatin in vivo, suggesting a mechanism through which recognition of unmethylated CpG determines the localization of both PRC1 and PRC2 at canonical and atypical target sites. We discuss our data in light of emerging evidence suggesting that PcG recruitment is a default state at licensed chromatin sites, mediated by interplay between CpG hypomethylation and counteracting H3 tail modifications.

A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

Reading, writing and editing methylated lysines on histone tails: new insights from recent structural studies.

The phenotypes of different cell types are governed by their differential gene expression programmes, which are prominently influenced by epigenetic gene regulation featuring heritable chromatin states. Different epigenetic states are associated with distinctive patterns of post-translational modifications of the histone tails, which in turn influence the recruitment of chromatin-modifying effectors and local chromatin structure. Despite rapid advances in understanding how particular histone marks correlate with transcriptional output, many of the molecular details on how the maintenance and alteration of these modifications relate to fundamental processes such as replication, DNA repair, and transcription remain to be elucidated. Here, we review recent advances in the structural description of the reading, writing, and editing of two histone methylation marks with opposite functions: at histone H3 lysine 4 (H3K4)-associated with actively transcribed genes, and at histone H3 lysine 27 (H3K27)-a hallmark of silenced chromatin. These two marks are associated with trithorax and polycomb, respectively, prototypes of the genes involved in epigenetic inheritance in Drosophila. We also briefly discuss some recent examples of how the readout of particular marks is influenced by the presence of other modifications.

Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells.

The solubility and stability of recombinant proteins are increased by their fusion to NusA.

The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20mg of fusion protein per litre of culture and 2.5-5mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins.

Dimerization properties of a Xenopus laevis kinesin-II carboxy-terminal stalk fragment.

We have analysed the structural and physical properties of the carboxy-terminal stalk region of a kinesin-II, Xenopus kinesin-like protein 3A/B (Xklp3A/B), which we showed to be essential for heterodimerization in a previous work (De Marco et al., 2001). We expressed the corresponding A-stalk and B-stalk fragments and investigated their modes of interaction by analytical ultracentrifugation (AUC), circular dichroism spectroscopy, denaturation assays and electron microscopy. Co-expression of the A-stalk and B-stalk produced the properly folded, hetero-dimeric coiled coil at high yields. The dimeric nature of the complex was confirmed by AUC. We also found that the isolated A-stalk fragment forms a stable helix by itself and shows a significant tendency towards homodimer and higher-order complex formation. In the absence of the corresponding A-stalk fragment, the isolated B-stalk fragment remains partially unfolded, which suggests that the A-stalk provides a template structure for the B-stalk in order to recompose the complete heterodimeric coiled coil.