Globally altered epigenetic landscape and delayed osteogenic differentiation in H3.3-G34W-mutant giant cell tumor of bone.

The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.

Clr4 specificity and catalytic activity beyond H3K9 methylation.

In fission yeast, the catalytic activity of the protein lysine methyltransferase (PKMT) Clr4, the sole homolog of the mammalian SUV39H1 and SUV39H2 enzymes, majorly contributes to the formation of heterochromatin. The enzyme introduces histone 3 lysine 9 (H3K9) di- and tri-methylation, a central heterochromatic histone modification, and later it was also found to methylate the Mlo3 protein, which has a role in heterochromatin formation as well. Herein, we have investigated the substrate specificity of Clr4 using custom made mutational scanning peptide arrays. Our data show, that Clr4 recognizes an RK core motif, showing high preference for R8. In addition, it exhibits specific contacts at the S10, T11, G12 and G13 positions of the H3 peptide recognizing an R-K-SKRT-TCS-G sequence. Based on the specificity profile and in vitro methyltransferase assay targeted searches, 11 putative methylation sites in S. pombe proteins were identified from reported Clr4 interacting proteins including Mlo3. Peptide methylation was observed on Mlo3 and 7 novel target sites with strongest methylation signals on Spbc28F2.11 (HMG box-containing protein) at lysine 292 and Hrp3 (Chromodomain ATP-dep DNA helicase) at lysine 89. These data suggest that Clr4 has additional methylation substrates and it will be important to study the biological function of these novel methylation events. Furthermore, the specificity profile of Clr4 has been used to develop a quantitative method to compare and cluster specificity profiles of PKMTs. It shows that the specificity profile of Clr4 is most similar to that of the SUV39H2 enzyme, one of its human homologs. This approach will be helpful in the comparison of the recognition profiles of other families of PKMTs as well.

Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates.

Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1.

Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase.

It has been reported that the Numb protein is methylated at lysine 158 and 163 and that this methylation is introduced by the SET8 protein lysine methyltransferase [Dhami et al., (2013) Molecular Cell 50, 565-576]. We studied this methylation in vitro using peptide arrays and recombinant Numb protein as substrates. Numb peptides and protein were incubated with recombinant SET8 purified after expression in E. coli or human HEK293 cells. However, no methylation of Numb by SET8 was detectable. SET8 methylation of Histone H4 and p53 peptides and proteins, which were used as positive controls, was readily observed. While SET8 methylation of Numb in cells cannot be ruled out, based on our findings, more evidence is needed to support this claim. It appears likely that another not yet identified PKMT is responsible for the reported methylation of Numb in cells.

Activity and specificity of the human SUV39H2 protein lysine methyltransferase.

The SUV39H1 and SUV39H2 enzymes introduce H3K9me3, which is essential for the viability of mammalian cells. It was the aim of the present work to investigate the substrate specificity and product pattern of SUV39H2. Methylation of peptide SPOT arrays showed that SUV39H2 recognizes a long motif on H3 comprising T6-K14, with highly specific readout of R8, S10, T11 and G12 and partial specificity at T6, A7, G13 and K14. Modification of R8 and phosphorylation of S10 or T11 lead to a reduction or loss of SUV39H2 activity towards H3K9. The specificity of SUV39H2 differs from other H3K9 PKMTs, like Dim-5 or G9a, and these biochemical differences can be explained by the structures of the corresponding enzymes. Based on the specificity profile we identified additional non-histone candidate substrates in human proteins, but all of them were only weakly methylated by SUV39H2 at the peptide level. We conclude that SUV39H2 displays a high preference for the methylation of H3. Using the catalytic SET domain we show here that the enzyme prefers H3K9me0 as a substrate over H3K9me1 and H3K9me2 and it introduces the first two methyl groups into H3K9me0 in a processive reaction. SUV39H2 can transfer up to three methyl groups to lysine 9 of histone H3 but the last methylation reaction is much slower than the first two steps. We also demonstrate that the N324K mutant in the SET domain of SUV39H2 that has been shown to cause an inherited nasal skin disease in Labrador Retrievers renders SUV39H2 inactive. Differences in the circular dichroism spectra of wild type and mutant proteins indicated that the mutation causes slight structural changes.

Specificity analysis of protein lysine methyltransferases using SPOT peptide arrays.

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Non-radioactive protein lysine methyltransferase microplate assay based on reading domains.

New protein lysine methyltransferase (PKMT) assays are needed to facilitate screening for improved PKMT inhibitors, because PKMTs are mutated or overexpressed in several cancers. In cells, methylated lysine residues are recognized by reading domains such as the chromodomain of HP1ß, which bind to target proteins in a lysine-methylation-specific manner. Herein we describe a sensitive, robust, and non-radioactive high-throughput PKMT assay that employs the HP1ß chromodomain to detect the methylation of peptide substrates by the human SUV39H1 and SUV39H2 PKMTs. The assay has a very good dynamic range and high signal-to-noise ratio. It can be used to screen for PKMT inhibitors, as illustrated by analyzing the inhibition of SUV39H1 by chaetocin. The IC50 value of this inhibition was found to be 480 nM, which is close to its published value. Our data indicate that natural reading domains can be used as alternates to methyl-specific antibodies in PKMT assays. Reading domains can be produced recombinantly in E. coli at low cost and consistent quality, and they are accessible to protein design.

Exploring the topology of the Gid complex, the E3 ubiquitin ligase involved in catabolite-induced degradation of gluconeogenic enzymes.

In the yeast Saccharomyces cerevisiae, key regulatory enzymes of gluconeogenesis such as fructose-1,6-bisphosphatase are degraded via the ubiquitin proteasome system when cells are replenished with glucose. Polyubiquitination is carried out by the Gid complex, a multisubunit ubiquitin ligase that consists of seven different Gid (glucose-induced degradation-deficient) proteins. Under gluconeogenic conditions the E3 ligase is composed of six subunits (Gid1/Vid30, Gid2/Rmd5, Gid5/Vid28, Gid7, Gid8, and Gid9/Fyv10). Upon the addition of glucose the regulatory subunit Gid4/Vid24 appears, binds to the Gid complex, and triggers ubiquitination of fructose-1,6-bisphosphatase. All seven proteins are essential for this process; however, nothing is known about the arrangement of the subunits in the complex. Interestingly, each Gid protein possesses several remarkable motifs (e.g. SPRY, LisH, CTLH domains) that may play a role in protein-protein interaction. We, therefore, generated altered versions of individual Gid proteins by deleting or mutating these domains and performed co-immunoprecipitation experiments to analyze the interaction between distinct subunits. Thus, we were able to create an initial model of the topology of this unusual E3 ubiquitin ligase.