Tandem-affinity purification coupled to mass spectrometry reveals protein interaction network of putative methyltransferases.

<p>Purification of 10 TAP-tagged putative methyltransferases from ponasterone-inducible strains of HEK 293 cells. Eluted proteins were separated by SDS-PAGE. Gels were silver stained and cut in slices that were then trypsin digested before protein identification by LC-MS/MS. Tagged baits and major interactors are marked.</p

Computational analysis defines a novel family of putative protein methyltransferases.

<p>(A) Unrooted phylogenetic tree of a family of human putative methyltransferases distantly related to PRMTs. FAM86 represents a number of genetic variants (FAM86A, B1, B2, C, and D) whose duplication is observed only in primates. Branch lengths are not proportional to the actual evolutionary distances between the sequences. (B) Secondary structure organization of the Rossmann fold domain of PRMTs responsible for the methyltransferase activity. Arrows represent β strands and rectangles correspond to α helices, including typically ill-defined or inexistent α helix C. (C) Multiple sequence alignment of the Rossmann fold of all members within this family as generated by ClustalW2 (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). Red residues are small, hydrophobic, aromatic; blue are acidic; magenta are basic; and all other residues are green. Primary sequence alignment corresponds nicely with secondary structure prediction by Jpred3 (<a href="http://www.compbio.dundee.ac.uk/www-jpred/" target="_blank">http://www.compbio.dundee.ac.uk/www-jpred/</a>). Overhead β strands and α helices are shown as in (B). Conserved motif I, the site of S-adenosylmethionine binding, is also marked.</p

Methylation of VCP decreases the activity of its N-terminal ATPase domain.

<p>(A) Linear representation of a fragment of VCP encompassing its N-terminal and first ATPase domain employed in the ATPase assay. Proximity of the methylated lysine to the Walker B motif is highlighted above. (B) In vitro methylation assays of 1–481_VCP-His fragment by GST-METTL21D as compared to full length VCP. (C, D) Colorimetric assays to measure released phosphate (C) and relative ATPase activity (D) by the 1–481 fragment of VCP. The experiment was done in triplicate. Data from the last 3 time points (9 measurements in total for each condition) was compiled to generate the graph shown in (D). (E) In vitro GST pull-down assay of VCP-His with GST-METTL21D. In all experiments the effect of un methylatable VCP mutant K135R, catalytically inactive METTL21D mutant E73Q, and methylation inhibitor S-adenosylhomocysteine is shown.</p

KIN, VCP, and a number of hsp70 isoforms are each trimethylated on lysine residues by specific methyltransferases within this family.

<p>(A) Linear representation of all identified substrates with domain architecture. Residues delineating each domain are marked below. ZnF, Zinc Finger; WH, Winged Helix; SH3, Src Homology 3; SBD, substrate binding domain. Position of the methylated lysines is shown above. (B) Multiple sequence alignment of the region surrounding trimethylated lysines (boxed) in human VCP, KIN, and Hsp70 isoforms compared to paralogous genes in various organisms. Hs, <i>Homo sapiens</i>; Sc, <i>Saccharomyces cerevisiae</i>; Dm, <i>Drosophila melanogaster</i>; At, <i>Arabidopsis thaliana</i>; Pf, <i>Plasmodium falciparum</i>. Color code is as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003210#pgen-1003210-g001" target="_blank">Figure 1</a>. Strong and weak residue similarity are represented by a colon (:) and period (.), respectively, and asterisk (*) denotes identity. (C–E) In vitro methylation assays with tritium-labeled S-adenosylmethionine of KIN-His with GST-METTL22 (C), VCP-His with GST-METTL21D (D), and three His-tagged hsp70 isoforms (HSPA1, HSPA5, and HSPA8) with GST-METTL21A (E). In each case, substitution of the methylated lysine by an arginine leads to loss of methylation signal as detected by autoradiography. Coomassie staining of the gel shows total proteins loaded onto the gel and serves as control.</p

ASPSCR1 promotes methylation of VCP by METTL21D.

<p>(A) In vitro methylation assays of VCP-His, UBXN6-His, ASPSCR1-His and fragments of ASPSCR1 with GST-METTL21D. Various combinations of the UBX proteins were added to reactions containing VCP. Coomassie staining of the gel is shown. (B) Linear representation of ASPSCR1 showing domain architecture of the protein (UBL, UBiquitin-Like domain; SHP, SHP box; UBX, UBiquitin regulatory X domain; CC, Coiled-Coil domain) and localization of residue 280 which marks the boundary between the N- and C- terminal fragments used in these experiments. (C) In vitro GST pull-down assays of VCP-His, UBXN6-His, ASPSCR1-His and fragments of ASPSCR1 with GST-METTL21D. Combinations of full-length ASPSCR1 and its fragments were once again employed with VCP. (D) Linear representation of VCP showing domain architecture of the protein (including double Ψ barrel superfold and 4-stranded β barrel of the N-terminal domain, Walker A and B motifs, as well as 4 α helices bundle of ATPase domains D1 and D2 as well as linker regions L1 and L2 and C-terminal domain) and localization of the mutants used in this study as well as the site of methylation. (E) In vitro methylation assays of wild-type VCP-His and IBMPFD and ALS-causing mutations R155H, R159G and R191Q in presence or absence of ASPSCR1-His. (F) In vitro GST pull-down assays of the same combination of proteins as in (E).</p

Integrating DNA Methylation and Gene Expression data in Placenta Tissue to Predict Childhood Obesity

<p>Recent advances in genomic technologies have made it feasible to measure, on the same individual, multiple types of genomic activity such as genotypes, gene expression, DNA copy number, methylation and microRNA expression. However, in order to benefit from the increasing amounts of heterogeneous data and to obtain a more complete view of genomic functions, there is a great need for statistical and computationally efficient methods that allow us to combine this information in an intelligent way. Challenges with prediction models in this setting arise from the high-dimensional non-linear nature of the data, the large number of measurements compared to the few samples for whom they are collected, and the presence of complex interactions between the different types of data. Methods such as sparse regression, hierarchical clustering and principal component analysis can address any one of these challenges, but can not do so simultaneously. Kernel methods, which use matrices measuring the similarity between two individuals, offer a powerful way of simultaneously addressing these challenges without significantly increasing the computational burden. In this work, we investigate the benefits and challenges that arise from using kernel methods in the context of integrating DNA methylation, gene expression and phenotypic data in a sample of mother-child pairs from a prospective birth cohort. The goal of this study is to identify epigenetic marks observed at birth that help predict childhood obesity.</p

Discovery of Cell Compartment Specific Protein–Protein Interactions using Affinity Purification Combined with Tandem Mass Spectrometry

Affinity purification combined with tandem mass spectrometry (AP-MS/MS) is a well-established method used to discover interaction partners for a given protein of interest. Because most AP-MS/MS approaches are performed using the soluble fraction of whole cell extracts (WCE), information about the cellular compartments where the interactions occur is lost. More importantly, classical AP-MS/MS often fails to identify interactions that take place in the nonsoluble fraction of the cell, for example, on the chromatin or membranes; consequently, protein complexes that are less soluble are underrepresented. In this paper, we introduce a method called multiple cell compartment AP-MS/MS (MCC-AP-MS/MS), which identifies the interactions of a protein independently in three fractions of the cell: the cytoplasm, the nucleoplasm, and the chromatin. We show that this fractionation improves the sensitivity of the method when compared to the classical affinity purification procedure using soluble WCE while keeping a very high specificity. Using three proteins known to localize in various cell compartments as baits, the CDK9 subunit of transcription elongation factor P-TEFb, the RNA polymerase II (RNAP II)-associated protein 4 (RPAP4), and the largest subunit of RNAP II, POLR2A, we show that MCC-AP-MS/MS reproducibly yields fraction-specific interactions. Finally, we demonstrate that this improvement in sensitivity leads to the discovery of novel interactions of RNAP II carboxyl-terminal domain (CTD) interacting domain (CID) proteins with POLR2A

H2A.Z Is Unusually Localized in HZAD Genes

<div><p>(A) The raw (green) and smoothed (red) H2A.Z/H2B log₂ ratios, as determined by our microarray experiment, are shown across a 5-KB region around the <i>GIT1</i> genes. The genes present in that region are shown in blue.</p> <p>(B) Same as (A) but for a 5-KB region around the <i>SRB8</i> gene.</p> <p>(C) H2A.Z covers wider regions near telomeres. The size of the regions covered by H2A.Z (as determined by distance between the coordinates where the smoothed H2A.Z/H2B log₂ ratio reaches “0.1”) is plotted against the distance to telomeres. A moving average (window = 10 KB) was applied to the data.</p></div

H2A.Z Regulates Nucleosome Positioning

<div><p>(A) Nucleosome positioning map of genes associated with a Z locus. The data from Yuan et al. [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030384#pbio-0030384-b37" target="_blank">37</a>] were used to compute the nucleosome occupancy curve for all genes containing a Z locus aligned on their ATG. Peaks represent nucleosomes, and valleys represent linker regions. An NFR is detected approximately 200 bp upstream of ATGs as described by Yuan et al. [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030384#pbio-0030384-b37" target="_blank">37</a>]. The vertical thickness of the curves contains 1-SD error bars for the mean log₂ ratio.</p> <p>(B) Same as (A) but for genes containing no Z locus.</p> <p>(C) Indirect end-labeling of MNase-digested chromatin from WT and <i>htz1</i>Δ cells grown in the presence of glucose.</p> <p>(D) High-resolution LM-PCR analysis of MNase digested nucB–C mononucleosomes. Upper part: structure of the <i>GAL1</i> promoter and PCR probes used; left: nucB analysis probing the right (R), and left (L) boundaries in WT and <i>htz1</i>Δ (Δ) cells; right: same as left part of the figure, but the analysis is with nucC.</p></div

Genome-Wide Location Analysis of H2A.Z and H2A; a Zoom on Chromosome III

<div><p>(A) The location of the Z loci along Chromosome III is depicted by gray bars.</p> <p>(B) A zoom in a 120 KB region between position 196611–316611 shows the raw H2A.Z/H2B log₂ ratios for each probe in that region (green bars) and the smoothed data resulted from the Gaussian plot analysis of the raw data for H2A.Z/H2B log₂ ratios (red line). The position of the Z loci is shown by gray lines. The genes present in that regions are shown in blue.</p> <p>(C) Same as in (B) but for a zoom in region 237700–277700 (40 KB).</p> <p>(D) The size of the Z loci within the promoter of the <i>SRB8</i> gene as determined by Q-PCR analysis of ChIP experiments. The binding ratios for H2A.Z (red) and TFIIB (blue) are shown relative to the center of the probe that generates the maximum enrichment. The data were smoothed by a sliding median over three probes (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030384#sg001" target="_blank">Figure S1</a>). The size of the observed H2A.Z domain is about 250 bp larger than that of TFIIB. Since TFIIB covers about 10 bp of DNA, we can infer that H2A.Z covers about 260 bp of DNA at that locus (shaded area). More details can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030384#sd001" target="_blank">Protocol S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030384#sg001" target="_blank">Figure S1</a>.</p></div