Decoding chromatin states by proteomic profiling of nucleosome readers.

DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome. While many 'readers' of individual modifications have been described, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states.

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation.

ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. We herein show that an adenosine analogue with a terminal alkyne functionality at position 2 of the adenine (2-alkyne adenosine or 2YnAd) is suitable for selective enrichment, fluorescence detection and mass spectrometry proteomics analysis of the candidate ADP-ribosylome in mammalian cells.

Unbiased Mass Spectrometry Elucidation of the Targets and Mechanisms of Activity-Based Probes: A Case Study Involving Sulfonyl Fluorides.

The elucidation of protein/drug interactions remains a major challenge in drug discovery. Liquid chromatography-tandem mass spectrometry has emerged as a tremendously powerful technology for this endeavor, but its full potential has yet to be realized owing in part to unresolved challenges in data analysis. Herein, we demonstrate how tandem mass spectrometry can comprehensively map small molecule/peptide adducts when combined with unconstrained sequencing.

Multiple Levels of Control Determine How E4bp4/Nfil3 Regulates NK Cell Development.

The transcription factor E4bp4/Nfil3 has been shown to have a critical role in the development of all innate lymphoid cell types including NK cells. In this study, we show that posttranslational modifications of E4bp4 by either SUMOylation or phosphorylation have profound effects on both E4bp4 function and NK cell development. We examined the activity of E4bp4 mutants lacking posttranslational modifications and found that was a novel E4bp4 target gene.

Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites.

Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9.

Paired single residue-transposed Lys-N and Lys-C digestions for label-free identification of N-terminal and C-terminal MS/MS peptide product ions: ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry for peptide de novo sequencing.

Rationale: Paired Lys-N and Lys-C proteases produce peptides of identical mass and similar retention time, but different tandem mass spectra. Data from these parallel experiments provide constraints that are applied before data analysis. With this approach, we can find matched spectra before analysis, distinguish ion type, and determine residue level confidence.

Fullerene oxidation and clustering in solution induced by light.

We investigate the environmental stability of fullerene solutions by static and dynamic light scattering, FTIR, NMR and mass spectroscopies, and quantum chemical calculations. We find that visible light exposure of fullerene solutions in toluene, a good solvent, under ambient laboratory conditions results in C60 oxidation to form fullerene epoxides, and subsequently causes fullerene clustering in solution. The clusters grow with time, even in absence of further illumination, and can reach dimensions from ≈100 nm to the μm scale over ≈1 day.

Quantitative proteomic discovery of dynamic epigenome changes that control human cytomegalovirus (HCMV) infection.

This work represents the first comprehensive quantitative analysis of global histone post-translational modifications (PTMs) from a virus infection, namely human cytomegalovirus (HCMV) infection. We used a nanoLC-MS/MS platform to identify and quantify the dynamic histone H3 and H4 PTMs expressed during HCMV replication in primary fibroblasts. Specifically, we examined the changes in histone PTMs over a 96 h time course to sample the immediate early (IE), early (E), and late (L) stages of viral infection.

On the histone lysine methyltransferase activity of fungal metabolite chaetocin.

Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. 1 (chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU(VAR)3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by 1 and functionally related analogues.

A quantitative atlas of histone modification signatures from human cancer cells.

Background: An integral component of cancer biology is the understanding of molecular properties uniquely distinguishing one cancer type from another. One class of such properties is histone post-translational modifications (PTMs). Many histone PTMs are linked to the same diverse nuclear functions implicated in cancer development, including transcriptional activation and epigenetic regulation, which are often indirectly assayed with standard genomic technologies.

Quantitative dynamics of the link between cellular metabolism and histone acetylation.

Acetylation on the tails of histones plays an important role in controlling transcription initiation. Although the steady-state abundances of histone acetyl groups have been reported, the rate at which histones are acetylated and deacetylated on a residue-specific basis has not been quantitatively established. We added [(13)C]glucose to human cells and monitored the dynamic incorporation of (13)C-labeled acetyl groups onto specific histone lysines with quantitative mass spectrometry.

Proteomics in epigenetics: new perspectives for cancer research.

The involvement of epigenetic processes in the origin and progression of cancer is now widely appreciated. Consequently, targeting the enzymatic machinery that controls the epigenetic regulation of the genome has emerged as an attractive new strategy for therapeutic intervention. The development of epigenetic drugs requires a detailed knowledge of the processes that govern chromatin regulation.

Novel phosphorylation sites in the S. cerevisiae Cdc13 protein reveal new targets for telomere length regulation.

The S. cerevisiae Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS).

Proteogenomic characterization and mapping of nucleosomes decoded by Brd and HP1 proteins.

Background: Histone post-translational modifications (PTMs) constitute a branch of epigenetic mechanisms that can control the expression of eukaryotic genes in a heritable manner. Recent studies have identified several PTM-binding proteins containing diverse specialized domains whose recognition of specific PTM sites leads to gene activation or repression. Here, we present a high-throughput proteogenomic platform designed to characterize the nucleosomal make-up of chromatin enriched with a set of histone PTM binding proteins known as histone PTM readers.

PILOT_PROTEIN: identification of unmodified and modified proteins via high-resolution mass spectrometry and mixed-integer linear optimization.

A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized.

Histone H3 lysine 56 methylation regulates DNA replication through its interaction with PCNA.

Histone modifications play important roles in regulating DNA-based biological processes. Of the modified sites, histone H3 lysine 56 (H3K56) is unique in that it lies within the globular core domain near the entry-exit sites of the nucleosomal DNA superhelix and its acetylation state in yeast is a marker for newly synthesized histones in transcription, DNA repair, and DNA replication. We now report the presence of H3K56 monomethylation (H3K56me1) in mammalian cells and find that the histone lysine methytransferase G9a/KMT1C is required for H3K56me1 both in vivo and in vitro.

Novel protein identification methods for biomarker discovery via a proteomic analysis of periodontally healthy and diseased gingival crevicular fluid samples.

Aim: To identify possible novel biomarkers in gingival crevicular fluid (GCF) samples from chronic periodontitis (CP) and periodontally healthy individuals using high-throughput proteomic analysis.

Materials And Methods: Gingival crevicular fluid samples were collected from 12 CP and 12 periodontally healthy subjects. Samples were trypically digested with trypsin, eluted using high-performance liquid chromatography, and fragmented using tandem mass spectrometry (MS/MS).

A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells.

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity.

Global turnover of histone post-translational modifications and variants in human cells.

Background: Post-translational modifications (PTMs) on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression.

A novel framework for predicting in vivo toxicities from in vitro data using optimal methods for dense and sparse matrix reordering and logistic regression.

In this work, we combine the strengths of mixed-integer linear optimization (MILP) and logistic regression for predicting the in vivo toxicity of chemicals using only their measured in vitro assay data. The proposed approach utilizes a biclustering method based on iterative optimal reordering (DiMaggio, P. A.

The significance, development and progress of high-throughput combinatorial histone code analysis.

The physiological state of eukaryotic DNA is chromatin. Nucleosomes, which consist of DNA in complex with histones, are the fundamental unit of chromatin. The post-translational modifications (PTMs) of histones play a critical role in the control of gene transcription, epigenetics and other DNA-templated processes.

Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown.

Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other histone molecules is currently uncharacterized.

Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein A1a.

Transcriptional states are formed and maintained by the interaction and post-translational modification (PTM) of several chromatin proteins, such as histones and high mobility group (HMG) proteins. Among these, HMGA1a, a small heterochromatin-associated nuclear protein has been shown to be post-translationally modified, and some of these PTMs have been linked to apoptosis and cancer. In cancerous cells, HMGA1a PTMs differ between metastatic and nonmetastatic cells, suggesting the existence of an HMGA1a PTM code analogous to the "histone code.