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. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.

We present a novel method utilizing "saltless" pH gradient weak cation exchange-hydrophilic interaction liquid chromatography directly coupled to electron transfer dissociation (ETD) mass spectrometry for the automated on-line high throughput characterization of hypermodified combinatorial histone codes. This technique, performed on a low resolution mass spectrometer, displays an improvement over existing methods with an ϳ100-fold reduction in sample requirements and analysis time. The scheme presented is capable of identifying all of the major combinatorial histone codes present in a sample in a 2-h analysis. The large N-terminal histone peptides are eluted by the pH and organic solvent weak cation exchangehydrophilic interaction liquid chromatography gradient and directly introduced via nanoelectrospray ionization into a benchtop linear quadrupole ion trap mass spectrometer equipped with ETD. Each polypeptide is sequenced, and the modification sites are identified by ETD fragmentation. The isobaric trimethyl and acetyl modifications are resolved chromatographically and confidently distinguished by the synthesis of mass spectrometric and chromatographic information. We demonstrate the utility of the method by complete characterization of human histone H3.2 and histone H4 from butyrate-treated cells, but it is generally applicable to the analysis of highly modified peptides. We find this methodology very useful for chromatographic separation of isomeric species that cannot be separated well by any other chromatographic means, leading to less complicated tandem mass spectra. The improved separation and increased sensitivity generated novel information about much less abundant forms. In this method demonstration we report over 200 H3. Eukaryotic nuclear DNA is nominally compacted into chromatin fibers by use of nucleosomes consisting of a 146-bp section of DNA wrapped around a core of histone proteins (1). Dynamic post-translational modifications (PTMs) 1 of the histones, primarily in the accessible N-terminal region or histone "tail," are an important but not fully understood component of dynamic gene regulation, epigenetic inheritance of cellular memory, genomic stability, and other nuclear mechanisms (2-7). An overwhelming number of studies point to the existence of a histone code of biological logic written on these proteins through these PTMs that are read by a diverse array of "effector" proteins leading to distinct biological events (3). Many single PTM sites on various histone proteins have been decidedly linked to specific physiological processes, such as histone H3 Lys-9 trimethylation (H3K9me3), which is associated with heterochromatin formation (one mode of gene silencing). Nevertheless what effect multiple modifications occurring in combination may have on modulating the histone code signal remains to be determined. Significant progress has been made toward understanding histone modifications using antibody-based histone modification detection methods and by bottom up mass spectrometry (4 -6). However...

Aim To identify possible novel biomarkers in gingival crevicular fluid (GCF) samples from chronic periodontitis (CP) and periodontally healthy individuals by high-throughput proteomic analysis. Materials and Methods GCF samples were collected from twelve CP and twelve periodontally healthy subjects. Samples were trypically digested, eluted using high-performance liquid chromatography, and fragmented using tandem mass spectrometry (MS/MS). MS/MS were analyzed using PILOT_PROTEIN to identify all unmodified proteins within the samples. Results Using the database derived from Homo sapiens taxonomy and all bacterial taxonomies, 432 human (120 new) and 30 bacterial proteins were identified. The human proteins, angiotensinogen, clusterin and thymidine phosphorylase were identified as biomarker candidates based on their high-scoring only in samples from periodontal health. Similarly, neutrophil defensin-1, carbonic anhydrase-1 and elongation factor-1 gamma were associated with CP. Candidate bacterial biomarkers include 33 kDa chaperonin, iron uptake protein A2 and phosphoenolpyruvate carboxylase (health-associated) and ribulose biphosphate carboxylase, a probable succinyl-CoA:3-ketoacid-coenzyme A transferase, or DNA-directed RNA polymerase subunit beta (CP-associated). Most of these human and bacterial proteins have not been previously evaluated as biomarkers of periodontal conditions and require further investigation. Conclusions The proposed methods for large-scale comprehensive proteomic analysis may lead to the identification of novel biomarkers of periodontal disease.

BackgroundAn 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. Thus, there is a need for a comprehensive and quantitative profiling of cancer lines focused on their chromatin modification states.ResultsTo complement genomic expression profiles of cancer lines, we report the proteomic classification of 24 different lines, the majority of which are cancer cells, by quantifying the abundances of a large panel of single and combinatorial histone H3 and H4 PTMs, and histone variants. Concurrent to the proteomic analysis, we performed transcriptomic analysis on histone modifying enzyme abundances as a proxy for quantifying their activity levels. While the transcriptomic and proteomic results were generally consistent in terms of predicting histone PTM abundance from enzyme abundances, several PTMs were regulated independently of the modifying enzyme expression. In addition, combinatorial PTMs containing H3K27 methylation were especially enriched in breast cell lines. Knockdown of the predominant H3K27 methyltransferase, enhancer of zeste 2 (EZH2), in a mouse mammary xenograft model significantly reduced tumor burden in these animals and demonstrated the predictive utility of proteomic techniques.ConclusionsOur proteomic and genomic characterizations of the histone modification states provide a resource for future investigations of the epigenetic and non-epigenetic determinants for classifying and analyzing cancer cells.

Background: Site-specific in vivo dynamics of histone acetylation have not been analyzed in a quantitative manner. Results: Histone acetylation turnover varies depending on the histone residue and presence of neighboring modifications. Conclusion: Acetylation of histones is a dynamic process that involves the dual action of HATs and HDACs to affect chromatin. Significance: Acetylation turnover can be quantitatively measured in many cellular processes.