Optical High Content Nanoscopy of Epigenetic Marks Decodes Phenotypic Divergence in Stem Cells.

While distinct stem cell phenotypes follow global changes in chromatin marks, single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics, we developed a novel approach, termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States), to discern chromatin organizational changes, demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness, thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall, EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks.

The histone lysine methyltransferase KMT2D sustains a gene expression program that represses B cell lymphoma development.

The gene encoding the lysine-specific histone methyltransferase KMT2D has emerged as one of the most frequently mutated genes in follicular lymphoma and diffuse large B cell lymphoma; however, the biological consequences of KMT2D mutations on lymphoma development are not known. Here we show that KMT2D functions as a bona fide tumor suppressor and that its genetic ablation in B cells promotes lymphoma development in mice. KMT2D deficiency also delays germinal center involution and impedes B cell differentiation and class switch recombination. Integrative genomic analyses indicate that KMT2D affects methylation of lysine 4 on histone H3 (H3K4) and expression of a set of genes, including those in the CD40, JAK-STAT, Toll-like receptor and B cell receptor signaling pathways. Notably, other KMT2D target genes include frequently mutated tumor suppressor genes such as TNFAIP3, SOCS3 and TNFRSF14. Therefore, KMT2D mutations may promote malignant outgrowth by perturbing the expression of tumor suppressor genes that control B cell-activating pathways.

LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2.

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.

EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra.

Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification. However, analysis of histones with this method is even more challenging because of the large number and variety of isobaric histone peptides and the high dynamic range of histone peptide abundances. Here, we introduce EpiProfile, a software tool that discriminates isobaric histone peptides using the distinguishing fragment ions in their tandem mass spectra and extracts the chromatographic area under the curve using previous knowledge about peptide retention time. The accuracy of EpiProfile was evaluated by analysis of mixtures containing different ratios of synthetic histone peptides. In addition to label-free quantification of histone peptides, EpiProfile is flexible and can quantify different types of isotopically labeled histone peptides. EpiProfile is unique in generating layouts (i.e. relative retention time) of histone peptides when compared with manual quantification of the data and other programs (such as Skyline), filling the need of an automatic and freely available tool to quantify labeled and non-labeled modified histone peptides. In summary, EpiProfile is a valuable nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry-based quantification tool for histone peptides, which can also be adapted to analyze nonhistone protein samples.

Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom-up proteomics PTM analysis.

MS-based proteomics has become the most utilized tool to characterize histone PTMs. Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (<6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in RP LC, and such procedure also hampers the discovery of natural propionylation events. In this work we tested 12 commercially available anhydrides, selected based on their safety and hydrophobicity. Performance was evaluated in terms of yield of the reaction, MS/MS fragmentation efficiency, and drift in retention time using the following samples: (i) a synthetic unmodified histone H3 tail, (ii) synthetic modified histone peptides, and (iii) a histone extract from cell lysate. Results highlighted that seven of the selected anhydrides increased peptide retention time as compared to propionic, and several anhydrides such as benzoic and valeric led to high MS/MS spectra quality. However, propionic anhydride derivatization still resulted, in our opinion, as the best protocol to achieve high MS sensitivity and even ionization efficiency among the analyzed peptides.

Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLL-rearranged leukemia.

Enhancer of zeste homolog 2 (EZH2) and related EZH1 control gene expression and promote tumorigenesis via methylating histone H3 at lysine 27 (H3K27). These methyltransferases are ideal therapeutic targets due to their frequent hyperactive mutations and overexpression found in cancer, including hematopoietic malignancies. Here, we characterized a set of small molecules that allow pharmacologic manipulation of EZH2 and EZH1, which include UNC1999, a selective inhibitor of both enzymes, and UNC2400, an inactive analog compound useful for assessment of off-target effect. UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged leukemia cells. UNC1999-induced transcriptome alterations overlap those following knockdown of embryonic ectoderm development, a common cofactor of EZH2 and EZH1, demonstrating UNC1999's on-target inhibition. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to derepression of polycomb targets including Cdkn2a. Gene derepression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. UNC2400 does not induce such effects. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9. Collectively, our study provides the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of polycomb repressive complex 2 (PRC2)-EZH2 and PRC2-EZH1 by small-molecule compounds as a novel therapeutics for MLL-rearranged leukemia.

A proteomic glimpse into the initial global epigenetic changes during HIV infection.

HIV-1 replication requires the insertion of viral DNA into the host genome, which is catalyzed by HIV-1 integrase. This integration event can lead to vast changes in the chromatin landscape and gene transcription. In this study, we sought to correlate the extensive changes of histone PTM abundances with the equally dynamic shifts in host transcriptional activity. To fully capture the changes that were occurring during the course of HIV-infection, we performed time-courses in which we extracted both histones and mRNA from HIV-infected, UV-inactivated HIV-infected and mock-infected SUP-T1 cells. We then analyzed the alterations to histone PTM profiles using nano-LC-MS/MS, as well as the expression of chromatin-associated enzymes, such as histone deacetylases, acetyltransferases, demethylases, methyltransferases, and histone chaperone proteins. As expected, we observed major changes in histone PTM abundances, which we linked to massive fluctuations in mRNA expression of associated chromatin enzymes. However, we find few differences between HIV and HIVUV (UV-inactivated) infection, which suggests that initial histone PTM changes during HIV infection are from the host in response to the infection, and not due to the HIV virus manipulating the transcriptional machinery. We believe that these preliminary experiments can provide a basis for future forays into targeted manipulations of histone PTM-regulated aspects of HIV progression through its replication cycle.

Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation.

Histone acetylation plays important roles in gene regulation, DNA replication, and the response to DNA damage, and it is frequently deregulated in tumors. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl coenzyme A (acetyl-CoA) availability mediated by oncogenic metabolic reprogramming. Here, we demonstrate that acetyl-CoA is dynamically regulated by glucose availability in cancer cells and that the ratio of acetyl-CoA:coenzyme A within the nucleus modulates global histone acetylation levels. In vivo, expression of oncogenic Kras or Akt stimulates histone acetylation changes that precede tumor development. Furthermore, we show that Akt's effects on histone acetylation are mediated through the metabolic enzyme ATP-citrate lyase and that pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors. The data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in cancer cells.

SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.