the contribution of mass spectrometry based proteomics to understanding epigenetics

Chromatin is a macromolecular complex composed of DNA and histones that regulate gene expression and nuclear architecture. The concerted action of DNA methylation, histone post-translational modifications and chromatin-associated proteins control the epigenetic regulation of the genome, ultimately determining cell fate and the transcriptional outputs of differentiated cells. Deregulation of this complex machinery leads to disease states, and exploiting epigenetic drugs is becoming increasingly attractive for therapeutic intervention. Mass spectrometry (MS)-based proteomics emerged as a powerful tool complementary to genomic approaches for epigenetic research, allowing the unbiased and comprehensive analysis of histone post-translational modifications and the characterization of chromatin constituents and chromatin-associated proteins. Furthermore, MS holds great promise for epigenetic biomarker discovery and represents a useful tool for deconvolution of epigenetic drug targets. Here, we will provide an ov..

Targeting Eph receptors with chemical compounds and peptides

The Eph receptors are a large family of tyrosine kinases involved in many biological processes and have recently emerged as promising drug target candidates. Indeed, they have been implicated in numerous pathological processes, including cancer progression and metastasis, pathological forms of angiogenesis and inhibition of spinal cord regeneration after injury. Therefore, inhibitors of Eph receptors function could be useful for diverse medical applications, in addition to helping elucidate the mechanism of receptor-ligand interaction. In this study we applied two different screening approaches, high throughput screening and combinatorial library screening, to the identification of chemical compounds that target EphA4 and EphA2. Two 2,5-dimethylpyrrolyl benzoic acid derivatives were identified in the high throughput screen for EphA4 antagonists. These compounds selectively inhibit ephrin binding to EphA4 and EphA2 and the characteristic biological activities of the two receptors, showing for the first time that the Eph receptors can be successfully targeted by small molecules. By combinatorial library screening, we also identified platinum(II) tetraamines as potential selective inhibitors of EphA2 activity. Finally, we modified TNYL-RAW, a potent peptide antagonist for EphB4, in order to improve its stability and delay its in vivo clearance from the blood circulation by coupling it with poly ethylene glycol (PEG) or the Fc portion of human IgG

enrichment of histones from patient samples for mass spectrometry based analysis of post translational modifications

Abstract Aberrations in histone post-translational modifications (PTMs) have been implicated with the development of numerous pathologies, including cancer. Therefore, profiling histone PTMs in patient samples could provide information useful for the identification of epigenetic biomarkers, as well as the discovery of potential novel targets. While antibody-based methods have been traditionally employed to analyze histone PTM in clinical samples, mass spectrometry (MS) can provide a more comprehensive, unbiased and quantitative view on histones and their PTMs. To combine the power of MS-based methods and the potential offered by histone PTM profiling of clinical samples, we have recently developed a series of methods for the extraction and enrichment of histones from different types of patient samples, including formalin-fixed paraffin-embedded tissues, fresh- and optimal cutting temperature-frozen tissues, and primary cells. Here, we provide a detailed description of these protocols, together with indications on the expected results and the most suitable workflow to be used downstream of each procedure

PAT-H-MS coupled with laser microdissection to study histone post-translational modifications in selected cell populations from pathology samples

Background: Aberrations in histone post-translational modifications (hPTMs) have been linked with various pathologies, including cancer, and could not only represent useful biomarkers but also suggest possible targetable epigenetic mechanisms. We have recently developed an approach, termed pathology tissue analysis of histones by mass spectrometry (PAT-H-MS), that allows performing a comprehensive and quantitative analysis of histone PTMs from formalin-fixed paraffin-embedded pathology samples. Despite its great potential, the application of this technique is limited by tissue heterogeneity. Methods: In this study, we further implemented the PAT-H-MS approach by coupling it with techniques aimed at reducing sample heterogeneity and selecting specific portions or cell populations within the samples, such as manual macrodissection and laser microdissection (LMD). Results: When applied to the analysis of a small set of breast cancer samples, LMD-PAT-H-MS allowed detecting more marked changes between luminal A-like and triple negative patients as compared with the classical approach. These changes included not only the already known H3 K27me3 and K9me3 marks, but also H3 K36me1, which was found increased in triple negative samples and validated on a larger cohort of patients, and could represent a potential novel marker distinguishing breast cancer subtypes. Conclusions: These results show the feasibility of applying techniques to reduce sample heterogeneity, including laser microdissection, to the PAT-H-MS protocol, providing new tools in clinical epigenetics and opening new avenues for the comprehensive analysis of histone post-translational modifications in selected cell populations

Domain Model Explains Propagation Dynamics and Stability of Histone H3K27 and H3K36 Methylation Landscapes

Chromatin states must be maintained during cell proliferation to uphold cellular identity and genome integrity. Inheritance of histone modifications is central in this process. However, the histone modification landscape is challenged by incorporation of new unmodified histones during each cell cycle, and the principles governing heritability remain unclear. We take a quantitative computational modeling approach to describe propagation of histone H3K27 and H3K36 methylation states. We measure combinatorial H3K27 and H3K36 methylation patterns by quantitative mass spectrometry on subsequent generations of histones. Using model comparison, we reject active global demethylation and invoke the existence of domains defined by distinct methylation endpoints. We find that H3K27me3 on pre-existing histones stimulates the rate of de novo H3K27me3 establishment, supporting a read-write mechanism in timely chromatin restoration. Finally, we provide a detailed quantitative picture of the mutual antagonism between H3K27 and H3K36 methylation and propose that it stabilizes epigenetic states across cell division

The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses.

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi

Detection and quantification of the histone code in the fungal genus Aspergillus

In eukaryotes, the combination of different histone post-translational modifications (PTMs) - the histone code - impacts the chromatin organization as compact and transcriptionally silent heterochromatin or accessible and transcriptionally active euchromatin. Although specific histone PTMs have been studied in fungi, an overview of histone PTMs and their relative abundance is still lacking. Here, we used mass spectrometry to detect and quantify histone PTMs in three fungal species belonging to three distinct taxonomic sections of the genus Aspergillus (Aspergillus niger, Aspergillus nidulans (two strains), and Aspergillus fumigatus). We overall detected 23 different histone PTMs, including a majority of lysine methylations and acetylations, and 23 co-occurrence patterns of multiple histone PTMs. Among those, we report for the first time the detection of H3K79me1, H3K79me2, and H4K31ac in Aspergilli. Although all three species harbour the same PTMs, we found significant differences in the relative abundance of H3K9me1/2/3, H3K14ac, H3K36me1 and H3K79me1, as well as the co-occurrence of acetylation on both K18 and K23 of histone H3 in a strain-specific manner. Our results provide novel insights about the underexplored complexity of the histone code in filamentous fungi, and its functional implications on genome architecture and gene regulation

EphA2 is a functional receptor for the growth factor progranulin.

Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases

Activation of Endogenous Retroviruses and Induction of Viral Mimicry by MEK1/2 Inhibition in Pancreatic Cancer

While pancreatic ductal adenocarcinomas (PDACs) are addicted to KRAS-activating mutations, inhibitors of downstream KRAS effectors, such as the MEK1/2 kinase inhibitor trametinib, are devoid of therapeutic effects. However, the extensive rewiring of regulatory circuits driven by the attenuation of the KRAS pathway may induce vulnerabilities of therapeutic relevance. An in-depth molecular analysis of the transcriptional and epigenomic alterations occurring in PDAC cells in the initial hours after MEK1/2 inhibition by trametinib unveiled the induction of endogenous retroviruses (ERVs) escaping epigenetic silencing, leading to the production of double-stranded RNAs and the increased expression of interferon (IFN) genes. We tracked ERV activation to the early induction of the transcription factor ELF3, which extensively bound and activated nonsilenced retroelements and synergized with IRF1 (interferon regulatory factor 1) in the activation of IFNs and IFN-stimulated genes. Trametinib-induced viral mimicry in PDAC may be exploited in the rational design of combination therapies in immuno-oncology

Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context

The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment.National Science Foundation (U.S.) (NSF EFRI grant 735997)National Institutes of Health (U.S.) (NIH Cell Migration Consortium grant GM06346)National Institutes of Health (U.S.) (NIH Cell Decision Processes Center grant GM68762)National Institutes of Health (U.S.) (NIH grant GM69668)National Institutes of Health (U.S.) (NIH grant GM81336