Mechanistical studies of two functions of HMGB1 protein : facilitation of nucleosome sliding and translocation from the nucleus to the extracellular medium

This thesis develops two lines of investigation that focus on HMGBl protein from different points of view. In Section 3, acétylation of lysine is identified as the molecular switch that controls movement of HMGBl from the nucleus to the cytoplasm and Section 4 investigates its role in chromatin remodelling. High Mobility Group protein HMGBl is a chromatin component that, when leaked out by necrotic cells, triggers inflammation. HMGBl can also be secreted by activated monocytes and macrophages and fimctions as a late mediator of inflammation. Secretion of a nuclear protein must require a tightly controlled relocation programme. It was found that in all cells HMGBl shuttles actively between nucleus and cytoplasm due to two nuclear localisation signals (NLSs) and two nuclear export signals (NESs) within its sequence. Analysis of HMGBl samples, extracted under specific conditions, using iso-electric focussing/SDS 2- dimensional gels, followed by mass spectrometry, demonstrated that HMGBl is extensively acetylated (up to 10 modification sites per molecule) and that acétylation of the two NLSs inhibits nuclear import, causing cytoplasmic accumulation of the protein. This provides the first step in directing the protein to the secretion pathway, since cytosolic HMGBl is then concentrated by default into secretory lysosomes and finally secreted when monocytic cells receive an appropriate second signal. Multiple acétylation of lysine sidechains was thereby defined as the molecular switch redirecting the nuclear protein HMGBl to the cytoplasm and subsequently to secretion. The second project investigated the potential role of HMGBl in nucleosome sliding. Nucleosome remodelling complexes containing the ATP-ase ISWl, such as ACF, contribute to chromatin remodelling by converting chemical energy into the sliding of nucleosomes on DNA. ISWl interacts with DNA at the sites of its entry into the nucleosome, where it alters histone/DNA interactions that may lead to the relocation of DNA relative to the associated histone octamer. This work elaborated on this concept: if the rate-limiting step in nucleosome sliding is the distortion of linker DNA, a protein that can generate and/or stabilise such distortions might facilitate sliding. HMGBl is able to transiently bend DNA, so is a good candidate to help “lubricate” nucleosome sliding. Using gel-shift assays, it was found that transient interaction of HMGBl with nucleosomal DNA, at sites overlapping with ISWl binding sites, enhanced ACF-induced sliding. The ACFl subunit of ACF was required to render the remodelling process sensitive to HMGBl action since no enhancement was detected when the sliding assay was performed using ISWl alone. In contrast, an HMGBl derivative lacking the acidic tail that interacts with chromatin in a more dynamic way, had a strong inhibitory effect on sliding. These data suggest that HMGBl is able to increase chromatin ‘fluidity’ by generating strategic DNA bends, or ‘bulges’, which are profitably used by ACF to induce nucleosome sliding. Moreover they support a “local loop” model of sliding and identify HMGBl as a potential regulator of ATP-dependent nucleosome remodelling processes. This work was published in the EMBO Journal on 16th December 2002

Dual role of PRMT1-dependent arginine methylation in cellular responses to genotoxic stress

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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..

Biochemical systems approaches for the analysis of histone modification readout.

Abstract Chromatin is the macromolecular nucleoprotein complex that governs the organization of genetic material in the nucleus of eukaryotic cells. In chromatin, DNA is packed with histone proteins into nucleosomes. Core histones are prototypes of hyper-modified proteins, being decorated by a large number of site-specific reversible and irreversible post-translational modifications (PTMs), which contribute to the maintenance and modulation of chromatin plasticity, gene activation, and a variety of other biological processes and disease states. The observations of the variety, frequency and co-occurrence of histone modifications in distinct patterns at specific genomic loci have led to the idea that hPTMs can create a molecular barcode, read by effector proteins that translate it into a specific transcriptional state, or process, on the underlying DNA. However, despite the fact that this histone-code hypothesis was proposed more than 10 years ago, the molecular details of its working mechanisms are only partially characterized. In particular, two questions deserve specific investigation: how the different modifications associate and synergize into patterns and how these PTM configurations are read and translated by multi-protein complexes into a specific functional outcome on the genome. Mass spectrometry (MS) has emerged as a versatile tool to investigate chromatin biology, useful for both identifying and validating hPTMs, and to dissect the molecular determinants of histone modification readout systems. We review here the MS techniques and the proteomics methods that have been developed to address these fundamental questions in epigenetics research, emphasizing approaches based on the proteomic dissection of distinct native chromatin regions, with a critical evaluation of their present challenges and future potential. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function

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

Yin Yang 1 extends the Myc-related transcription factors network in embryonic stem cells

The Yin Yang 1 (YY1) transcription factor is a master regulator of development, essential for early embryogenesis and adult tissues formation. YY1 is the mammalian orthologue of Pleiohomeotic, one of the transcription factors that binds Polycomb DNA response elements in Drosophila melanogaster and mediates Polycomb group proteins (PcG) recruitment to DNA. Despite several publications pointing at YY1 having a similar role in mammalians, others showed features of YY1 that are not compatible with PcG functions. Here, we show that, in mouse Embryonic Stem (ES) cells, YY1 has genome-wide PcG-independent activities while it is still stably associated with the INO80 chromatin-remodeling complex, as well as with novel RNA helicase activities. YY1 binds chromatin in close proximity of the transcription start site of highly expressed genes. Loss of YY1 functions preferentially led to a down-regulation of target genes expression, as well as to an up-regulation of several small non-coding RNAs, suggesting a role for YY1 in regulating small RNA biogenesis. Finally, we found that YY1 is a novel player of Myc-related transcription factors and that its coordinated binding at promoters potentiates gene expression, proposing YY1 as an active component of the Myc transcription network that links ES to cancer cells

Mass Spectrometry-Based Proteomics to Unveil the Non-coding RNA World

The interaction between non-coding RNAs (ncRNAs) and proteins is crucial for the stability, localization and function of the different classes of ncRNAs. Although ncRNAs, when embedded in various ribonucleoprotein (RNP) complexes, control the fundamental processes of gene expression, their biological functions and mechanisms of action are still largely unexplored. Mass Spectrometry (MS)-based proteomics has emerged as powerful tool to study the ncRNA world: on the one hand, by identifying the proteins interacting with distinct ncRNAs; on the other hand, by measuring the impact of ncRNAs on global protein levels. Here, we will first provide a concise overview on the basic principles of MS-based proteomics for systematic protein identification and quantification; then, we will recapitulate the main approaches that have been implemented for the screening of ncRNA interactors and the dissection of ncRNA-protein complex composition. Finally, we will describe examples of various proteomics strategies developed to characterize the effect of ncRNAs on gene expression, with a focus on the systematic identification of microRNA (miRNA) targets

Site-specific acetylation of ISWI by GCN5

<p>Abstract</p> <p>Background</p> <p>The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition.</p> <p>Results</p> <p>We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the <it>Drosophila </it>remodelling ATPase ISWI at a single, conserved lysine, K753, <it>in vivo </it>and <it>in vitro</it>. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF.</p> <p>Conclusion</p> <p>Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RK^T/_SxGx(K^ac)xP^R/_Kdiffers from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles.</p

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

The High Mobility Group (Hmg) Boxes of the Nuclear Protein Hmg1 Induce Chemotaxis and Cytoskeleton Reorganization in Rat Smooth Muscle Cells

HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a Gi/o protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage