33 research outputs found
Quantitative proteomics methods for the analysis of histone post-translational modifications
Les histones sont des protĂ©ines nuclĂ©aires hautement conservĂ©es chez les cellules des eucaryotes. Elles permettent dâorganiser et de compacter lâADN sous la forme de nuclĂ©osomes, ceux-ci representant les sous unitĂ©s de base de la chromatine. Les histones peuvent ĂȘtre modifiĂ©es par de nombreuses modifications post-traductionnelles (PTMs) telles que lâacĂ©tylation, la mĂ©thylation et la phosphorylation. Ces modifications jouent un rĂŽle essentiel dans la rĂ©plication de lâADN, la transcription et lâassemblage de la chromatine. Lâabondance de ces modifications peut varier de facon significative lors du developpement des maladies incluant plusieurs types de cancer. Par exemple, la perte totale de la trimĂ©thylation sur H4K20 ainsi que lâacĂ©tylation sur H4K16 sont des marqueurs tumoraux spĂ©cifiques a certains types de cancer chez lâhumain. Par consĂ©quent, lâĂ©tude de ces modifications et des Ă©vĂ©nements determinant la dynamique des leurs changements dâabondance sont des atouts importants pour mieux comprendre les fonctions cellulaires et molĂ©culaires lors du dĂ©veloppement de la maladie.
De maniĂšre gĂ©nĂ©rale, les modifications des histones sont Ă©tudiĂ©es par des approches biochimiques telles que les immuno-buvardage de type Western ou les mĂ©thodes dâimmunoprĂ©cipitation de la chromatine (ChIP). Cependant, ces approches prĂ©sentent plusieurs inconvĂ©nients telles que le manque de spĂ©cificitĂ© ou la disponibilitĂ© des anticorps, leur coĂ»t ou encore la difficultĂ© de les produire et de les valider. Au cours des derniĂšres dĂ©cennies, la spectromĂ©trie de masse (MS) sâest avĂ©rĂ©e ĂȘtre une mĂ©thode performante pour la caractĂ©risation et la quantification des modifications dâhistones. La MS offre de nombreux avantages par rapport aux techniques traditionnelles. Entre autre, elle permet dâeffectuer des analyses reproductibles, spĂ©cifiques et facilite lâetude dâun large spectre de PTMs en une seule analyse. Dans cette thĂšse, nous prĂ©senterons le dĂ©veloppement et lâapplication de nouveaux outils analytiques pour lâidentification et Ă la quantification des PTMs modifiant les histones.
Dans un premier temps, une mĂ©thode a Ă©tĂ© dĂ©veloppĂ©e pour mesurer les changements dâacĂ©tylation spĂ©cifiques Ă certains sites des histones. Cette mĂ©thode combine lâanalyse des histones intactes et les mĂ©thodes de sĂ©quençage peptidique afin de dĂ©terminer les changements dâacĂ©tylation suite Ă la rĂ©action in vitro par lâhistone acĂ©tyltransfĂ©rase (HAT) de levure Rtt109 en prĂ©sence de ses chaperonnes (Asf1 ou Vps75).
Dans un second temps, nous avons dĂ©veloppĂ© une mĂ©thode dâanalyse des peptides isomĂ©riques des histones. Cette mĂ©thode combine la LC-MS/MS Ă haute rĂ©solution et un nouvel outil informatique appelĂ© Iso-PeptidAce qui permet de dĂ©convoluer les spectres mixtes de peptides isomĂ©riques. Nous avons Ă©valuĂ© Iso-PeptidAce avec un mĂ©lange de peptides synthĂ©tiques isomĂ©riques. Nous avons Ă©galement validĂ© les performances de cette approche avec des histones isolĂ©es de cellules humaines Ă©rythroleucĂ©miques (K562) traitĂ©es avec des inhibiteurs dâhistones dĂ©sacĂ©tylases (HDACi) utilisĂ©s en clinique, et des histones de Saccharomyces cerevisiae liĂ©es au facteur dâassemblage de la chromatine (CAF-1) purifiĂ©es par chromatographie dâaffinitĂ©. Enfin, en utilisant la mĂ©thode prĂ©sentĂ©e prĂ©cĂ©demment, nous avons fait une analyse approfondie de la spĂ©cificitĂ© de plusieurs HATs et HDACs chez Schizosaccharomyces pombe. Nous avons donc dĂ©terminĂ© les niveaux dâacĂ©tylation dâhistones purifiĂ©es Ă partir de cellules contrĂŽles ou de souches mutantes auxquelles il manque une HAT ou HDAC. Notre analyse nous a permis de valider plusieurs cibles connues des HATs et HDACs et dâen identifier de nouvelles. Nos donnĂ©es ont Ă©galement permis de dĂ©finir le rĂŽle des diffĂ©rentes HATs et HDACs dans le maintien de lâĂ©quilibre dâacĂ©tylation des histones. Dans lâensemble, nous anticipons que les mĂ©thodes dĂ©crites dans cette thĂšse permettront de rĂ©soudre certains dĂ©fis rencontrĂ©s dans lâĂ©tude de la chromatine. De plus, ces donnĂ©es apportent de nouvelles connaissances pour lâĂ©laboration dâĂ©tudes gĂ©nĂ©tiques et biochimiques utilisant S. pombe.Histones are highly conserved, basic proteins found in eukaryotic cell nuclei. They organize and package DNA strands into nucleosome core particles (NCPs), the fundamental repeating units of eukaryotic chromatin. The histones are subject to a wide variety of posttranslational modifications (PTMs) including acetylation, methylation and phosphorylation. These PTMs play an essential role in DNA-replication, transcription, and chromatin assembly. Alterations in histone PTM abundances have been implicated in several types of cancer. For example, the global loss of trimethylation at H4K20 and acetylation at H4K16 is a hallmark of human cancers. Thus, characterization of histone PTMs and their dynamics is extremely useful for elucidating normal cellular functions and molecular pathways that lead to diseases.
Traditionally, histone PTMs are analyzed using antibody-based approaches such as western blot and chromatin immunoprecipitation (ChIP) assays. These methods, however, suffer from several limitations including antibody cross-reactivity, epitope occlusion, and the cost and difficulty in producing and validating antibodies. Over the last decade, mass spectrometry (MS) has emerged as a powerful technique for the characterization and quantification of histone PTMs. MS offers several advantages over the traditional approaches including reproducibility, specificity, and ability to rapidly analyze numerous PTMs in a single experiment. In this thesis, the development and applications of novel analytical tools for the identification and quantification of histone PTMs are presented.
First, a method useful for measuring the global and site specific changes in histone acetylation is described. This method combines intact mass analysis and peptide sequencing approaches to study the global and site specific changes in histone acetylation during in vitro assays with yeast Rtt109 and its chaperone (Asf1 or Vps75). Second, a method for analysis of isomeric histone peptides is presented. This method combines a high resolution LC-MS/MS with a novel bioinformatics tool called Iso-PeptidAce to deconvolute mixed spectra of co-eluting isomeric peptides. We benchmarked Iso-PeptidAce using mixtures of synthetic isomeric peptides. We demonstrated its capability in histones isolated from human erythroleukemic (K562) cells treated with clinically relevant histone deacetylase inhibitors (HDACi) and in affinity-purified S. cerevisiae histones bound to chromatin assembly factor-1 (CAF-1). Third, by employing the above methods, an in-depth quantitative analysis of the substrate specificities of several fission yeast HATs and HDACs was assessed. We determined the acetylation site occupancy of multiple lysines in histones isolated from a control or mutant strains lacking specific HAT or HDAC activities. Our analysis identified several known and novel HAT and HDAC target sites. Our data also defined the division of labor between the different HATs and HDACs in maintaining the steady-state level of histone acetylation. Overall, we anticipate that the methods described in this thesis will address some of the existing challenges facing the chromatin field. Moreover, the data presented will provide valuable insights for future genetic and biochemical studies involving the fission yeast
A synthetic biology approach to probing nucleosome symmetry
The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell
Regulation of the DNA Damage Response and Gene Expression by the Dot1L Histone Methyltransferase and the 53Bp1 Tumour Suppressor
Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined.Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1Lâ»/â» and wild type cells are equally resistant to ionising radiation, whereas 53Bp1â»/â»/Dot1Lâ»/â» cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the mRNA levels of over 1200 genes involved in diverse biological functions. These results, combined with the previously reported list of differentially expressed genes in mouse ES cells knocked down for Dot1L, demonstrates surprising cell type and species conservation of Dot1L-dependent gene expression. In 53Bp1â»/â» cells, over 300 genes, many with functions in immune responses and apoptosis, were differentially expressed. To date, this is the first global analysis of gene expression in a 53Bp1-deficient cell line.Taken together, our results uncover a negative role for Dot1L and H3K79 methylation in the DNA damage response in the absence of 53Bp1. They also enlighten the roles of Dot1L and 53Bp1 in gene expression and the control of DNA double-strand repair pathways in the context of chromatin
Oxidative footprinting and mass spectrometry based structural characterization of mAb 14F7-NeuGc GM3 ganglioside complex
The Characterization of anti body-antigen interactions provider Crucial information on the structural basis of the specificity of binding of two antibodies Their target anti-gens. In this project, an attempt was made two characterize the binding interactions Between a monoclonal anti-body (Mab) Designated as 14F7 and NeuGc-GM3 ganglioside antigen. The 14F7 strongly recognize the ganglioside antigen Fri human melanoma and breast tumors. The crystal structure of the Fab fragment 14F7 has Been solved by X-ray crystallography and bonding the two ganglioside Predicted by docking and MD Simulations. However, despite Significant efforts, so far no experimental structural analysis of the 14F7-NeuGc-GM3 complex is available. Such information Would ask extremely valuable for developing new and improved antibodies for cancer immunotherapy.
There Parents, in this thesis, an alternative approach was overtaken two get experimental data. The binding interactions Within the complex were examined Using a newly Developed MS-based oxidative footprinting technique. Briefly, the anti-body sample, with and without the ligand, was overexposed two an excimer laser source in order two oxidize solvent accessible amino acid side chains. The oxidized samples were digested with trypsin simply. Finally, the tryptic fragments were analyzed by MALDI-TOF MS and ESI-LTQ-FT MS.
Sequencing of the MS / MS spectra and database searching identified two residue, Trp325 of the VL-CDR3 and Met112 of the VH-CDR3, That were oxidized in Both the free and ligand-bound 14F7. The Percentage of oxidation Of The peptides containing the residue were quantified based on the areas under the selected ion chromatograms of the oxidized and non-oxidized forms of the peptides. The Data Suggest That the Extent of oxidation of Both peptides is significantly lower in the ligand-bound 14F7. More over, an analysis of the spectral intensities and side chain solvent accessibilities confirmed That residue Met112 and Trp325 are protected from radical oxidation upon ligand binding the two anti-body. A new docking model of the complex is presented That is consis tent with all experimental data. The combined result, in contrast two the previous model, Suggest That the ligand volume in close proximity the two light chain, in Addition two bonding the two heavy chain CDR3 of the anti body. This information Can now ask unusually for Further Development
of anti-tumor anti-body with improved potency and Affinity for the target tumor anti-gens
Unraveling Site-Specific and Combinatorial Histone Modifications Using High-Resolution Mass Spectrometry in Histone Deacetylase Mutants of Fission Yeast
Histone
deacetylases (HDACs) catalyze the removal of acetylation
marks from lysine residues on histone and nonhistone substrates. Their
activity is generally associated with essential cellular processes
such as transcriptional repression and heterochromatin formation.
Interestingly, abnormal activity of HDACs has been reported in various
types of cancers, which makes them a promising therapeutic target
for cancer treatment. In the current study, we aim to understand the
mechanisms underlying the function of HDACs using an in-depth quantitative
analysis of changes in histone acetylation levels in <i>Schizosaccharomyces
pombe</i> (<i>S. pombe</i>) lacking major HDAC activities.
We employed a targeted quantitative mass spectrometry approach to
profile changes of acetylation and methylation at multiple lysine
residues on the N-terminal tail of histones H3 and H4. Our analyses
identified a number of histone acetylation sites that are significantly
affected by <i>S. pombe</i> HDAC mutations. We discovered
that mutation of the Class I HDAC known as Clr6 causes a major increase
in the abundance of triacetylated H4 molecules at K5, K8, and K12.
A <i>clr6â1</i> hypomorphic mutation also increased
the abundance of multiple acetyl-lysines in histone H3. In addition,
our study uncovered a few crosstalks between histone acetylation and
methylation upon deletion of HDACs Hos2 and Clr3. We anticipate that
the results from this study will greatly improve our current understanding
of the mechanisms involved in HDAC-mediated gene regulation and heterochromatin
assembly
Interplay Between Histone H3 Lysine 56 Deacetylation and Chromatin Modifiers in Response to DNA Damage
Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells.
Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism
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Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection
The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair
Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection
Targeted detection and quantitation of histone modifications from 1,000 cells.
Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations