Investigation translationnelle de la chromatine

DNA is organized via histones, the leading players in the compaction of genetic material. Technological evolution has favored the discovery of many protein variants. However, the annotation of the latter was unconventional, complicating their identification by mass spectrometry. Thus, I developed a comprehensive database, called MS_HistoneDB, dedicated to the detection of histone variants by mass spectrometry. MS_HistoneDB allows the use of murine and human samples. Also, the use of immunological tests makes it difficult to discriminate at the protein level of almost similar variants. Thus, I developed a targeted mass spectrometry analysis method to detect and quantify histone variants in a multiplex assay. This methodology has been applied in the investigation of chromatin during spermatogenesis, in mouse models, physiological or pathological mimicking male infertility.Another aspect of my work focused on proteins that bind modified forms of histones. Thus, I studied the "readers" of the BET family (Bromodomain and Extra-terminal domain). These proteins are recruited on chromatin via their bromodomains, a specific module recognizing acetylated histones. Their extra-terminal domain acts as a recruitment platform for transcriptional regulators. These proteins are conserved in the yeast Saccharomyces cerevisiae, and also in fungal pathogens responsible for invasive infections, where the only member is called Bdf1. Thus, I studied the extra-terminal domain of Bdf1 and demonstrated that it is essential for yeast survival. Then, I explored the molecular mechanisms involved. Finally, selective inhibitors are being developed in pathogenic yeast species. All of this work paves the way for the development of a new therapeutic class of antifungals.L’ADN s’organise via les histones, les principaux acteurs dans la compaction du matériel génétique. L’évolution technologique a favorisé la découverte de nombreux variants protéique. Toutefois, l’annotation de ces derniers s’est faite de manière non conventionnelle, compliquant leur identification par spectrométrie de masse. Ainsi, j’ai développé une banque de données exhaustive, intitulée MS_HistoneDB, dédiée à la détection des variants d’histone par spectrométrie de masse. MS_HistoneDB permet l’utilisation d’échantillons murin et humain. En outre, l’utilisation de tests immunologiques permet difficilement de discriminer au niveau protéique des variants quasi-similaire. Ainsi, j’ai développé une méthode d’analyse de spectrométrie de masse ciblée pour détecter et quantifier les variants d’histones en un essai multiplexe. Cette méthodologie a été appliqué dans l’investigation de la chromatine au cours de la spermatogenèse, dans des modèles murins, physiologique ou pathologique mimant l’infertilité masculine.Un autre aspect de mon travail s’est intéressé aux protéines liant les formes modifiées des histones. Ainsi, j’ai étudié les « readers »de la famille BET (Bromodomaine et Extra-terminal domain). Ces protéines sont recrutées sur la chromatine via leurs bromodomaines, module spécifique reconnaissant les histones acétylées. Leur domaine extra terminal joue le rôle de plateforme de recrutement de régulateurs transcriptionnels. Ces protéines sont conservées chez la levure Saccharomyces cerevisiae,et également chez les pathogènes fongiques responsables d’infections invasives, où l’unique membre est appelé Bdf1. Ainsi, j’ai étudié le domaine extra-terminale de Bdf1 et démontré qu’il est essentiel à la survie des levures. Puis, j’ai exploré les mécanismes moléculaires impliqués. Enfin, des inhibiteurs sélectifs sont en cours de développement dans des espèces de levure pathogène. L’ensemble de ces travaux ouvrent la voie au développement d’une nouvelle classe thérapeutique d’antifongiques

Translational epigenetics applied to histone variants and chromatin readers

L’ADN s’organise via les histones, les principaux acteurs dans la compaction du matériel génétique. L’évolution technologique a favorisé la découverte de nombreux variants protéique. Toutefois, l’annotation de ces derniers s’est faite de manière non conventionnelle, compliquant leur identification par spectrométrie de masse. Ainsi, j’ai développé une banque de données exhaustive, intitulée MS_HistoneDB, dédiée à la détection des variants d’histone par spectrométrie de masse. MS_HistoneDB permet l’utilisation d’échantillons murin et humain. En outre, l’utilisation de tests immunologiques permet difficilement de discriminer au niveau protéique des variants quasi-similaire. Ainsi, j’ai développé une méthode d’analyse de spectrométrie de masse ciblée pour détecter et quantifier les variants d’histones en un essai multiplexe. Cette méthodologie a été appliqué dans l’investigation de la chromatine au cours de la spermatogenèse, dans des modèles murins, physiologique ou pathologique mimant l’infertilité masculine.Un autre aspect de mon travail s’est intéressé aux protéines liant les formes modifiées des histones. Ainsi, j’ai étudié les « readers »de la famille BET (Bromodomaine et Extra-terminal domain). Ces protéines sont recrutées sur la chromatine via leurs bromodomaines, module spécifique reconnaissant les histones acétylées. Leur domaine extra terminal joue le rôle de plateforme de recrutement de régulateurs transcriptionnels. Ces protéines sont conservées chez la levure Saccharomyces cerevisiae,et également chez les pathogènes fongiques responsables d’infections invasives, où l’unique membre est appelé Bdf1. Ainsi, j’ai étudié le domaine extra-terminale de Bdf1 et démontré qu’il est essentiel à la survie des levures. Puis, j’ai exploré les mécanismes moléculaires impliqués. Enfin, des inhibiteurs sélectifs sont en cours de développement dans des espèces de levure pathogène. L’ensemble de ces travaux ouvrent la voie au développement d’une nouvelle classe thérapeutique d’antifongiques.DNA is organized via histones, the leading players in the compaction of genetic material. Technological evolution has favored the discovery of many protein variants. However, the annotation of the latter was unconventional, complicating their identification by mass spectrometry. Thus, I developed a comprehensive database, called MS_HistoneDB, dedicated to the detection of histone variants by mass spectrometry. MS_HistoneDB allows the use of murine and human samples. Also, the use of immunological tests makes it difficult to discriminate at the protein level of almost similar variants. Thus, I developed a targeted mass spectrometry analysis method to detect and quantify histone variants in a multiplex assay. This methodology has been applied in the investigation of chromatin during spermatogenesis, in mouse models, physiological or pathological mimicking male infertility.Another aspect of my work focused on proteins that bind modified forms of histones. Thus, I studied the "readers" of the BET family (Bromodomain and Extra-terminal domain). These proteins are recruited on chromatin via their bromodomains, a specific module recognizing acetylated histones. Their extra-terminal domain acts as a recruitment platform for transcriptional regulators. These proteins are conserved in the yeast Saccharomyces cerevisiae, and also in fungal pathogens responsible for invasive infections, where the only member is called Bdf1. Thus, I studied the extra-terminal domain of Bdf1 and demonstrated that it is essential for yeast survival. Then, I explored the molecular mechanisms involved. Finally, selective inhibitors are being developed in pathogenic yeast species. All of this work paves the way for the development of a new therapeutic class of antifungals

Investigation translationnelle de la chromatine

DNA is organized via histones, the leading players in the compaction of genetic material. Technological evolution has favored the discovery of many protein variants. However, the annotation of the latter was unconventional, complicating their identification by mass spectrometry. Thus, I developed a comprehensive database, called MS_HistoneDB, dedicated to the detection of histone variants by mass spectrometry. MS_HistoneDB allows the use of murine and human samples. Also, the use of immunological tests makes it difficult to discriminate at the protein level of almost similar variants. Thus, I developed a targeted mass spectrometry analysis method to detect and quantify histone variants in a multiplex assay. This methodology has been applied in the investigation of chromatin during spermatogenesis, in mouse models, physiological or pathological mimicking male infertility.Another aspect of my work focused on proteins that bind modified forms of histones. Thus, I studied the "readers" of the BET family (Bromodomain and Extra-terminal domain). These proteins are recruited on chromatin via their bromodomains, a specific module recognizing acetylated histones. Their extra-terminal domain acts as a recruitment platform for transcriptional regulators. These proteins are conserved in the yeast Saccharomyces cerevisiae, and also in fungal pathogens responsible for invasive infections, where the only member is called Bdf1. Thus, I studied the extra-terminal domain of Bdf1 and demonstrated that it is essential for yeast survival. Then, I explored the molecular mechanisms involved. Finally, selective inhibitors are being developed in pathogenic yeast species. All of this work paves the way for the development of a new therapeutic class of antifungals.L’ADN s’organise via les histones, les principaux acteurs dans la compaction du matériel génétique. L’évolution technologique a favorisé la découverte de nombreux variants protéique. Toutefois, l’annotation de ces derniers s’est faite de manière non conventionnelle, compliquant leur identification par spectrométrie de masse. Ainsi, j’ai développé une banque de données exhaustive, intitulée MS_HistoneDB, dédiée à la détection des variants d’histone par spectrométrie de masse. MS_HistoneDB permet l’utilisation d’échantillons murin et humain. En outre, l’utilisation de tests immunologiques permet difficilement de discriminer au niveau protéique des variants quasi-similaire. Ainsi, j’ai développé une méthode d’analyse de spectrométrie de masse ciblée pour détecter et quantifier les variants d’histones en un essai multiplexe. Cette méthodologie a été appliqué dans l’investigation de la chromatine au cours de la spermatogenèse, dans des modèles murins, physiologique ou pathologique mimant l’infertilité masculine.Un autre aspect de mon travail s’est intéressé aux protéines liant les formes modifiées des histones. Ainsi, j’ai étudié les « readers »de la famille BET (Bromodomaine et Extra-terminal domain). Ces protéines sont recrutées sur la chromatine via leurs bromodomaines, module spécifique reconnaissant les histones acétylées. Leur domaine extra terminal joue le rôle de plateforme de recrutement de régulateurs transcriptionnels. Ces protéines sont conservées chez la levure Saccharomyces cerevisiae,et également chez les pathogènes fongiques responsables d’infections invasives, où l’unique membre est appelé Bdf1. Ainsi, j’ai étudié le domaine extra-terminale de Bdf1 et démontré qu’il est essentiel à la survie des levures. Puis, j’ai exploré les mécanismes moléculaires impliqués. Enfin, des inhibiteurs sélectifs sont en cours de développement dans des espèces de levure pathogène. L’ensemble de ces travaux ouvrent la voie au développement d’une nouvelle classe thérapeutique d’antifongiques

Proteomic Analysis of Histone Variants and Their PTMs: Strategies and Pitfalls

International audienc

MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones

Abstract Background Histones and histone variants are essential components of the nuclear chromatin. While mass spectrometry has opened a large window to their characterization and functional studies, their identification from proteomic data remains challenging. Indeed, the current interpretation of mass spectrometry data relies on public databases which are either not exhaustive (Swiss-Prot) or contain many redundant entries (UniProtKB or NCBI). Currently, no protein database is ideally suited for the analysis of histones and the complex array of mammalian histone variants. Results We propose two proteomics-oriented manually curated databases for mouse and human histone variants. We manually curated >1700 gene, transcript and protein entries to produce a non-redundant list of 83 mouse and 85 human histones. These entries were annotated in accordance with the current nomenclature and unified with the \u201cHistoneDB2.0 with Variants\u201d database. This resource is provided in a format that can be directly read by programs used for mass spectrometry data interpretation. In addition, it was used to interpret mass spectrometry data acquired on histones extracted from mouse testis. Several histone variants, which had so far only been inferred by homology or detected at the RNA level, were detected by mass spectrometry, confirming the existence of their protein form. Conclusions Mouse and human histone entries were collected from different databases and subsequently curated to produce a non-redundant protein-centric resource, MS_HistoneDB. It is dedicated to the proteomic study of histones in mouse and human and will hopefully facilitate the identification and functional study of histone variants

Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

International audienceBackgroundHistones organize DNA into chromatin through a variety of processes. Among them, a vast diversity of histone variants can be incorporated into chromatin and finely modulate its organization and functionality. Classically, the study of histone variants has largely relied on antibody-based assays. However, antibodies have a limited efficiency to discriminate between highly similar histone variants.ResultsIn this study, we established a mass spectrometry-based analysis to address this challenge. We developed a targeted proteomics method, using selected reaction monitoring or parallel reaction monitoring, to quantify a maximum number of histone variants in a single multiplexed assay, even when histones are present in a crude extract. This strategy was developed on H2A and H2B variants, using 55 peptides corresponding to 25 different histone sequences, among which a few differ by a single amino acid. The methodology was then applied to mouse testis extracts in which almost all histone variants are expressed. It confirmed the abundance profiles of several testis-specific histones during successive stages of spermatogenesis and the existence of predicted H2A.L.1 isoforms. This methodology was also used to explore the over-expression pattern of H2A.L.1 isoforms in a mouse model of male infertility.ConclusionsOur results demonstrate that targeted proteomics is a powerful method to quantify highly similar histone variants and isoforms. The developed method can be easily transposed to the study of human histone variants, whose abundance can be deregulated in various diseases

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes

International audienceEpigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetyla-tion and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification

MOESM4 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 4. Details of the SRM transitions for each signature peptide. SRM assay parameters including precursor and fragment ion type, charge state, elution time as well as raw data are provided in Suppl. data. (*) Indicates peptides monitored only in their endogenous form

MOESM9 of Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Additional file 9. Rules used to select or reject peptides using their transition profiles. The validation of the best transitions was performed using a signal-to-noise ratio (> 5) and a perfect co-elution of the heavy standard peptide with the endogenous peptide. Three fragment ions (F1, F2, and F3) are represented for the heavy and the endogenous peptides. a All fragment ions can be integrated because the heavy and endogenous fragment ions co-elute in the same intensity order. b In that case, only F2 can be integrated because the ratio heavy/endogenous is different for F1 and F3. c The fragment F2 is contaminated by another analyte eluting at a slightly later time; it has to be excluded from the analysis. d Here, the signal-to-noise ratio is below five, no fragment ion can be integrated. e. The endogenous peptide traces do not co-elute with the heavy peptide traces