Robert Schneider: Chromatin Dynamics and Epigenetics

2022
Ruiz Tejada Segura, M.L. ; Abou Moussa, E. ; Garabello, E. ; Nakahara, T.S. ; Makhlouf, M. ; Mathew, L.S. ; Wang, L. ; Valle, F. ; Huang, S.S.Y. ; Mainland, J.D. ; Caselle, M. ; Osella, M. ; Lorenz, S. ; Reisert, J. ; Logan, D.W. ; Malnic, B. ; Scialdone, A. ; Saraiva, L.R.
Cell Rep. 38:110547 (2022)
The sense of smell helps us navigate the environment, but its molecular architecture and underlying logic remain understudied. The spatial location of odorant receptor genes (Olfrs) in the nose is thought to be independent of the structural diversity of the odorants they detect. Using spatial transcriptomics, we create a genome-wide 3D atlas of the mouse olfactory mucosa (OM). Topographic maps of genes differentially expressed in space reveal that both Olfrs and non-Olfrs are distributed in a continuous and overlapping fashion over at least five broad zones in the OM. The spatial locations of Olfrs correlate with the mucus solubility of the odorants they recognize, providing direct evidence for the chromatographic theory of olfaction. This resource resolves the molecular architecture of the mouse OM and will inform future studies on mechanisms underlying Olfr gene choice, axonal pathfinding, patterning of the nervous system, and basic logic for the peripheral representation of smell.
Wissenschaftlicher Artikel
Scientific Article
Günsel, G.G. ; Conlon, T.M. ; Jeridi, A. ; Kim, R. ; Ertüz, Z. ; Lang, N.J. ; Ansari, M. ; Novikova, M. ; Jiang, D. ; Strunz, M. ; Gaianova, M. ; Hollauer, C. ; Gabriel, C. ; Angelidis, I. ; Doll, S. ; Pestoni, J. ; Edelmann, S.L. ; Kohlhepp, M.S. ; Guillot, A. ; Bassler, K. ; Van Eeckhoutte, H.P. ; Kayalar, Ö. ; Konyalilar, N. ; Kanashova, T. ; Rodius, S. ; Ballester-Lopez, C. ; Genes Robles, C.M. ; Smirnova, N.F. ; Rehberg, M. ; Agarwal, C. ; Krikki, I. ; Piavaux, B. ; Verleden, S.E. ; Vanaudenaerde, B. ; Königshoff, M. ; Dittmar, G. ; Bracke, K.R. ; Schultze, J.L. ; Watz, H. ; Eickelberg, O. ; Stöger, T. ; Burgstaller, G. ; Tacke, F. ; Heissmeyer, V. ; Rinkevich, Y. ; Bayram, H. ; Schiller, H. B. ; Conrad, M. ; Schneider, R. ; Yildirim, A.Ö.
Nat. Commun. 13:1303 (2022)
Extravasation of monocytes into tissue and to the site of injury is a fundamental immunological process, which requires rapid responses via post translational modifications (PTM) of proteins. Protein arginine methyltransferase 7 (PRMT7) is an epigenetic factor that has the capacity to mono-methylate histones on arginine residues. Here we show that in chronic obstructive pulmonary disease (COPD) patients, PRMT7 expression is elevated in the lung tissue and localized to the macrophages. In mouse models of COPD, lung fibrosis and skin injury, reduced expression of PRMT7 associates with decreased recruitment of monocytes to the site of injury and hence less severe symptoms. Mechanistically, activation of NF-κB/RelA in monocytes induces PRMT7 transcription and consequential mono-methylation of histones at the regulatory elements of RAP1A, which leads to increased transcription of this gene that is responsible for adhesion and migration of monocytes. Persistent monocyte-derived macrophage accumulation leads to ALOX5 over-expression and accumulation of its metabolite LTB4, which triggers expression of ACSL4 a ferroptosis promoting gene in lung epithelial cells. Conclusively, inhibition of arginine mono-methylation might offer targeted intervention in monocyte-driven inflammatory conditions that lead to extensive tissue damage if left untreated.
Wissenschaftlicher Artikel
Scientific Article
Iturbide Martinez De Albeniz, A. ; Ruiz Tejada Segura, M.L. ; Noll, C. ; Schorpp, K.K. ; Rothenaigner, I. ; Lubatti, G. ; Agami, A. ; Hadian, K. ; Scialdone, A. ; Torres-Padilla, M.E.
Nat. Struct. Mol. Biol. 29:282 (2022)
In the version of this article initially published, the surname of author Mayra L. Ruiz Tejada Segura was misspelled as Ruiz Tejeda Segura. The error has been corrected in the online version of the article.
Millán-Zambrano, G. ; Burton, A. ; Bannister, A.J. ; Schneider, R.
Nat. Rev. Genet., DOI: 10.1038/s41576-022-00468-7 (2022)
Much has been learned since the early 1960s about histone post-translational modifications (PTMs) and how they affect DNA-templated processes at the molecular level. This understanding has been bolstered in the past decade by the identification of new types of histone PTM, the advent of new genome-wide mapping approaches and methods to deposit or remove PTMs in a locally and temporally controlled manner. Now, with the availability of vast amounts of data across various biological systems, the functional role of PTMs in important processes (such as transcription, recombination, replication, DNA repair and the modulation of genomic architecture) is slowly emerging. This Review explores the contribution of histone PTMs to the regulation of genome function by discussing when these modifications play a causative (or instructive) role in DNA-templated processes and when they are deposited as a consequence of such processes, to reinforce and record the event. Important advances in the field showing that histone PTMs can exert both direct and indirect effects on genome function are also presented.
Review
Review
2021
Skalska, L. ; Begley, V. ; Beltran, M. ; Lukauskas, S. ; Khandelwal, G. ; Faull, P. ; Bhamra, A. ; Tavares, M. ; Wellman, R. ; Tvardovskiy, A. ; Foster, B. ; Ruiz de Los Mozos, I. ; Herrero, J. ; Surinova, S. ; Snijders, A.P. ; Bartke, T. ; Jenner, R.G.
Mol. Cell 81, 2944-2959.e10 (2021)
A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.
Wissenschaftlicher Artikel
Scientific Article
Nitsch, S. ; Zorro Shahidian, L. ; Schneider, R.
EMBO Rep. 22:e52774 (2021)
In eukaryotic cells, DNA is tightly packed with the help of histone proteins into chromatin. Chromatin architecture can be modified by various post-translational modifications of histone proteins. For almost 60 years now, studies on histone lysine acetylation have unraveled the contribution of this acylation to an open chromatin state with increased DNA accessibility, permissive for gene expression. Additional complexity emerged from the discovery of other types of histone lysine acylations. The acyl group donors are products of cellular metabolism, and distinct histone acylations can link the metabolic state of a cell with chromatin architecture and contribute to cellular adaptation through changes in gene expression. Currently, various technical challenges limit our full understanding of the actual impact of most histone acylations on chromatin dynamics and of their biological relevance. In this review, we summarize the state of the art and provide an overview of approaches to overcome these challenges. We further discuss the concept of subnuclear metabolic niches that could regulate local CoA availability and thus couple cellular metabolisms with the epigenome.
Review
Review
Iturbide Martinez De Albeniz, A. ; Ruiz Tejada Segura, M.L. ; Noll, C. ; Schorpp, K.K. ; Rothenaigner, I. ; Ruiz-Morales, E.R. ; Lubatti, G. ; Agami, A. ; Hadian, K. ; Scialdone, A. ; Torres-Padilla, M.E.
Nat. Struct. Mol. Biol. 28, 521-532 (2021)
Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.
Wissenschaftlicher Artikel
Scientific Article
Santos-Rosa, H. ; Millán-Zambrano, G. ; Han, N. ; Leonardi, T. ; Klimontova, M. ; Nasiscionyte, S. ; Pandolfini, L. ; Tzelepis, K. ; Bartke, T. ; Kouzarides, T.
Mol. Cell 81, 2793-2807.e8 (2021)
DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.
Wissenschaftlicher Artikel
Scientific Article
Belousov, K.I. ; Filatov, N.A. ; Kukhtevich, I. ; Kantsler, V. ; Evstrapov, A.A. ; Bukatin, A.S.
Sci. Rep. 11:8797 (2021)
Nowadays droplet microfluidics is widely used to perform high throughput assays and for the synthesis of micro- and nanoparticles. These applications usually require packaging several reagents into droplets and their mixing to start a biochemical reaction. For rapid mixing microfluidic devices usually require additional functional elements that make their designs more complex. Here we perform a series of 2D numerical simulations, followed by experimental studies, and introduce a novel asymmetric flow-focusing droplet generator, which enhances mixing during droplet formation due to a 2D or 3D asymmetric vortex, located in the droplet formation area of the microfluidic device. Our results suggest that 2D numerical simulations can be used for qualitative analysis of two-phase flows and droplet generation process in quasi-two-dimensional devices, while the relative simplicity of such simulations allows them to be easily applied to fairly complicated microfluidic geometries. Mixing inside droplets formed in the asymmetric generator occurs up to six times faster than in a conventional symmetric one. The best mixing efficiency is achieved in a specific range of droplet volumes, which can be changed by scaling the geometry of the device. Thus, the droplet generator suggested here can significantly simplify designs of microfluidic devices because it enables both the droplet formation and fast mixing of the reagents within droplets. Moreover, it can be used to precisely estimate reaction kinetics.
Wissenschaftlicher Artikel
Scientific Article
Zorro Shahidian, L. ; Haas, M. ; Le Gras, S. ; Nitsch, S. ; Mourao, A. ; Geerlof, A. ; Margueron, R. ; Michaelis, J. ; Daujat, S. ; Schneider, R.
EMBO Rep. 22:e51009 (2021)
Histone post-translational modifications (PTMs) are key players in chromatin regulation. The identification of novel histone acylations raises important questions regarding their role in transcription. In this study, we characterize the role of an acylation on the lateral surface of the histone octamer, H3K122 succinylation (H3K122succ), in chromatin function and transcription. Using chromatin succinylated at H3K122 in in vitro transcription assays, we show that the presence of H3K122succ is sufficient to stimulate transcription. In line with this, we found in our ChIP assays H3K122succ enriched on promoters of active genes and H3K122succ enrichment scaling with gene expression levels. Furthermore, we show that the co-activators p300/CBP can succinylate H3K122 and identify sirtuin 5 (SIRT5) as a new desuccinylase. By applying single molecule FRET assays, we demonstrate a direct effect of H3K122succ on nucleosome stability, indicating an important role for histone succinylation in modulating chromatin dynamics. Together, these data provide the first insights into the mechanisms underlying transcriptional regulation by H3K122succ.
Wissenschaftlicher Artikel
Scientific Article
Petryk, N. ; Bultmann, S. ; Bartke, T. ; Defossez, P.-A.
Nucleic Acids Res. 49, 3020–3032 (2021)
DNA methylation is essential to development and cellular physiology in mammals. Faulty DNA methylation is frequently observed in human diseases like cancer and neurological disorders. Molecularly, this epigenetic mark is linked to other chromatin modifications and it regulates key genomic processes, including transcription and splicing. Each round of DNA replication generates two hemi-methylated copies of the genome. These must be converted back to symmetrically methylated DNA before the next S-phase, or the mark will fade away; therefore the maintenance of DNA methylation is essential. Mechanistically, the maintenance of this epigenetic modification takes place during and after DNA replication, and occurs within the very dynamic context of chromatin re-assembly. Here, we review recent discoveries and unresolved questions regarding the mechanisms, dynamics and fidelity of DNA methylation maintenance in mammals. We also discuss how it could be regulated in normal development and misregulated in disease.
Review
Review
2020
Bheda, P. ; Aguilar-Gómez, D. ; Kukhtevich, I. ; Becker, J. ; Charvin, G. ; Kirmizis, A. ; Schneider, R.
STAR Protoc. 1:100228 (2020)
The budding yeast Saccharomyces cerevisiae is an excellent model organism to dissect the maintenance and inheritance of phenotypes due to its asymmetric division. This requires following individual cells over time as they go through divisions to define pedigrees. Here, we provide a detailed protocol for collecting and analyzing time-lapse imaging data of yeast cells. The microfluidics protocol can achieve improved time resolution for single-cell tracking to enable characterization of maintenance and inheritance of phenotypes. For complete details on the use and execution of this protocol, please refer to Bheda et al. (2020a).
Wissenschaftlicher Artikel
Scientific Article
Ignatova, V.V. ; Kaiser, S. ; Ho, J.S.Y. ; Bing, X. ; Stolz, P. ; Tan, Y.X. ; Lee, C.L. ; Gay, F.P.H. ; Lastres, P.R. ; Gerlini, R. ; Rathkolb, B. ; Aguilar-Pimentel, J.A. ; Sanz-Moreno, A. ; Klein-Rodewald, T. ; Calzada-Wack, J. ; Ibragimov, E. ; Valenta, M. ; Lukauskas, S. ; Pavesi, A. ; Marschall, S. ; Leuchtenberger, S. ; Fuchs, H. ; Gailus-Durner, V. ; Hrabě de Angelis, M. ; Bultmann, S. ; Rando, O.J. ; Guccione, E. ; Kellner, S.M. ; Schneider, R.
Sci. Adv. 6:eaaz4551 (2020)
Recently, covalent modifications of RNA, such as methylation, have emerged as key regulators of all aspects of RNA biology and have been implicated in numerous diseases, for instance, cancer. Here, we undertook a combination of in vitro and in vivo screens to test 78 potential methyltransferases for their roles in hepatocellular carcinoma (HCC) cell proliferation. We identified methyltransferase-like protein 6 (METTL6) as a crucial regulator of tumor cell growth. We show that METTL6 is a bona fide transfer RNA (tRNA) methyltransferase, catalyzing the formation of 3-methylcytidine at C32 of specific serine tRNA isoacceptors. Deletion of Mettl6 in mouse stem cells results in changes in ribosome occupancy and RNA levels, as well as impaired pluripotency. In mice, Mettl6 knockout results in reduced energy expenditure. We reveal a previously unknown pathway in the maintenance of translation efficiency with a role in maintaining stem cell self-renewal, as well as impacting tumor cell growth profoundly.
Wissenschaftlicher Artikel
Scientific Article
Bheda, P. ; Kirmizis, A. ; Schneider, R.
Curr. Genet. 66, 1029–1035 (2020)
Transcriptional reinduction memory is a phenomenon whereby cells "remember" their transcriptional response to a previous stimulus such that subsequent encounters with the same stimulus can result in altered gene expression kinetics. Chromatin structure is thought to play a role in certain transcriptional memory mechanisms, leading to questions as to whether and how memory can be actively maintained and inherited to progeny through cell division. Here we summarize efforts towards dissecting chromatin-based transcriptional memory inheritance ofGALgenes inSaccharomyces cerevisiae. We focus on methods and analyses ofGAL(as well asMALandINO) memory in single cells and discuss the challenges in unraveling the underlying mechanisms in yeast and higher eukaryotes.
Review
Review
Strunz, M. ; Simon, L. ; Ansari, M. ; Kathiriya, J.J. ; Angelidis, I. ; Mayr, C. ; Tsidiridis, G. ; Lange, M. ; Mattner, L. ; Yee, M. ; Ogar, P. ; Sengupta, A. ; Kukhtevich, I. ; Schneider, R. ; Zhao, Z. ; Voss, C. ; Stöger, T. ; Neumann, J.H.L. ; Hilgendorff, A. ; Behr, J. ; O'Reilly, M. ; Lehmann, M. ; Burgstaller, G. ; Königshoff, M. ; Chapman, H.A. ; Theis, F.J. ; Schiller, H. B.
Nat. Commun. 11:3559 (2020)
The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8+transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell-cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states. Injury repair is characterized by the generation of transient cell states important for tissue recovery. Here, the authors present a single cell RNA-seq map of recovery from bleomycin lung injury in mice and uncover a Krt8+ transitional stem cell state that precedes the regeneration of AT1 cells and persists in human lung fibrosis.
Wissenschaftlicher Artikel
Scientific Article
Kukhtevich, I. ; Lohrberg, N. ; Padovani, F. ; Schneider, R. ; Schmoller, K.M.
Nat. Commun. 11:2952 (2020)
The formation and maintenance of subcellular structures and organelles with a well-defined size is a key requirement for cell function, yet our understanding of the underlying size control mechanisms is limited. While budding yeast cell polarization and subsequent assembly of a septin ring at the site of bud formation has been successfully used as a model for biological self-assembly processes, the mechanisms that set the size of the septin ring at the bud neck are unknown. Here, we use live-cell imaging and genetic manipulation of cell volume to show that the septin ring diameter increases with cell volume. This cell-volume-dependence largely accounts for modulations of ring size due to changes in ploidy and genetic manipulation of cell polarization. Our findings suggest that the ring diameter is set through the dynamic interplay of septin recruitment and Cdc42 polarization, establishing it as a model for size homeostasis of self-assembling organelles. Budding yeast cell polarization is known to self-assemble, but it is still not clear what controls the size of the resulting septin ring. Here the authors show that the septin ring diameter is set by cell volume, ensuring that larger cells have larger rings.
Wissenschaftlicher Artikel
Scientific Article
Bheda, P. ; Aguilar-Gomez, D. ; Becker, N.B. ; Becker, J. ; Stavrou, E. ; Kukhtevich, I. ; Höfer, T. ; Maerkl, S. ; Charvin, G. ; Marr, C. ; Kirmizis, A. ; Schneider, R.
Mol. Cell 78, 915-925 (2020)
Transcriptional memory of gene expression enables adaptation to repeated stimuli across many organisms. However, the regulation and heritability of transcriptional memory in single cells and through divisions remains poorly understood. Here, we combined microfluidics with single-cell live imaging to monitor Saccharomyces cerevisiae galactokinase 1 (GAL1) expression over multiple generations. By applying pedigree analysis, we dissected and quantified the maintenance and inheritance of transcriptional reinduction memory in individual cells through multiple divisions. We systematically screened for loss- and gain-of-memory knockouts to identify memory regulators in thousands of single cells. We identified new loss-of-memory mutants, which affect memory inheritance into progeny. We also unveiled a gain-of-memory mutant, elp6 Delta, and suggest that this new phenotype can be mediated through decreased histone occupancy at the GAL1 promoter. Our work uncovers principles of maintenance and inheritance of gene expression states and their regulators at the single-cell level.
Wissenschaftlicher Artikel
Scientific Article
Bheda, P.
Mol. Metab. 38:100955 (2020)
Background: Organisms can be primed by metabolic exposures to continue expressing response genes even once the metabolite is no longer available, and can affect the speed and magnitude of responsive gene expression during subsequent exposures. This “metabolic transcriptional memory” can have a profound impact on the survivability of organisms in fluctuating environments. Scope of review: Here I present several examples of metabolic transcriptional memory in the microbial world and discuss what is known so far regarding the underlying mechanisms, which mainly focus on chromatin modifications, protein inheritance, and broad changes in metabolic network. From these lessons learned in microbes, some insights into the yet understudied human metabolic memory can be gained. I thus discuss the implications of metabolic memory in disease progression in humans – i.e., the memory of high blood sugar exposure and the resulting effects on diabetic complications. Major conclusions: Carbon source shifts from glucose to other less preferred sugars such as lactose, galactose, and maltose for energy metabolism as well as starvation of a signal transduction precursor sugar inositol are well-studied examples of metabolic transcriptional memory in Escherichia coli and Saccharomyces cerevisiae. Although the specific factors guiding metabolic transcriptional memory are not necessarily conserved from microbes to humans, the same basic mechanisms are in play, as is observed in hyperglycemic memory. Exploration of new metabolic transcriptional memory systems as well as further detailed mechanistic analyses of known memory contexts in microbes is therefore central to understanding metabolic memory in humans, and may be of relevance for the successful treatment of the ever-growing epidemic of diabetes.
Review
Review
Bartke, T. ; Schneider, R.
Mol. Metab. 38:100987 (2020)
Review
Review
Strunz, M. ; Simon, L. ; Ansari, M. ; Mattner, L. ; Angelidis, I. ; Mayr, C. ; Kathiriya, J. ; Yee, M. ; Ogar, P. ; Voss, C. ; Stöger, T. ; Kukhtevich, I. ; Schneider, R. ; Lehmann, M. ; Koenigshoff, M. ; Burgstaller, G. ; O'Reilly, M. ; Chapman, H. ; Theis, F.J. ; Schiller, H. B.
Wound Repair Regen. 28, A7-A7 (2020)
Meeting abstract
Meeting abstract
Ignatova, V.V. ; Stolz, P. ; Kaiser, S. ; Gustafsson, T.H. ; Lastres, P.R. ; Sanz-Moreno, A. ; Cho, Y.-L. ; Amarie, O.V. ; Aguilar-Pimentel, J.A. ; Klein-Rodewald, T. ; Calzada-Wack, J. ; Becker, L. ; Marschall, S. ; Kraiger, M.J. ; Garrett, L. ; Seisenberger, C. ; Hölter, S.M. ; Borland, K. ; Van De Logt, E. ; Jansen, P.W.T.C. ; Baltissen, M.P. ; Valenta, M. ; Vermeulen, M. ; Wurst, W. ; Gailus-Durner, V. ; Fuchs, H. ; Hrabě de Angelis, M. ; Rando, O.J. ; Kellner, S.M. ; Bultmann, S. ; Schneider, R.
Genes Dev. 34, 715-729 (2020)
Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.
Wissenschaftlicher Artikel
Scientific Article
2019
Beltran, M. ; Tavares, M. ; Justin, N. ; Khandelwal, G. ; Ambrose, J. ; Foster, B. ; Worlock, K.B. ; Tvardovskiy, A. ; Kunzelmann, S. ; Herrero, J. ; Bartke, T. ; Gamblin, S.J. ; Wilson, J.R. ; Jenner, R.G.
Nat. Struct. Mol. Biol., DOI: 10.1038/s41594-019-0341-8 (2019)
In the version of this article initially published, Fig. 4 included some errors. In Fig. 4c, the color for the left bar in each set of three bars (green) was incorrect; the correct color is orange (H2AK119ub, as in key). In Fig. 4d, top row, the downward error bars for H3K27me3 in the top middle plot (Fgf11 B) were incorrect; the correct s.d. in the negative direction is smaller for each. In Fig. 4d, bottom row, the far left downward error bar for HA-dCas9 in the left plot (Fgf11 A) was incorrect; the correct s.d. in the negative direction is larger. The errors have been corrected in the HTML and PDF versions of the article. (Figure presented.).
Beltran, M. ; Tavares, M. ; Justin, N. ; Khandelwal, G. ; Ambrose, J. ; Foster, B. ; Worlock, K.B. ; Kunzelmann, S. ; Herrero, J. ; Bartke, T. ; Gamblin, S.J. ; Wilson, J.R. ; Jenner, R.G.
Nat. Struct. Mol. Biol. 26, 899-909 (2019)
Polycomb repressive complex 2 (PRC2) maintains repression of cell-type-specific genes but also associates with genes ectopically in cancer. While it is currently unknown how PRC2 is removed from genes, such knowledge would be useful for the targeted reversal of deleterious PRC2 recruitment events. Here, we show that G-tract RNA specifically removes PRC2 from genes in human and mouse cells. PRC2 preferentially binds G tracts within nascent precursor mRNA (pre-mRNA), especially within predicted G-quadruplex structures. G-quadruplex RNA evicts the PRC2 catalytic core from the substrate nucleosome. In cells, PRC2 transfers from chromatin to pre-mRNA upon gene activation, and chromatin-associated G-tract RNA removes PRC2, leading to H3K27me3 depletion from genes. Targeting G-tract RNA to the tumor suppressor gene CDKN2A in malignant rhabdoid tumor cells reactivates the gene and induces senescence. These data support a model in which pre-mRNA evicts PRC2 during gene activation and provides the means to selectively remove PRC2 from specific genes.
Wissenschaftlicher Artikel
Scientific Article
Mrozek-Gorska, P. ; Buschle, A. ; Pich, D. ; Schwarzmayr, T. ; Fechtner, R. ; Scialdone, A. ; Hammerschmidt, W.
Proc. Natl. Acad. Sci. U.S.A. 116, 16046-16055 (2019)
Epstein-Barr virus (EBV) is a human tumor virus and a model of herpesviral latency. The virus efficiently infects resting human B lymphocytes and induces their continuous proliferation in vitro, which mimics certain aspects of EBV's oncogenic potential in vivo. How lymphoblastoid cell lines (LCLs) evolve from the infected lymphocytes is uncertain. We conducted a systematic time-resolved longitudinal study of cellular functions and transcriptional profiles of newly infected naive primary B lymphocytes. EBV reprograms the cells comprehensively and globally. Rapid and extensive transcriptional changes occur within 24 h and precede any metabolic and phenotypic changes. Within 72 h, the virus activates the cells, changes their phenotypes with respect to cell size, RNA, and protein content, and induces metabolic pathways to cope with the increased demand for energy, supporting an efficient cell cycle entry on day 3 postinfection. The transcriptional program that EBV initiates consists of 3 waves of clearly discernable clusters of cellular genes that peak on day 2, 3, or 4 and regulate RNA synthesis, metabolic pathways, and cell division, respectively. Upon onset of cell doublings on day 4, the cellular transcriptome appears to be completely reprogrammed to support the proliferating cells, but 3 additional clusters of EBV-regulated genes fine-tune cell signaling, migration, and immune response pathways, eventually. Our study reveals that more than 11,000 genes are regulated upon EBV infection as naive B cells exit quiescence to enter a germinal center-like differentiation program, which culminates in immortalized, proliferating cells that partially resemble plasmablasts and early plasma cells.
Wissenschaftlicher Artikel
Scientific Article
Pellegrino, J. ; Shahjahanpour, A. ; Lukauskas, S. ; Olayo Alarcon, R. ; Ramakrishnan, A. ; Colomé-Tatché, M. ; Schneider, R.
Poster: CSH meeting: mechanisms of metabolic signaling, 14 May 2019, Cold Spring Harbour, NY, USA. (2019)
Ignatova, V.V. ; Jansen, P.W.T.C. ; Baltissen, M.P. ; Vermeulen, M. ; Schneider, R.
Sci. Rep. 9:6584 (2019)
Human methytransferase like proteins (METTL) are part of a large protein family characterized by the presence of binding domains for S-adenosyl methionine, a co-substrate for methylation reactions. Despite the fact that members of this protein family were shown or predicted to be DNA, RNA or protein methyltransferases, most METTL proteins are still poorly characterized. Identification of complexes in which these potential enzymes act could help to understand their function(s) and substrate specificities. Here we systematically studied interacting partners of METTL protein family members in HeLa cells using label-free quantitative mass spectrometry. We found that, surprisingly, many of the METTL proteins appear to function outside of stable complexes whereas others including METTL7B, METTL8 and METTL9 have high-confidence interaction partners. Our study is the first systematic and comprehensive overview of the interactome of METTL protein family that can provide a crucial resource for further studies of these potential novel methyltransferases.
Wissenschaftlicher Artikel
Scientific Article
Bartke, T. ; Groth, A.
Mol. Cell. Oncol. 6:1605820 (2019)
Mutations caused by DNA damage are a main driver of cancer. We discovered that recognition of newly synthesised histone H4 directs breast cancer type 1 susceptibility protein (BRCA1) to post-replicative chromatin. The switch from mutagenic to error-free DNA double strand break repair by homologous recombination is therefore controlled by chromatin.
Sonstiges: Meinungsartikel
Other: Opinion
Nakamura, K. ; Saredi, G. ; Becker, J.R. ; Foster, B. ; Nguyen, N.V. ; Beyer, T.E. ; Cesa, L.C. ; Faull, P.A. ; Lukauskas, S. ; Frimurer, T. ; Chapman, J.R. ; Bartke, T. ; Groth, A.
Nat. Cell Biol. 21, 311-318 (2019)
Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining(1). HR supresses tumorigenesis(1), but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present(2). Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. (2-5)), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K2OmeO), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner(3)-as a reader of H4K2OmeO present on new histones in post-replicative chromatin(6). BARD1 ankyrin repeat domain mutations disabling H4K2OmeO recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K2OmeO is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.
Wissenschaftlicher Artikel
Scientific Article
Campagne, A. ; Lee, M.K. ; Zielinski, D. ; Michaud, A. ; Le Corre, S. ; Dingli, F. ; Chen, H. ; Shahidian, L.Z. ; Vassilev, I. ; Servant, N. ; Loew, D. ; Pasmant, E. ; Postel-Vinay, S. ; Wassef, M. ; Margueron, R.
Nat. Commun. 10:348 (2019)
In Drosophila, a complex consisting of Calypso and ASX catalyzes H2A deubiquitination and has been reported to act as part of the Polycomb machinery in transcriptional silencing. The mammalian homologs of these proteins (BAP1 and ASXL1/2/3, respectively), are frequently mutated in various cancer types, yet their precise functions remain unclear. Using an integrative approach based on isogenic cell lines generated with CRISPR/Cas9, we uncover an unanticipated role for BAP1 in gene activation. This function requires the assembly of an enzymatically active BAP1-associated core complex (BAP1. com) containing one of the redundant ASXL proteins. We investigate the mechanism underlying BAP1. com-mediated transcriptional regulation and show that it does not participate in Polycomb-mediated silencing. Instead, our results establish that the function of BAP1. com is to safeguard transcriptionally active genes against silencing by the Polycomb Repressive Complex 1.
Wissenschaftlicher Artikel
Scientific Article
Brockers, K. ; Schneider, R.
Epigenomics 11, 363-366 (2019)
Editorial
Editorial
2018
Foster, B. ; Stolz, P. ; Mulholland, C.B. ; Montoya, A. ; Kramer, H. ; Bultmann, S. ; Bartke, T.
Mol. Cell 72, 739-752 (2018)
The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the "backside'' of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.
Wissenschaftlicher Artikel
Scientific Article
Andrau, J.-C. ; Geyer, M. ; Hermeking, H. ; Hölzel, M. ; Imhof, A. ; Murphy, S. ; Schneider, R.
Poster: 40 years Myc, 50 years Polymerase II, and 65 years Dirk Eick!, 9 November 2018, Planegg-Martinsried. (2018)
Zorzan, E. ; Da Ros, S. ; Giantin, M. ; Zorro Shahidian, L. ; Guerra, G. ; Palumbo, M. ; Sissi, C. ; Dacasto, M.
J. Pharmacol. Exp. Ther. 367, 461-472 (2018)
G-quadruplexes (G4) are nucleic acid secondary structures frequently assumed by G-rich sequences located mostly at telomeres and proto-oncogenes promoters. Recently, we identified, in canine KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) promoter, two G-rich sequences able to fold into G4: d_kit1 and d_kit2_A16. In this study, an anthraquinone (AQ1) and an anthracene derivative (AN6), known to stabilize the G4 structures of the corresponding human h_kit1 and h_kit2, were tested on the canine G4 and in two canine mast cell tumor (MCT) cell lines (C2 and NI-1) to verify their capability to down-regulate KIT expression. The cytotoxicity of AQ1 and AN6 was determined using the Alamar Blue test; also the constitutive expression of KIT and other proto-oncogenes containing G4 structures in their promoter (BCL2, VEGF alpha, VEGFR2, KRAS, and TERT) was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Then the time- and dose-dependent effects of both ligands on target gene expression were assessed by qRT-PCR. All target genes were constitutively expressed up to 96 hours of culture. Both ligands decreased KIT mRNA levels and c-kit protein amount, and AN6 was comparatively fairly more effective. DNA interaction studies and a dual-luciferase gene reporter assay performed on a noncancerous canine cell line (Madin-Darby Canine Kidney cells) proved that this down-regulation was the result of the interaction of AN6 with KIT proximal promoter. Interestingly, our results only partially overlap with those previously obtained in human cell lines, where AQ1 was found as the most effective compound. These preliminary data might suggest AN6 as a promising candidate for the selective targeting of canine KIT-dependent tumors.
Wissenschaftlicher Artikel
Scientific Article
Makowski, M.M. ; Grawe, C. ; Foster, B. ; Nguyen, N.V. ; Bartke, T. ; Vermeulen, M.
Nat. Commun. 9:1653 (2018)
Interaction proteomics studies have provided fundamental insights into multimeric biomolecular assemblies and cell-scale molecular networks. Significant recent developments in mass spectrometry-based interaction proteomics have been fueled by rapid advances in label-free, isotopic, and isobaric quantitation workflows. Here, we report a quantitative protein-DNA and protein-nucleosome binding assay that uses affinity purifications from nuclear extracts coupled with isobaric chemical labeling and mass spectrometry to quantify apparent binding affinities proteome-wide. We use this assay with a variety of DNA and nucleosome baits to quantify apparent binding affinities of monomeric and multimeric transcription factors and chromatin remodeling complexes.
Wissenschaftlicher Artikel
Scientific Article
Nieborak, A. ; Schneider, R.
Mol. Metab., DOI: 10.1016/j.molmet.2018.01.007 (2018)
Background: To maintain homeostasis, cells need to coordinate the expression of their genes. Epigenetic mechanisms controlling transcription activation and repression include DNA methylation and post-translational modifications of histones, which can affect the architecture of chromatin and/or create 'docking platforms' for multiple binding proteins. These modifications can be dynamically set and removed by various enzymes that depend on the availability of key metabolites derived from different intracellular pathways. Therefore, small metabolites generated in anabolic and catabolic processes can integrate multiple external and internal stimuli and transfer information on the energetic state of a cell to the transcriptional machinery by regulating the activity of chromatin-modifying enzymes. Scope of review: This review provides an overview of the current literature and concepts on the connections and crosstalk between key cellular metabolites, enzymes responsible for their synthesis, recycling, and conversion and chromatin marks controlling gene expression. Major conclusions: Whereas current evidence indicates that many chromatin-modifying enzymes respond to alterations in the levels of their cofactors, cosubstrates, and inhibitors, the detailed molecular mechanisms and functional consequences of such processes are largely unresolved. A deeper investigation of mechanisms responsible for altering the total cellular concentration of particular metabolites, as well as their nuclear abundance and accessibility for chromatin-modifying enzymes, will be necessary to better understand the crosstalk between metabolism, chromatin marks, and gene expression.
Review
Review
2017
Kebede, A.F. ; Nieborak, A. ; Shahidian, L.Z. ; Le Gras, S. ; Richter, F.M. ; Gomez, D.A. ; Baltissen, M.P. ; Meszaros, G. ; Magliarelli, H.F. ; Taudt, A. ; Margueron, R. ; Colomé-Tatché, M. ; Ricci, R. ; Daujat, S. ; Vermeulen, M. ; Mittler, G. ; Schneider, R.
Nat. Struct. Mol. Biol. 24, 1048–1056 (2017)
Histones are highly covalently modified, but the functions of many of these modifications remain unknown. In particular, it is unclear how histone marks are coupled to cellular metabolism and how this coupling affects chromatin architecture. We identified histone H3 Lys14 (H3K14) as a site of propionylation and butyrylation in vivo and carried out the first systematic characterization of histone propionylation. We found that H3K14pr and H3K14bu are deposited by histone acetyltransferases, are preferentially enriched at promoters of active genes and are recognized by acylation-state-specific reader proteins. In agreement with these findings, propionyl-CoA was able to stimulate transcription in an in vitro transcription system. Notably, genome-wide H3 acylation profiles were redefined following changes to the metabolic state, and deletion of the metabolic enzyme propionyl-CoA carboxylase altered global histone propionylation levels. We propose that histone propionylation, acetylation and butyrylation may act in combination to promote high transcriptional output and to couple cellular metabolism with chromatin structure and function.
Wissenschaftlicher Artikel
Scientific Article
Izzo, A. ; Ziegler-Birling, C. ; Hill, P.W.S. ; Brondani, L. ; Hajkova, P. ; Torres-Padilla, M.E. ; Schneider, R.
J. Cell Biol. 216, 3017-3028 (2017)
Crown. In mammals, histone H1 consists of a family of related proteins, including five replication-dependent (H1.1-H1.5) and two replication-independent (H1.10 and H1.0) subtypes, all expressed in somatic cells. To systematically study the expression and function of H1 subtypes, we generated knockin mouse lines in which endogenous H1 subtypes are tagged. We focused on key developmental periods when epigenetic reprogramming occurs: early mouse embryos and primordial germ cell development. We found that dynamic changes in H1 subtype expression and localization are tightly linked with chromatin remodeling and might be crucial for transitions in chromatin structure during reprogramming. Although all somatic H1 subtypes are present in the blastocyst, each stage of preimplantation development is characterized by a different combination of H1 subtypes. Similarly, the relative abundance of somatic H1 subtypes can distinguish male and female chromatin upon sex differentiation in developing germ cells. Overall, our data provide new insights into the chromatin changes underlying epigenetic reprogramming. We suggest that distinct H1 subtypes may mediate the extensive chromatin remodeling occurring during epigenetic reprogramming and that they may be key players in the acquisition of cellular totipotency and the establishment of specific cellular states.
Wissenschaftlicher Artikel
Scientific Article
Scahill, C.M. ; Digby, Z. ; Sealy, I.M. ; Wojciechowska, S. ; White, R.J. ; Collins, J.E. ; Stemple, D.L. ; Bartke, T. ; Mathers, M.E. ; Patton, E.E. ; Busch-Nentwich, E.M.
PLoS Genet. 13:e1006959 (2017)
KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology.
Wissenschaftlicher Artikel
Scientific Article
Col, E. ; Hoghoughi, N. ; Dufour, S. ; Penin, J. ; Koskas, S. ; Faure, V. ; Ouzounova, M. ; Hernandez-Vargash, H. ; Reynoird, N. ; Daujat, S. ; Folco, E.J. ; Vigneron, M. ; Schneider, R. ; Verdel, A. ; Khochbin, S. ; Herceg, Z. ; Caron, C. ; Vourc'h, C.
Sci. Rep. 7:5418 (2017)
The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin. Both events are under the control of Heat Shock Factor I (HSF1). Here we show that under heat shock, HSF1 recruits major cellular acetyltransferases, GCN5, TIP60 and p300 to pericentric heterochromatin leading to a targeted hyperacetylation of pericentric chromatin. Redistribution of histone acetylation toward pericentric region in turn directs the recruitment of Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, BRD4, which are required for satellite III transcription by RNAP II. Altogether we uncover here a critical role for HSF1 in stressed cells relying on the restricted use of histone acetylation signaling over pericentric heterochromatin (HC).
Wissenschaftlicher Artikel
Scientific Article
Chendeb, M. ; Schneider, R. ; Davidson, I. ; Fadloun, A.
Oncotarget 8, 38239-38250 (2017)
In gene therapy, effective and selective suicide gene expression is crucial. We exploited the endogenous Long INterspersed Element-1 (L1) machinery often reactivated in human cancers to integrate the Herpes Simplex Virus Thymidine Kinase (HSV-TK) suicide gene selectively into the genome of cancer cells. We developed a plasmid-based system directing HSV-TK expression only when reverse transcribed and integrated in the host genome via the endogenous L1 ORF1/2 proteins and an Alu element. Delivery of these new constructs into cells followed by Ganciclovir (GCV) treatment selectively induced mortality of L1 ORF1/2 protein expressing cancer cells, but had no effect on primary cells that do not express L1 ORF1/2. This novel strategy for selective targeting of tumour cells provides high tolerability as the HSV-TK gene cannot be expressed without reverse transcription and integration, and high selectivity as these processes take place only in cancer cells expressing high levels of functional L1 ORF1/2.
Wissenschaftlicher Artikel
Scientific Article
2016
Pradeepa, M.M. ; Grimes, G.R. ; Kumar, Y. ; Olley, G. ; Taylor, G.C.A. ; Schneider, R. ; Bickmore, W.A.
Nat. Genet. 48, 681-686 (2016)
Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes(1). These modifications include acetylation of histone H3 at lysine 27 (H3K27ac), which blocks Polycomb-mediated trimethylation of H3K27 (H3K27me3)(2). H3K27ac is also widely used to identify active enhancers(3,4), and the assumption has been that profiling H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of histone H3 (lysine 64 (H3K64ac) and lysine 122 (H3K122ac)) marks active gene promoters and also a subset of active enhancers. Moreover, we find a new class of active functional enhancers that is marked by H3K122ac but lacks H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than has previously been considered.
Wissenschaftlicher Artikel
Scientific Article
Bheda, P. ; Jing, H. ; Wolberger, C. ; Lin, H.
Annu. Rev. Biochem. 85, 405-429 (2016)
Sirtuins are NAD(+)-dependent enzymes universally present in all organisms, where they play central roles in regulating numerous biological processes. Although early studies showed that sirtuins deacetylated lysines in a reaction that consumes NAD(+), more recent studies have revealed that these enzymes can remove a variety of acyl-lysine modifications. The specificities for varied acyl modifications may thus underlie the distinct roles of the different sirtuins within a given organism. Additional contributions to sirtuin function may also result from structural variations both within and flanking the conserved catalytic domain. This review summarizes the structure, chemistry, and substrate specificity of sirtuins with a focus on how different sirtuins recognize distinct substrates and thus carry out specific functions. Expected final online publication date for the Annual Review of Biochemistry Volume 85 is June 02, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Review
Review
Lawrence, M. ; Daujat, S. ; Schneider, R.
Trends Genet. 32, 42-56 (2016)
The DNA of each cell is wrapped around histone octamers, forming so-called 'nucleosomal core particles'. These histone proteins have tails that project from the nucleosome and many residues in these tails can be post-translationally modified, influencing all DNA-based processes, including chromatin compaction, nucleosome dynamics, and transcription. In contrast to those present in histone tails, modifications in the core regions of the histones had remained largely uncharacterised until recently, when some of these modifications began to be analysed in detail. Overall, recent work has shown that histone core modifications can not only directly regulate transcription, but also influence processes such as DNA repair, replication, stemness, and changes in cell state. In this review, we focus on the most recent developments in our understanding of histone modifications, particularly those on the lateral surface of the nucleosome. This region is in direct contact with the DNA and is formed by the histone cores. We suggest that these lateral surface modifications represent a key insight into chromatin regulation in the cell. Therefore, lateral surface modifications form a key area of interest and a focal point of ongoing study in epigenetics.
Review
Review
Izzo, A. ; Schneider, R.
Biochim. Biophys. Acta-Gene Regul. Mech. 1859, 486-495 (2016)
BACKGROUND: Linker histone H1 is a structural component of chromatin. It exists as a family of related proteins known as variants and/or subtypes. H1.1, H1.2, H1.3, H1.4 and H1.5 are present in most somatic cells, whereas other subtypes are mainly expressed in more specialized cells. SCOPE OF REVIEW: H1 subtypes have been shown to have unique functions in chromatin structure and dynamics. This can occur at least in part via specific post-translational modifications of distinct H1 subtypes. However, while core histone modifications have been extensively studied, our knowledge of H1 modifications and their molecular functions has remained for a long time limited to phosphorylation. In this review we discuss the current state of knowledge of linker histone H1 modifications and where possible highlight functional differences in the modifications of distinct H1 subtypes. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: H1 histones are intensely post-translationally modified. These modifications are located in the N- and C-terminal tails as well as within the globular domain. Recently, advanced mass spectrometrical analysis revealed a large number of novel histone H1 subtype specific modification sites and types. H1 modifications include phosphorylation, acetylation, methylation, ubiquitination, and ADP ribosylation. They are involved in the regulation of all aspects of linker histone functions, however their mechanism of action is often only poorly understood. Therefore systematic functional characterization of H1 modifications will be necessary in order to better understand their role in gene regulation as well as in higher-order chromatin structure and dynamics. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan.
Wissenschaftlicher Artikel
Scientific Article
Kabra, D.G. ; Pfuhlmann, K. ; García-Cáceres, C. ; Schriever, S.C. ; Casquero García, V. ; Kebede, A.F. ; Fuente-Martin, E. ; Trivedi, C. ; Heppner, K. ; Uhlenhaut, N.H. ; Legutko, B. ; Kabra, U.D. ; Gao, Y. ; Yi, C.-X. ; Quarta, C. ; Clemmensen, C. ; Finan, B. ; Müller, T.D. ; Meyer, C.W. ; Paez-Pereda, M. ; Stemmer, K. ; Woods, S.C. ; Perez-Tilve, D. ; Schneider, R. ; Olson, E.N. ; Tschöp, M.H. ; Pfluger, P.T.
Nat. Commun. 7:10782 (2016)
Hypothalamic leptin signalling has a key role in food intake and energy-balance control and is often impaired in obese individuals. Here we identify histone deacetylase 5 (HDAC5) as a regulator of leptin signalling and organismal energy balance. Global HDAC5 KO mice have increased food intake and greater diet-induced obesity when fed high-fat diet. Pharmacological and genetic inhibition of HDAC5 activity in the mediobasal hypothalamus increases food intake and modulates pathways implicated in leptin signalling. We show HDAC5 directly regulates STAT3 localization and transcriptional activity via reciprocal STAT3 deacetylation at Lys685 and phosphorylation at Tyr705. In vivo, leptin sensitivity is substantially impaired in HDAC5 loss-of-function mice. Hypothalamic HDAC5 overexpression improves leptin action and partially protects against HFD-induced leptin resistance and obesity. Overall, our data suggest that hypothalamic HDAC5 activity is a regulator of leptin signalling that adapts food intake and body weight to our dietary environment.
Wissenschaftlicher Artikel
Scientific Article
Izzo, A. ; Schneider, R.
Genome Biol. 17:8 (2016)
By performing high-throughput chromosome conformation capture analyses in embryonic stem cells depleted of the linker histone H1, Geeven and colleagues have uncovered exciting new evidence concerning a role for this histone in modulating three-dimensional genome architecture and chromatin organization. Please see link to Research article: http://www.genomebiology.com/2015/16/1/289
Wissenschaftlicher Artikel
Scientific Article
Quarta, C. ; Schneider, R. ; Tschöp, M.H.
Cell 164, 341-342 (2016)
Heritable epigenetic mechanisms might contribute to the worldwide increase in the prevalence of obesity. Dalgaard et al. identify an epigenetic molecular switch that controls body weight control. The discovery suggests the existence of mammalian polyphenism in energy metabolism and might have implications for strategies to limit the obesity epidemic.
Editorial
Editorial
2015
Hergeth, S.P. ; Schneider, R.
EMBO Rep. 16, 1439-1453 (2015)
The linker histone H1 family members are a key component of chromatin and bind to the nucleosomal core particle around the DNA entry and exit sites. H1 can stabilize both nucleosome structure and higher-order chromatin architecture. In general, H1 molecules consist of a central globular domain with more flexible tail regions at both their N- and C-terminal ends. The existence of multiple H1 subtypes and a large variety of posttranslational modifications brings about a considerable degree of complexity and makes studying this protein family challenging. Here, we review recent progress in understanding the function of linker histones and their subtypes beyond their role as merely structural chromatin components. We summarize current findings on the role of H1 in heterochromatin formation, transcriptional regulation and embryogenesis with a focus on H1 subtypes and their specific modifications.
Wissenschaftlicher Artikel
Scientific Article
Cohen, I.R. ; Peleg, R. ; Voronov, E. ; Tomas, M. ; Tudor, C. ; Wegner, M. ; Brodabi, L. ; Freudenberg, M. ; Mittler, G. ; Ferrando-May, E. ; Dinarello, C.A. ; Apte, R.N. ; Schneider, R.
Sci. Rep. 5:14756 (2015)
Environmental signals can be translated into chromatin changes, which alter gene expression. Here we report a novel concept that cells can signal chromatin damage from the nucleus back to the surrounding tissue through the cytokine interleukin-1alpha (IL-1α). Thus, in addition to its role as a danger signal, which occurs when the cytokine is passively released by cell necrosis, IL-1α could directly sense DNA damage and act as signal for genotoxic stress without loss of cell integrity. Here we demonstrate localization of the cytokine to DNA-damage sites and its subsequent secretion. Interestingly, its nucleo-cytosolic shuttling after DNA damage sensing is regulated by histone deacetylases (HDAC) and IL-1α acetylation. To demonstrate the physiological significance of this newly discovered mechanism, we used IL-1α knockout mice and show that IL-1α signaling after UV skin irradiation and DNA damage is important for triggering a sterile inflammatory cascade in vivo that contributes to efficient tissue repair and wound healing.
Wissenschaftlicher Artikel
Scientific Article
Kebede, A.F. ; Schneider, R. ; Daujat, S.
FEBS J. 282, 1658-1674 (2015)
N-terminal tails of histones are easily accessible outside of the nucleosomal core particle and post-translational modifications (PTMs) of these tails have been the focus of attention in the past 15-20 years. By recruiting (or excluding) specific readers, histone modifications can regulate chromatin dynamics and, by extension, DNA-dependent processes. However, until very recently, the direct impact of histone PTMs on nucleosome structure and thus on chromatin function has remained somewhat elusive. Recent findings of novel sites and types of histone PTMs located within the globular domain of histones and, in particular, on the lateral surface of the histone octamer have changed this. As a result of their structurally important location in close proximity to the DNA molecule, this new class of histone PTMs can have a direct impact on chromatin function. Depending on their precise position at the nucleosome lateral surface (e.g. near the DNA entry/exit sites or in the dyad region), histone PTMs can regulate nucleosome structure and/or stability differently. We review recent progress on how histone PTMs can influence DNA unwrapping and/or nucleosome disassembly and shed light on how these types of novel modifications contribute mechanistically to the regulation of transcriptional activity.
Wissenschaftlicher Artikel
Scientific Article
Ziegler-Birling, C. ; Daujat, S. ; Schneider, R. ; Torres-Padilla, M.E.
Epigenetics, DOI: 10.1080/15592294.2015.1103424 (2015)
In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of three key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all three acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming.
Wissenschaftlicher Artikel
Scientific Article
Sijare, F. ; Geißler, A.L. ; Fichter, C.D. ; Hergeth, S.P. ; Bogatyreva, L. ; Hauschke, D. ; Schneider, R. ; Werner, M. ; Lassmann, S.
Virchows Arch. 466, 503-515 (2015)
Experimental model systems identified phosphorylation of linker histone variant H1.4 at Ser 27 (H1.4S27p) as a novel mitotic mark set by Aurora B kinase. Here, we examined expression of Aurora B and H1.4S27p in colorectal carcinoma (CRC) cell lines (HCT116, DLD1, Caco-2, HT29) and tissue specimens (n = 36), in relation to microsatellite instability (MSI) status and ploidy. In vitro, Aurora B (pro-/meta-/anaphase) and H1.4S27p (pro-/metaphase) were localized in mitotic figures. The proportion of labeled mitoses was significantly different between cell lines for Aurora B (p = 0.019) but not for H1.4S27p (p = 0.879). For Aurora B, these differences were not associated with an altered Aurora B gene copy number (FISH) or messenger RNA (mRNA) expression level (qRT-PCR). Moreover, Aurora B expression and H1.4S27 phosphorylation were no longer coordinated during metaphase in aneuploid HT29 cells (p = 0.039). In CRCs, immunoreactivity for Aurora B or H1.4S27p did not correlate with T- or N-stage, grade, or MSI status. However, metaphase labeling of H1.4S27p was significantly higher in diploid than in aneuploid CRCs (p = 0.011). Aurora B was significantly correlated with H1.4S27p-positive metaphases in MSI (p = 0.010) or diploid (p = 0.003) CRCs. Finally, combined classification of MSI status and ploidy revealed a significant positive correlation of Aurora B with H1.4S27p in metaphases of diploid/MSI (p = 0.010) and diploid/microsatellite-stable (MSS; p = 0.031) but not of aneuploid/MSS (p = 0.458) CRCs. The present study underlines the functional link of Aurora B expression and H1.4S27p during specific phases of mitosis in diploid and/or MSI-positive CRCs in vitro and in situ. Importantly, the study shows that the coordination between Aurora B expression and phosphorylation of H1.4 at Ser 27 is lost in cycling aneuploid CRC cells.
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Scientific Article
2014
di Cerbo, V. ; Mohn, F. ; Ryan, D.P. ; Montellier, E. ; Kacem, S. ; Tropberger, P. ; Kallis, E. ; Holzner, M. ; Hoerner, L. ; Feldmann, A. ; Richter, F.M. ; Bannister, A.J. ; Mittler, G. ; Michaelis, J. ; Khochbin, S. ; Feil, R. ; Schuebeler, D. ; Owen-Hughes, T. ; Daujat, S. ; Schneider, R.
eLife 3:e01632 (2014)
Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states.
Wissenschaftlicher Artikel
Scientific Article
Biterge, B. ; Richter, F.M. ; Mittler, G. ; Schneider, R.
Sci. Rep. 4:6674 (2014)
Histone modifications play crucial roles in modulating chromatin function and transcriptional activity. Due to their long half-life, histones can, in addition to post-translational modifications, also accumulate spontaneous chemical alterations, which can affect their functionality and require either protein repair or degradation. One of the major sources of such protein damage or ageing is the conversion of aspartate into isoaspartate residues that can then be methylated. Here, we characterize a novel histone modification, the methylation of histone H4 at aspartate 24 (H4D24me). We generated H4D24me specific antibodies and showed that H4D24me is ubiquitously present in different mouse and human cells. Our in vitro and in vivo data identified PCMT1 (Protein L-isoaspartate O-methyltransferase), an enzyme involved in protein repair, as a novel H4D24 specific histone methyltransferase. Furthermore, we demonstrated that VprBP (HIV-1 viral protein R (Vpr)-binding protein), a chromo domain-containing protein, specifically recognizes H4D24me potentially implicating H4D24me in H4 degradation. Thus, this work links for the first time a histone modification with histone protein aging and histone homeostasis, suggesting novel functions for histone modifications beyond transcriptional regulation.
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Scientific Article
Bheda, P. ; Schneider, R.
Trends Cell Biol. 24, 712-723 (2014)
Mechanistically, how epigenetic states are inherited through cellular divisions remains an important open question in the chromatin field and beyond. Defining the heritability of epigenetic states and the underlying chromatin-based mechanisms within a population of cells is complicated due to cell heterogeneity combined with varying levels of stability of these states; thus, efforts must be focused toward single-cell analyses. The approaches presented here constitute the forefront of epigenetics research at the single-cell level using classic and innovative methods to dissect epigenetics mechanisms from the limited material available in a single cell. This review further outlines exciting future avenues of research to address the significance of epigenetic heterogeneity and the contributions of microfluidics technologies to single-cell isolation and analysis.
Wissenschaftlicher Artikel
Scientific Article
Biterge, B. ; Schneider, R.
Cell Tissue Res. 356, 457-466 (2014)
Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.
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Scientific Article
2013
Tropberger, P. ; Pott, S. ; Keller, C. ; Kamieniarz-Gdula, K. ; Caron, M. ; Richter, F.M. ; Li, G. ; Mittler, G. ; Liu, E.T. ; Bühler, M. ; Margueron, R. ; Schneider, R.
Cell 152, 859-872 (2013)
Histone modifications are key regulators of chromatin function. However, little is known to what extent histone modifications can directly impact on chromatin. Here, we address how a modification within the globular domain of histones regulates chromatin function. We demonstrate that H3K122ac can be sufficient to stimulate transcription and that mutation of H3K122 impairs transcriptional activation, which we attribute to a direct effect of H3K122ac on histone-DNA binding. In line with this, we find that H3K122ac defines genome-wide genetic elements and chromatin features associated with active transcription. Furthermore, H3K122ac is catalyzed by the coactivators p300/CBP and can be induced by nuclear hormone receptor signaling. Collectively, this suggests that transcriptional regulators elicit their effects not only via signaling to histone tails but also via direct structural perturbation of nucleosomes by directing acetylation to their lateral surface.
Wissenschaftlicher Artikel
Scientific Article
Izzo, A. ; Kamieniarz-Gdula, K. ; Ramírez, F. ; Noureen, N. ; Kind, J. ; Manke, T. ; van Steensel, B. ; Schneider, R.
Cell Rep. 3, 2142-2154 (2013)
Human cells contain five canonical, replication-dependent somatic histone H1 subtypes (H1.1, H1.2, H1.3, H1.4, and H1.5). Although they are key chromatin components, the genomic distribution of the H1 subtypes is still unknown, and their role in chromatin processes has thus far remained elusive. Here, we map the genomic localization of all somatic replication-dependent H1 subtypes in human lung fibroblasts using an integrative DNA adenine methyltransferase identification (DamID) analysis. We find in general that H1.2 to H1.5 are depleted from CpG-dense regions and active regulatory regions. H1.1 shows a DamID binding profile distinct from the other subtypes, suggesting a unique function. H1 subtypes can mark specific domains and repressive regions, pointing toward a role for H1 in three-dimensional genome organization. Our work integrates H1 subtypes into the epigenome maps of human cells and provides a valuable resource to refine our understanding of the significance of H1 and its heterogeneity in the control of genome function.
Wissenschaftlicher Artikel
Scientific Article
Tropberger, P. ; Schneider, R.
Nat. Struct. Mol. Biol. 20, 657-661 (2013)
Histones have two structurally and functionally distinct domains: globular domains forming the nucleosomal core around which DNA is wrapped and unstructured tails protruding from the nucleosomal core. Whereas post-translational modifications (PTMs) in histone tails are well studied, much less is currently known about histone-core PTMs. Many core PTMs map to residues located on the lateral surface of the histone octamer, close to the DNA, and they have the potential to alter intranucleosomal histone-DNA interactions. Here we discuss recent advances in understanding the function of lateral-surface PTMs. Whereas modifications in the histone tails might have limited structural impact on the nucleosome itself and function as signals to recruit specific binding proteins, PTMs in the lateral surface can have a direct structural effect on nucleosome and chromatin dynamics, even in the absence of specific binding proteins, which adds a twist to the debate on the functionality and causality of PTMs.
Wissenschaftlicher Artikel
Scientific Article
di Cerbo, V. ; Schneider, R.
Brief. Funct. Genomic. Proteomic. 12, 231-243 (2013)
Lysine N-ε-acetylation is a post-translational modification that regulates the function of histone and non-histone proteins. In several malignancies, histone acetyltransferase (HAT) activities are disturbed as a consequence of various genetic or epigenetic alterations. In particular, HATs can function as tumor suppressors, helping cells control cellular proliferation and cell cycle, and also as oncogenes, because abnormal acetylation can activate malignant proteins and contribute to cancer. An impaired acetylation profile can be indicative of a pathological process, and thus evaluation of histone acetylation could be used as a predictive index of patient survival or therapy outcome. Therefore, epigenetic therapy might be a very effective strategy to defeat cancer. With the use of histone deacetylase inhibitors and acetylation modulators (e.g. HAT inhibitors, bromodomain inhibitors), we are paving the way for a future epigenetic drug control of human diseases.
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Scientific Article
Fejer, G. ; Wegner, M.D. ; Györy, I. ; Cohen, I.R. ; Engelhard, P. ; Voronov, E. ; Manke, T. ; Ruzsics, Z. ; Dölken, L. ; Prazeres da Costa, O. ; Branzk, N. ; Huber, M. ; Prasse, A. ; Schneider, R. ; Apte, R.N. ; Galanos, C. ; Freudenberg, M.A.
Proc. Natl. Acad. Sci. U.S.A. 110, E2191-E2198 (2013)
Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF-induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.
Wissenschaftlicher Artikel
Scientific Article
Waldmann, T. ; Schneider, R.
Curr. Opin. Cell Biol. 25, 184-189 (2013)
Cancer is one of the most common human diseases. It is long known that mutations in key regulator genes are hallmarks of all cancer types. Apart from these classical genetic pathways there is more and more evidence that also epigenetic alterations are crucially involved in tumourigenesis. In this review we discuss and summarise recent findings of mechanisms responsible for cancer formation apart from the classic genetic mutations. Furthermore, we show how epigenetic and genetic mechanisms could depend on each other and contribute together to cancer formation. We focus mainly on post-translational histone modifications since they are one of the major epigenetic mechanisms regulating gene expression and when they are imbalanced this can result in cancer.
Wissenschaftlicher Artikel
Scientific Article
Lange, U.C. ; Siebert, S. ; Wossidlo, M. ; Weiss, T. ; Ziegler-Birling, C. ; Walter, J. ; Torres-Padilla, M.E. ; Daujat, S. ; Schneider, R.
Nat. Commun. 4:2233 (2013)
To ensure genome stability, pericentromeric regions are compacted in a dense heterochromatic structure through a combination of specific 'epigenetic' factors and modifications. A cascadal pathway is responsible for establishing pericentromeric chromatin involving chromatin modifiers and 'readers', such as H3K9 histone methyltransferases (Suv)39h and heterochromatin protein 1. Here we define how H3K64me3 on the lateral surface of the histone octamer integrates within the heterochromatinization cascade. Our data suggest that enrichment of H3K64me3 at pericentromeric chromatin foci is dependent on H3K9me3 but independent of a number of central factors such as heterochromatin protein 1, DNA methyltransferases and Suv4-20h histone methyltransferases. Our results support a model in which pericentromeric heterochromatin foci are formed along distinct pathways upon H3K9 trimethylation, involving H3K64me3 to potentially stabilize DNA-histone interactions, as well as sequential recruitment of repressive histone tail and DNA modifications. We hence suggest that multiple mechanisms ensure heterochromatin integrity at pericentromeres, with H3K64me3 as an important factor.
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Scientific Article
2012
Kamieniarz, K. ; Izzo, A. ; Dundr, M. ; Tropberger, P. ; Ozretic, L. ; Kirfel, J. ; Scheer, E. ; Tropel, P. ; Wisniewski, J.R. ; Tora, L. ; Viville, S. ; Buettner, R. ; Schneider, R.
Genes Dev. 26, 797-802 (2012)
The linker histone H1 is a key player in chromatin organization, yet our understanding of the regulation of H1 functions by post-translational modifications is very limited. We provide here the first functional characterization of H1 acetylation. We show that H1.4K34 acetylation (H1.4K34ac) is mediated by GCN5 and is preferentially enriched at promoters of active genes, where it stimulates transcription by increasing H1 mobility and recruiting a general transcription factor. H1.4K34ac is dynamic during spermatogenesis and marks undifferentiated cells such as induced pluripotent stem (iPS) cells and testicular germ cell tumors. We propose a model for H1.4K34ac as a novel regulator of chromatin function with a dual role in transcriptional activation.
Wissenschaftlicher Artikel
Scientific Article
Öberg, C. ; Izzo, A. ; Schneider, R. ; Wrange, Ö. ; Belikov, S.
J. Mol. Biol. 419, 183-197 (2012)
Linker histone H1 is located on the surface of the nucleosome where it interacts with the linker DNA region and stabilizes the 30-nm chromatin fiber. Vertebrates have several different, relatively conserved subtypes of H1; however, the functional reason for this is unclear. We have previously shown that H1 can be reconstituted in Xenopus oocytes, cells that lack somatic H1, by cytosolic mRNA injection and incorporated into in vivo assembled chromatin. Using this assay, we have expressed individual H1 subtypes in the oocytes to study their effect on chromatin structure using nucleosomal repeat length (NRL) as readout. We have compared chicken differentiation-specific histone H5, Xenopus differentiation-specific xH1(0) and the somatic variant xH1A as well as the ubiquitously expressed human somatic subtypes hH1.2, hH1.3, hH1.4 and hH1.5. This shows that all subtypes, except for human H1.5, result in a saturable increase in NRL. hH1.4 results in an increase of approximately 13-20 bp as does xH1(0) and xH1A. chH5 gives rise to the same or slightly longer increase compared to hH1.4. Interestingly, both hH1.2 and hH1.3 show a less extensive increase of only 4.5-7 bp in the NRL, thus yielding the shortest increase of the studied subtypes. We show for the first time in an in vivo system lacking H1 background that ubiquitously expressed and redundant H1 subtypes that coexist in most types of cells of higher eukaryotes differ in their effects on the nucleosomal spacing in vivo. This suggests that H1 subtypes have different roles in the organization and functioning of the chromatin fiber.
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Scientific Article
Peifer, M. ; Fernández-Cuesta, L. ; Sos, M.L. ; George, J. ; Seidel, D. ; Kasper, L.H. ; Plenker, D. ; Leenders, F. ; Sun, R. ; Zander, T. ; Menon, R. ; Koker, M. ; Dahmen, I. ; Müller, C. ; di Cerbo, V. ; Schildhaus, H.U. ; Altmüller, J. ; Baessmann, I. ; Becker, C. ; de Wilde, B. ; Vandesompele, J. ; Böhm, D. ; Ansén, S. ; Gabler, F. ; Wilkening, I. ; Heynck, S. ; Heuckmann, J.M. ; Lu, X. ; Carter, S.L. ; Cibulskis, K. ; Banerji, S. ; Getz, G. ; Park, K.S. ; Rauh, D. ; Grütter, C. ; Fischer, M. ; Pasqualucci, L. ; Wright, G. ; Wainer, Z. ; Russell, P. ; Petersen, I. ; Chen, Y. ; Stoelben, E. ; Ludwig, C. ; Schnabel, P.A. ; Hoffmann, H. ; Muley, T. ; Brockmann, M.M. ; Engel-Riedel, W. ; Muscarella, L.A. ; Fazio, V.M. ; Groen, H.J.M. ; Timens, W. ; Sietsma, H. ; Thunnissen, E. ; Smit, E. ; Heideman, D.A. ; Snijders, P.J. ; Cappuzzo, F. ; Ligorio, C. ; Damiani, S. ; Field, J. ; Solberg, S. ; Brustugun, O.T. ; Lund-Iversen, M. ; Sänger, J. ; Clement, J.H. ; Soltermann, A. ; Moch, H. ; Weder, W. ; Solomon, B. ; Soria, J.C. ; Validire, P. ; Besse, B. ; Brambilla, E. ; Brambilla, C. ; Lantuejoul, S. ; Lorimier, P. ; Schneider, P.M. ; Hallek, M. ; Pao, W. ; Meyerson, M. ; Sage, J. ; Shendure, J. ; Schneider, R. ; Büttner, R. ; Wolf, J. ; Nürnberg, P. ; Perner, S. ; Heukamp, L.C. ; Brindle, P.K. ; Haas, S. ; Thomas, R.K.
Nat. Genet. 44, 1104-1110 (2012)
Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.
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Scientific Article
Talbert, P.B. ; Ahmad, K. ; Almouzni, G. ; Ausió, J. ; Berger, F. ; Bhalla, P.L. ; Bonner, W.M. ; Cande, W.Z. ; Chadwick, B.P. ; Chan, S.W. ; Cross, G.A. ; Cui, L. ; Dimitrov, S.I. ; Doenecke, D. ; Eirin-López, J.M. ; Gorovsky, M.A. ; Hake, S.B. ; Hamkalo, B.A. ; Holec, S. ; Jacobsen, S.E. ; Kamieniarz, K. ; Khochbin, S. ; Ladurner, A.G. ; Landsman, D. ; Latham, J.A. ; Loppin, B. ; Malik, H.S. ; Marzluff, W.F. ; Pehrson, J.R. ; Postberg, J. ; Schneider, R. ; Singh, M.B. ; Smith, M.M. ; Thompson, E.E. ; Torres-Padilla, M.E. ; Tremethick, D.J. ; Turner, B.M. ; Waterborg, J.H. ; Wollmann, H. ; Yelagandula, R. ; Zhu, B.M. ; Henikoff, S.
Epigenetics Chromatin 5:7 (2012)
Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.
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Scientific Article
2011
Hergeth, S.P. ; Dundr, M. ; Tropberger, P. ; Zee, B.M. ; Garcia, B.A. ; Daujat, S. ; Schneider, R.
J. Cell Sci. 124, 1623-1628 (2011)
The linker histone H1 plays an essential role in maintaining and establishing higher-order chromatin structure. As with core histones, histone H1 is also extensively covalently modified. We showed previously that phosphorylation of S27 in human histone H1.4 (H1.4S27-P), prevents binding of heterochromatin protein 1 (HP1) family members (officially known as chromobox protein homologs) to the neighboring dimethylated K26. Here, we present the first functional characterization of H1.4S27-P in vivo and in vitro. We show that H1.4S27 phosphorylation is cell-cycle-regulated and its levels peak on metaphase chromosomes. We identify further Aurora B as the kinase phosphorylating H1.4S27. We demonstrate that histone H1.4 is the only somatic linker histone variant targeted by Aurora B and that Aurora B exclusively phosphorylates S27. Adjacent K26 dimethylation can regulate Aurora B activity towards S27, uncovering a crosstalk between these modifications. Finally, our fluorescence recovery after photobleaching (FRAP) analysis on histone H1.4 mutants suggests a role of S27 phosphorylation in the regulation of histone H1.4 mobility and chromatin binding in mitosis.
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Scientific Article
Kappes, F. ; Waldmann, T. ; Mathew, V. ; Yu, J. ; Zhang, L. ; Khodadoust, M.S. ; Chinnaiyan, A.M. ; Luger, K. ; Erhardt, S. ; Schneider, R. ; Markovitz, D.M.
Genes Dev. 25, 673-678 (2011)
Heterochromatin integrity is crucial for genome stability and regulation of gene expression, but the factors involved in mammalian heterochromatin biology are only incompletely understood. Here we identify the oncoprotein DEK, an abundant nuclear protein with a previously enigmatic in vivo function, as a Suppressor of Variegation [Su(var)] that is crucial to global heterochromatin integrity. We show that DEK interacts directly with Heterochromatin Protein 1 α (HP1α) and markedly enhances its binding to trimethylated H3K9 (H3K9me3), which is key for maintaining heterochromatic regions. Loss of Dek in Drosophila leads to a Su(var) phenotype and global reduction in heterochromatin. Thus, these findings show that DEK is a key factor in maintaining the balance between heterochromatin and euchromatin in vivo.
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Scientific Article
Cohen, I.R. ; Poreba, E. ; Kamieniarz, K. ; Schneider, R.
Genes Cancer 2, 631-647 (2011)
Covalent modifications of histones can regulate all DNA-dependent processes. In the last few years, it has become more and more evident that histone modifications are key players in the regulation of chromatin states and dynamics as well as in gene expression. Therefore, histone modifications and the enzymatic machineries that set them are crucial regulators that can control cellular proliferation, differentiation, plasticity, and malignancy processes. This review discusses the biology and biochemistry of covalent histone posttranslational modifications (PTMs) and evaluates the dual role of their modifiers in cancer: as oncogenes that can initiate and amplify tumorigenesis or as tumor suppressors.
Wissenschaftlicher Artikel
Scientific Article
Waldmann, T. ; Izzo, A. ; Kamieniarz, K. ; Richter, F.M. ; Vogler, C. ; Sarg, B. ; Lindner, H. ; Young, N.L. ; Mittler, G. ; Garcia, B.A. ; Schneider, R.
Epigenetics Chromatin 4:11 (2011)
BACKGROUND: Covalent histone modifications are central to all DNA-dependent processes. Modifications of histones H3 and H4 are becoming well characterised, but knowledge of how H2A modifications regulate chromatin dynamics and gene expression is still very limited. RESULTS: To understand the function of H2A modifications, we performed a systematic analysis of the histone H2A methylation status. We identified and functionally characterised two new methylation sites in H2A: R11 (H2AR11) and R29 (H2AR29). Using an unbiased biochemical approach in combination with candidate assays we showed that protein arginine methyltransferase (PRMT) 1 and PRMT6 are unique in their ability to catalyse these modifications. Importantly we found that H2AR29me2 is specifically enriched at genes repressed by PRMT6, implicating H2AR29me2 in transcriptional repression. CONCLUSIONS: Our data establishes R11 and R29 as new arginine methylation sites in H2A. We identified the specific modifying enzymes involved, and uncovered a novel functional role of H2AR29me2 in gene silencing in vivo. Thus this work reveals novel insights into the function of H2A methylation and in the mechanisms of PRMT6-mediated transcriptional repression.
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Scientific Article
2010
Weiss, T. ; Hergeth, S.P. ; Zeissler, U. ; Izzo, A. ; Tropberger, P. ; Zee, B.M. ; Dundr, M. ; Garcia, B.A. ; Daujat, S. ; Schneider, R.
Epigenetics Chromatin 3:7 (2010)
BACKGROUND: The linker histone H1 has a key role in establishing and maintaining higher order chromatin structure and in regulating gene expression. Mammals express up to 11 different H1 variants, with H1.2 and H1.4 being the predominant ones in most somatic cells. Like core histones, H1 has high levels of covalent modifications; however, the full set of modifications and their biological role are largely unknown. RESULTS: In this study, we used a candidate screen to identify enzymes that methylate H1 and to map their corresponding methylation sites. We found that the histone lysine methyltransferases G9a/KMT1C and Glp1/KMT1D methylate H1.2 in vitro and in vivo, and we mapped this novel site to lysine 187 (H1.2K187) in the C-terminus of H1. This H1.2K187 methylation is variant-specific. The main target for methylation by G9a in H1.2, H1.3, H1.5 and H1.0 is in the C-terminus, whereas H1.4 is preferentially methylated at K26 (H1.4K26me) in the N-terminus. We found that the readout of these marks is different; H1.4K26me can recruit HP1, but H1.2K187me cannot. Likewise, JMJD2D/KDM4 only reverses H1.4K26 methylation, clearly distinguishing these two methylation sites. Further, in contrast to C-terminal H1 phosphorylation, H1.2K187 methylation level is steady throughout the cell cycle. CONCLUSIONS: We have characterised a novel methylation site in the C-terminus of H1 that is the target of G9a/Glp1 both in vitro and in vivo. To our knowledge, this is the first demonstration of variant-specific histone methylation by the same methyltransferases, but with differing downstream readers, thereby supporting the hypothesis of H1 variants having specific functions.
Wissenschaftlicher Artikel
Scientific Article
Infantino, S. ; Benz, B. ; Waldmann, T. ; Jung, M. ; Schneider, R. ; Reth, M.
J. Exp. Med. 207, 711-719 (2010)
Signals processed through the B cell antigen receptor (BCR) control both the proliferation and differentiation of B lymphocytes. How these different signaling modes are established at the BCR is poorly understood. We show that a conserved arginine in the tail sequence of the Igalpha subunit of the BCR is methylated by the protein arginine methyltransferase 1. This modification negatively regulates the calcium and PI-3 kinase pathways of the BCR while promoting signals leading to B cell differentiation. Thus, Igalpha arginine methylation can play an important role in specifying the outcome of BCR signaling.
Wissenschaftlicher Artikel
Scientific Article
Lange, U.C. ; Schneider, R.
Bioessays 32, 659-668 (2010)
During mammalian development, maintenance of cell fate through mitotic divisions require faithful replication not only of the DNA but also of a particular epigenetic state. Germline cells have the capacity of erasing this epigenetic memory at crucial times during development, thereby resetting their epigenome. Certain marks, however, appear to escape this reprogramming, which allows their transmission to the offspring and potentially guarantees transgenerational epigenetic inheritance. Here we discuss the molecular requirements for faithful transmission of epigenetic information and our current knowledge about the transmission of epigenetic information through generations.
Wissenschaftlicher Artikel
Scientific Article
Izzo, A. ; Schneider, R.
Brief. Funct. Genomic. Proteomic. 9, 429-443 (2010)
Eukaryotic chromatin can be highly dynamic and can continuously exchange between an open transcriptionally active conformation and a compacted silenced one. Post-translational modifications of histones have a pivotal role in regulating chromatin states, thus influencing all chromatin dependent processes. Methylation is currently one of the best characterized histone modification and occurs on arginine and lysine residues. Histone methylation can regulate other modifications (e.g. acetylation, phosphorylation and ubiquitination) in order to define a precise functional chromatin environment. In this review we focus on histone methylation and demethylation, as well as on the enzymes responsible for setting these marks. In particular we are describing novel concepts on the interdependence of histone modifications marks and discussing the molecular mechanisms governing this cross-talks.
Wissenschaftlicher Artikel
Scientific Article
Plazas-Mayorca, M.D. ; Bloom, J.S. ; Zeissler, U. ; Leroy, G. ; Young, N.L. ; DiMaggio, P.A. ; Krugylak, L. ; Schneider, R. ; Garcia, B.A.
Mol. Biosyst. 6, 1719-1729 (2010)
Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other histone molecules is currently uncharacterized. In this study, we comprehensively analyze the effects of G9a/Glp1 knockdown on the most abundant histone modifications through both Bottom Up and Middle Down mass spectrometry-based proteomics. In addition to the expected decrease in H3K9me1/me2 we find that other degrees of methylation on K9 are affected by the reduction of G9a/Glp1 activity, particularly when K9 methylation occurs in combination with K14 acetylation. In line with this, an increase in K14 acetylation upon G9a knockdown was observed across all H3 variants (H3.1, H3.2 and H3.3), hinting at the potential existence of a binary switch between K9 methylation and K14 acetylation. Interestingly, we also detect changes in the abundance of other modifications (such as H3K79me2) in response to lowered levels of G9a/Glp1 suggesting histone PTM cross-talk amongst the H3 variants. In contrast, we find that G9a/Glp1 knockdown produces little effect on the levels of histone H4 PTMs, indicating low to no trans-histone PTM crosstalk. Lastly, we determined gene expression profiles of control and G9a/Glp1 knockdown cells, and we find that the G9a/Glp1 knockdown influences several genes, including DNA binding proteins and key factors in chromatin. Our results provide new insights into the intra- and inter- histone cross-regulation of histone K9 methylation and its potential downstream gene targets.
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Scientific Article
Vogler, C. ; Huber, C. ; Waldmann, T. ; Ettig, R. ; Braun, L. ; Izzo, A. ; Daujat, S. ; Chassignet, I. ; Lopez-Contreras, A.J. ; Fernandez-Capetillo, O. ; Dundr, M. ; Rippe, K. ; Längst, G. ; Schneider, R.
PLoS Genet. 6:e1001234 (2010)
The tails of histone proteins are central players for all chromatin-mediated processes. Whereas the N-terminal histone tails have been studied extensively, little is known about the function of the H2A C-terminus. Here, we show that the H2A C-terminal tail plays a pivotal role in regulating chromatin structure and dynamics. We find that cells expressing C-terminally truncated H2A show increased stress sensitivity. Moreover, both the complete and the partial deletion of the tail result in increased histone exchange kinetics and nucleosome mobility in vivo and in vitro. Importantly, our experiments reveal that the H2A C-terminus is required for efficient nucleosome translocation by ISWI-type chromatin remodelers and acts as a novel recognition module for linker histone H1. Thus, we suggest that the H2A C-terminal tail has a bipartite function: stabilisation of the nucleosomal core particle, as well as mediation of the protein interactions that control chromatin dynamics and conformation.
Wissenschaftlicher Artikel
Scientific Article
Tropberger, P. ; Schneider, R.
Epigenetics 5, 112-117 (2010)
Post-translational modifications (PTM) of histones are key regulators of chromatin function. New mass spectrometrical technologies have revealed that PTMs are not restricted to the histone tails, but can also be found in the globular domains, especially at the DNA-binding surface of the nucleosomes. Recent work on this new group of epigenetic marks showed that these modifications have not only the potential to alter the physical properties of the nucleosome, but may act as signals that regulate the recruitment of effector proteins to chromatin as well.
Wissenschaftlicher Artikel
Scientific Article
2009
Daujat, S. ; Weiss, T. ; Mohn, F. ; Lange, U.C. ; Ziegler-Birling, C. ; Zeissler, U. ; Lappe, M. ; Schübeler, D. ; Torres-Padilla, M.E. ; Schneider, R.
Nat. Struct. Mol. Biol. 16, 777-781 (2009)
Histone modifications are central to the regulation of all DNA-dependent processes. Lys64 of histone H3 (H3K64) lies within the globular domain at a structurally important position. We identify trimethylation of H3K64 (H3K64me3) as a modification that is enriched at pericentric heterochromatin and associated with repeat sequences and transcriptionally inactive genomic regions. We show that this new mark is dynamic during the two main epigenetic reprogramming events in mammals. In primordial germ cells, H3K64me3 is present at the time of specification, but it disappears transiently during reprogramming. In early mouse embryos, it is inherited exclusively maternally; subsequently, the modification is rapidly removed, suggesting an important role for H3K64me3 turnover in development. Taken together, our findings establish H3K64me3 as a previously uncharacterized histone modification that is preferentially localized to repressive chromatin. We hypothesize that H3K64me3 helps to 'secure' nucleosomes, and perhaps the surrounding chromatin, in an appropriately repressed state during development.
Wissenschaftlicher Artikel
Scientific Article
Kamieniarz, K. ; Schneider, R.
Chem. Biol. 16, 1027-1029 (2009)
In the recent issue of Molecular Cell, Neumann et al. dissect the effect of H3K56 acetylation on chromatin structure using a novel method for generation of acetylated proteins. This is a valuable addition to the toolkit for those interested in unraveling how posttranslational modifications regulate protein function.
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Scientific Article
2008
Hajkova, P. ; Ancelin, K. ; Waldmann, T. ; Lacoste, N. ; Lange, U.C. ; Cesari, F. ; Lee, C. ; Almouzni, G. ; Schneider, R. ; Surani, M.A.
Nature 452, 877-881 (2008)
A unique feature of the germ cell lineage is the generation of totipotency. A critical event in this context is DNA demethylation and the erasure of parental imprints in mouse primordial germ cells (PGCs) on embryonic day 11.5 (E11.5) after they enter into the developing gonads. Little is yet known about the mechanism involved, except that it is apparently an active process. We have examined the associated changes in the chromatin to gain further insights into this reprogramming event. Here we show that the chromatin changes occur in two steps. The first changes in nascent PGCs at E8.5 establish a distinctive chromatin signature that is reminiscent of pluripotency. Next, when PGCs are residing in the gonads, major changes occur in nuclear architecture accompanied by an extensive erasure of several histone modifications and exchange of histone variants. Furthermore, the histone chaperones HIRA and NAP-1 (NAP111), which are implicated in histone exchange, accumulate in PGC nuclei undergoing reprogramming. We therefore suggest that the mechanism of histone replacement is critical for these chromatin rearrangements to occur. The marked chromatin changes are intimately linked with genome-wide DNA demethylation. On the basis of the timing of the observed events, we propose that if DNA demethylation entails a DNA repair-based mechanism, the evident histone replacement would represent a repair-induced response event rather than being a prerequisite.
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Scientific Article
Izzo, A. ; Kamieniarz, K. ; Schneider, R.
Biol. Chem. 389, 333-343 (2008)
The linker histone H1 binds to the DNA entering and exiting the nucleosomal core particle and has an important role in establishing and maintaining higher order chromatin structures. H1 forms a complex family of related proteins with distinct species, tissue and developmental specificity. In higher eukaryotes all H1 variants have the same general structure, consisting of a central conserved globular domain and less conserved N-terminal and C-terminal tails. These tails are moderately conserved among species, but differ among variants, suggesting a specific function for each H1 variant. Due to compensatory mechanisms and to the lack of proper tools, it has been very difficult to study the biological role of individual variants in chromatin-mediated processes. Our knowledge about H1 variants is indeed limited, and in vitro and in vivo observations have often been contradictory. Therefore, H1 variants were considered to be functionally redundant. However, recent knockout studies and biochemical analyses in different organisms have revealed exciting new insights into the specificity and mechanisms of actions of the H1 family members. Here, we collect and compare the available literature about H1 variants and discuss possible specific roles that challenge the concept of H1 being a mere structural component of chromatin and a general repressor of transcription.
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Scientific Article
Laue, K. ; Daujat, S. ; Crump, J.G. ; Plaster, N. ; Roehl, H.H. ; Kimmel, C.B. ; Schneider, R. ; Hammerschmidt, M.
Development 135, 1935-1946 (2008)
The Trithorax group (TrxG) is composed of diverse, evolutionary conserved proteins that form chromatin-associated complexes accounting for epigenetic transcriptional memory. However, the molecular mechanisms by which particular loci are marked for reactivation after mitosis are only partially understood. Here, based on genetic analyses in zebrafish, we identify the multidomain protein Brpf1 as a novel TrxG member with a central role during development. brpf1 mutants display anterior transformations of pharyngeal arches due to progressive loss of anterior Hox gene expression. Brpf1 functions in association with the histone acetyltransferase Moz (Myst3), an interaction mediated by the N-terminal domain of Brpf1, and promotes histone acetylation in vivo. Brpf1 recruits Moz to distinct sites of active chromatin and remains at chromosomes during mitosis, mediated by direct histone binding of its bromodomain, which has a preference for acetylated histones, and its PWWP domain, which binds histones independently of their acetylation status. This is the first demonstration of histone binding for PWWP domains. Mutant analyses further show that the PWWP domain is absolutely essential for Brpf1 function in vivo. We conclude that Brpf1, coordinated by its particular set of domains, acts by multiple mechanisms to mediate Moz-dependent histone acetylation and to mark Hox genes for maintained expression throughout vertebrate development.
Wissenschaftlicher Artikel
Scientific Article
Lange, U.C. ; Adams, D.J. ; Lee, C. ; Barton, S.J. ; Schneider, R. ; Bradley, A. ; Surani, M.A.
Mol. Cell. Biol. 28, 4688-4696 (2008)
The family of interferon-inducible transmembrane proteins (Ifitm) consists of five highly sequence-related cell surface proteins, which are implicated in diverse cellular processes. Ifitm genes are conserved, widely expressed, and characteristically found in genomic clusters, such as the 67-kb Ifitm family locus on mouse chromosome 7. Recently, Ifitm1 and Ifitm3 have been suggested to mediate migration of early primordial germ cells (PGCs), a process that is little understood. To investigate Ifitm function during germ cell development, we used targeted chromosome engineering to generate mutants which either lack the entire Ifitm locus or carry a disrupted Ifitm3 gene only. Here we show that the mutations have no detectable effects on development of the germ line or on the generation of live young. Hence, contrary to previous reports, Ifitm genes are not essential for PGC migration. The Ifitm family is a striking example of a conserved gene cluster which appears to be functionally redundant during development.
Wissenschaftlicher Artikel
Scientific Article
2007
Wissmann, M. ; Yin, N. ; Müller, J.M. ; Greschik, H. ; Fodor, B.D. ; Jenuwein, T. ; Vogler, C. ; Schneider, R. ; Günther, T. ; Buettner, R. ; Metzger, E. ; Schule, R.
Nat. Cell Biol. 9, 347-353 (2007)
Posttranslational modifications of histones, such as methylation, regulate chromatin structure and gene expression. Recently, lysine-specific demethylase 1 (LSD1), the first histone demethylase, was identified. LSD1 interacts with the androgen receptor and promotes androgen-dependent transcription of target genes by ligand-induced demethylation of mono- and dimethylated histone H3 at Lys 9 (H3K9) only. Here, we identify the Jumonji C (JMJC) domain-containing protein JMJD2C as the first histone tridemethylase regulating androgen receptor function. JMJD2C interacts with androgen receptor in vitro and in vivo. Assembly of ligand-bound androgen receptor and JMJD2C on androgen receptor-target genes results in demethylation of trimethyl H3K9 and in stimulation of androgen receptor-dependent transcription. Conversely, knockdown of JMJD2C inhibits androgen-induced removal of trimethyl H3K9, transcriptional activation and tumour cell proliferation. Importantly, JMJD2C colocalizes with androgen receptor and LSD1 in normal prostate and in prostate carcinomas. JMJD2C and LSD1 interact and both demethylases cooperatively stimulate androgen receptor-dependent gene transcription. In addition, androgen receptor, JMJD2C and LSD1 assemble on chromatin to remove methyl groups from mono, di and trimethylated H3K9. Thus, our data suggest that specific gene regulation requires the assembly and coordinate action of demethylases with distinct substrate specificities.
Wissenschaftlicher Artikel
Scientific Article
Schneider, R. ; Grosschedl, R.
Genes Dev. 21, 3027-3043 (2007)
The organization of the genome in the nucleus of a eukaryotic cell is fairly complex and dynamic. Various features of the nuclear architecture, including compartmentalization of molecular machines and the spatial arrangement of genomic sequences, help to carry out and regulate nuclear processes, such as DNA replication, DNA repair, gene transcription, RNA processing, and mRNA transport. Compartmentalized multiprotein complexes undergo extensive modifications or exchange of protein subunits, allowing for an exquisite dynamics of structural components and functional processes of the nucleus. The architecture of the interphase nucleus is linked to the spatial arrangement of genes and gene clusters, the structure of chromatin, and the accessibility of regulatory DNA elements. In this review, we discuss recent studies that have provided exciting insight into the interplay between nuclear architecture, genome organization, and gene expression.
Review
Review
2006
Ancelin, K. ; Lange, U.C. ; Hajkova, P. ; Schneider, R. ; Bannister, A.J. ; Kouzarides, T. ; Surani, M.A.
Nat. Cell Biol. 8, 623-630 (2006)
Blimp1, a transcriptional repressor, has a crucial role in the specification of primordial germ cells (PGCs) in mice at embryonic day 7.5 (E7.5). This SET-PR domain protein can form complexes with various chromatin modifiers in a context-dependent manner. Here, we show that Blimp1 has a novel interaction with Prmt5, an arginine-specific histone methyltransferase, which mediates symmetrical dimethylation of arginine 3 on histone H2A and/or H4 tails (H2A/H4R3me2s). Prmt5 has been shown to associate with Tudor, a component of germ plasm in Drosophila melanogaster. Blimp1-Prmt5 colocalization results in high levels of H2A/H4 R3 methylation in PGCs at E8.5. However, at E11.5, Blimp1-Prmt5 translocates from the nucleus to the cytoplasm, resulting in the loss of H2A/H4 R3 methylation at the time of extensive epigenetic reprogramming of germ cells. Subsequently, Dhx38, a putative target of the Blimp1-Prmt5 complex, is upregulated. Interestingly, expression of Dhx38 is also seen in pluripotent embryonic germ cells that are derived from PGCs when Blimp1 expression is lost. Our study demonstrates that Blimp1 is involved in a novel transcriptional regulatory complex in the mouse germ-cell lineage.
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Scientific Article
Schneider, R.
In:. 2006. 379-386
2005
Metzger, E. ; Wissmann, M. ; Yin, N. ; Müller, J.M. ; Schneider, R. ; Peters, A.H. ; Günther, T. ; Buettner, R. ; Schule, R.
Nature 437, 436-439 (2005)
Gene regulation in eukaryotes requires the coordinate interaction of chromatin-modulating proteins with specific transcription factors such as the androgen receptor. Gene activation and repression is specifically regulated by histone methylation status at distinct lysine residues. Here we show that lysine-specific demethylase 1 (LSD1; also known as BHC110) co-localizes with the androgen receptor in normal human prostate and prostate tumour. LSD1 interacts with androgen receptor in vitro and in vivo, and stimulates androgen-receptor-dependent transcription. Conversely, knockdown of LSD1 protein levels abrogates androgen-induced transcriptional activation and cell proliferation. Chromatin immunoprecipitation analyses demonstrate that androgen receptor and LSD1 form chromatin-associated complexes in a ligand-dependent manner. LSD1 relieves repressive histone marks by demethylation of histone H3 at lysine 9 (H3-K9), thereby leading to de-repression of androgen receptor target genes. Furthermore, we identify pargyline as an inhibitor of LSD1. Pargyline blocks demethylation of H3-K9 by LSD1 and consequently androgen-receptor-dependent transcription. Thus, modulation of LSD1 activity offers a new strategy to regulate androgen receptor functions. Here, we link demethylation of a repressive histone mark with androgen-receptor-dependent gene activation, thus providing a mechanism by which demethylases control specific gene expression.
Wissenschaftlicher Artikel
Scientific Article
Daujat, S. ; Zeissler, U. ; Waldmann, T. ; Happel, N. ; Schneider, R.
J. Biol. Chem. 280, 38090-38095 (2005)
Histone lysine methylation can have positive or negative effects on transcription, depending on the precise methylation site. According to the "histone code" hypothesis these methylation marks can be read by proteins that bind them specifically and then regulate downstream events. Hetero-chromatin protein 1 (HP1), an essential component of heterochromatin, binds specifically to methylated Lys(9) of histone H3 (K9/H3). The linker histone H1.4 is methylated on Lys(26) (K26/H1.4), but the role of this methylation in downstream events remains unknown. Here we identify HP1 as a protein specifically recognizing and binding to methylated K26/H1.4. We demonstrate that the Chromo domain of HP1 is mediating this binding and that phosphorylation of Ser(27) on H1.4 (S27/H1.4) prevents HP1 from binding. We suggest that methylation of K26/H1.4 could have a role in tethering HP1 to chromatin and that this could also explain how HP1 is targeted to those regions of chromatin where it does not colocalize with methylated K9/H3. Our results provide the first experimental evidence for a "phospho switch" model in which neighboring phosphorylation reverts the effect of histone lysine methylation.
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Scientific Article
Bannister, A.J. ; Schneider, R. ; Myers, F.A. ; Thorne, A.W. ; Crane-Robinson, C. ; Kouzarides, T.
J. Biol. Chem. 280, 17732-17736 (2005)
Methylation of lysine 4 of histone H3 (K4/H3) is linked to transcriptional activity, whereas methylation of K9/H3 is tightly associated with gene inactivity. These are well characterized sites of methylation within histones, but there are numerous other, less characterized, sites of modification. In Saccharomyces cerevisiae, methylation of K36/H3 has been linked to active genes, but little is known about this methylation in higher eukaryotes. Here we analyzed for the first time the levels and spatial distribution of di- and tri-methyl (di- and tri-Me) K36/H3 in metazoan genes. We analyzed chicken genes that are developmentally regulated, constitutively active, or inactive. We found that active genes contain high levels of these modifications compared with inactive genes. Furthermore, in actively transcribed regions the levels of di- and tri-Me K36/H3 peak toward the 3' end of the gene. This is in striking contrast to the distributions of di- and tri-Me K4/H3, which peak early in actively transcribed regions. Thus, di/tri-Me K4/H3 and di/tri-Me K36/H3 are both useful markers of active genes, but their genic distribution indicates differing roles. Our data suggest that the unique spatial distribution of di- and tri-Me K36/H3 plays a role in transcriptional termination and/or early RNA processing.
Wissenschaftlicher Artikel
Scientific Article
2004
Schneider, R. ; Bannister, A.J. ; Myers, F.A. ; Thorne, A.W. ; Crane-Robinson, C. ; Kouzarides, T.
Nat. Cell Biol. 6, 73-77 (2004)
Lysine residues within histones can be mono-, di - or tri-methylated. In Saccharomyces cerevisiae tri-methylation of Lys 4 of histone H3 (K4/H3) correlates with transcriptional activity, but little is known about this methylation state in higher eukaryotes. Here, we examine the K4/H3 methylation pattern at the promoter and transcribed region of metazoan genes. We analysed chicken genes that are developmentally regulated, constitutively active or inactive. We found that the pattern of K4/H3 methylation shows similarities to S. cerevisiae. Tri-methyl K4/H3 peaks in the 5' transcribed region and active genes can be discriminated by high levels of tri-methyl K4/H3 compared with inactive genes. However, our results also identify clear differences compared to yeast, as significant levels of K4/H3 methylation are present on inactive genes within the beta-globin locus, implicating this modification in maintaining a 'poised' chromatin state. In addition, K4/H3 di-methylation is not genome-wide and di-methylation is not uniformly distributed throughout the transcribed region. These results indicate that in metazoa, di- and tri-methylation of K4/H3 is linked to active transcription and that significant differences exist in the genome-wide methylation pattern as compared with S. cerevisiae.
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Scientific Article
Schneider, R. ; Bannister, A.J. ; Weise, C. ; Kouzarides, T.
J. Biol. Chem. 279, 23859-23862 (2004)
The N-terminal tails of histones are central to the regulation of chromatin structure. They form a binding platform for multiple protein complexes, which in turn regulate DNA processes such as transcription. Using peptide mass fingerprinting we identified INHAT (inhibitor of acetyltransferases) as a specific histone H3 N-terminal tail-binding complex. INHAT comprises two essential subunits, SET and pp32. We demonstrate that both SET and pp32 bind directly to the N terminus of H3. The binding is differentially affected by various modifications within the H3 N terminus. In particular, single phosphorylations within the H3 tail abrogates binding of INHAT, as does the simultaneous acetylation of multiple lysine residues. The histone modifications that affect INHAT binding are therefore compatible with its known role in transcriptional repression. We suggest that the charge of the histone tail is a major determinant in allowing INHAT to bind chromatin and coordinate the activity of multiple histone acetyltransferases.
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Scientific Article
Cuthbert, G.L. ; Daujat, S. ; Snowden, A.W. ; Erdjument-Bromage, H. ; Hagiwara, T. ; Yamada, M. ; Schneider, R. ; Gregory, P.D. ; Tempst, P. ; Bannister, A.J. ; Kouzarides, T.
Cell 118, 545-553 (2004)
Methylation of arginine residues within histone H3 has been linked to active transcription. This modification appears on the estrogen-regulated pS2 promoter when the CARM1 methyltransferase is recruited during transcriptional activation. Here we describe a process, deimination, that converts histone arginine to citrulline and antagonizes arginine methylation. We show that peptidyl arginine deiminase 4 (PADI4) specifically deiminates, arginine residues R2, R8, R17, and R26 in the H3 tail. Deimination by PADI4 prevents arginine methylation by CARM1. Dimethylation of arginines prevents deimination by PADI4 although monomethylation still allows deimination to take place. In vivo targeting experiments on an endogenous promoter demonstrate that PADI4 can repress hormone receptor-mediated gene induction. Consistent with a repressive role for PADI4, this enzyme is recruited to the pS2 promoter following hormone induction when the gene is transcriptionally downregulated. The recruitment of PADI4 coincides with deimination of the histone H3 N-terminal tail. These results define deimination as a novel mechanism for antagonizing the transcriptional induction mediated by arginine methylation.
Wissenschaftlicher Artikel
Scientific Article
2003
Erhardt, S. ; Su, I.H. ; Schneider, R. ; Barton, S.J. ; Bannister, A.J. ; Perez-Burgos, L. ; Jenuwein, T. ; Kouzarides, T. ; Tarakhovsky, A. ; Surani, M.A.
Development 130, 4235-4248 (2003)
Enhancer of zeste 2 (Ezh2), a SET domain-containing protein, is crucial for development in many model organisms, including early mouse development. In mice, Ezh2 is detected as a maternally inherited protein in the oocyte but its function at the onset of development is unknown. We have used a conditional allele of Ezh2 to deplete the oocyte of this maternal inheritance. We show that the loss of maternal Ezh2 has a long-term effect causing severe growth retardation of neonates despite 'rescue' through embryonic transcription from the paternal allele. This phenotypic effect on growth could be attributed to the asymmetric localisation of the Ezh2/Eed complex and the associated histone methylation pattern to the maternal genome, which is disrupted in Ezh2 mutant zygotes. During subsequent development, we detect distinct histone methylation patterns in the trophectoderm and the pluripotent epiblast. In the latter where Oct4 expression continues from the zygote onwards, the Ezh2/Eed complex apparently establishes a unique epigenetic state and plasticity, which probably explains why loss of Ezh2 is early embryonic lethal and obligatory for the derivation of pluripotent embryonic stem cells. By contrast, in the differentiating trophectoderm cells where Oct4 expression is progressively downregulated Ezh2/Eed complex is recruited transiently to one X chromosome in female embryos at the onset of X-inactivation. This accumulation and the associated histone methylation are also lost in Ezh2 mutants, suggesting a role in X inactivation. Thus, Ezh2 has significant and diverse roles during early development, as well as during the establishment of the first differentiated cells, the trophectoderm, and of the pluripotent epiblast cells.
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Scientific Article
Santos-Rosa, H. ; Schneider, R. ; Bernstein, B.E. ; Karabetsou, N. ; Morillon, A. ; Weise, C. ; Schreiber, S.L. ; Mellor, J. ; Kouzarides, T.
Mol. Cell 12, 1325-1332 (2003)
Set1p methylates lysine 4 (K4) of histone H3 and regulates the expression of many genes in yeast. Here we use a biochemical approach to identify a protein, Isw1p, which recognizes chromatin preferentially when it is di- and trimethylated at K4 H3. We show that on certain actively transcribed genes, the Isw1p chromatin remodeling ATPase requires K4 H3 methylation to associate with chromatin in vivo. Analysis of one such gene, MET16, shows that the enzymatic activities of Set1p and Isw1p are functionally connected: Set1p methylation and Isw1p ATPase generate specific chromatin changes at the 5' end of the gene, are necessary for the correct distribution of RNA polymerase II over the coding region, and are required for the recruitment of the cleavage and polyadenylation factor Rna15p. These results indicate that K4 H3 methylation and Isw1p ATPase activity are intimately linked in regulating transcription of certain genes in yeast.
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Scientific Article
Auner, H. ; Buckle, M. ; Deufel, A. ; Kutateladze, T. ; Lazarus, L. ; Mavathur, R. ; Muskhelishvili, G. ; Pemberton, I. ; Schneider, R. ; Travers, A.
J. Mol. Biol. 331, 331-344 (2003)
The Escherichia coli DNA architectural protein FIS activates transcription from stable RNA promoters on entry into exponential growth and also reduces the level of negative supercoiling. Here we show that such a reduction decreases the activity of the tyrT promoter but that activation by FIS rescues tyrT transcription at non-optimal superhelical densities. Additionally we show that three different "up" mutations in the tyrT core promoter either abolish or reduce the dependence of tyrT transcription on both high negative superhelicity and FIS in vivo and infer that the specific sequence organisation of the core promoter couples the control of transcription initiation by negative superhelicity and FIS. In vitro all the mutations potentiate FIS-independent untwisting of the -10 region while at the wild-type promoter FIS facilitates this step. We propose that this untwisting is a crucial limiting step in the initiation of tyrT RNA synthesis. The tyrT core promoter structure is thus optimised to combine high transcriptional activity with acute sensitivity to at least three major independent regulatory inputs: negative superhelicity, FIS and ppGpp.
Wissenschaftlicher Artikel
Scientific Article
2002
Schneider, R. ; Bannister, A.J.
Methods 26, 226-232 (2002)
Protein N-methylation has recently emerged as a major mechanism involved in regulating gene transcription and epigenetic inheritance. Transcriptional coactivators and corepressors have both been shown to associate with protein N-methyltransferases. These enzymes methylate arginine or lysine residues within histones as well as other proteins, such as transcription factors. Here, we discuss the recent advances in our understanding of protein methylation and its involvement in gene expression. Protocols are presented for studying transcriptionally relevant protein methylation.
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Scientific Article
Bernstein, B.E. ; Humphrey, E.L. ; Erlich, R.L. ; Schneider, R. ; Bouman, P. ; Liu, J.S. ; Kouzarides, T. ; Schreiber, S.L.
Proc. Natl. Acad. Sci. U.S.A. 99, 8695-8700 (2002)
Posttranslational modifications of histone tails regulate chromatin structure and transcription. Here we present global analyses of histone acetylation and histone H3 Lys 4 methylation patterns in yeast. We observe a significant correlation between acetylation of histones H3 and H4 in promoter regions and transcriptional activity. In contrast, we find that dimethylation of histone H3 Lys 4 in coding regions correlates with transcriptional activity. The histone methyltransferase Set1 is required to maintain expression of these active, promoter-acetylated, and coding region-methylated genes. Global comparisons reveal that genomic regions deacetylated by the yeast enzymes Rpd3 and Hda1 overlap extensively with Lys 4 hypo- but not hypermethylated regions. In the context of recent studies showing that Lys 4 methylation precludes histone deacetylase recruitment, we conclude that Set1 facilitates transcription, in part, by protecting active coding regions from deacetylation.
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Scientific Article
Schneider, R. ; Bannister, A.J. ; Kouzarides, T.
Trends Biochem. Sci. 27, 396-402 (2002)
Enzymes that covalently modify histones control many cellular processes by affecting gene expression. A new class of these enzymes is the histone lysine methyltransferase family, whose catalytic activity lies within a conserved domain, the SET domain. This article surveys the evidence for a connection between SET-domain-containing proteins and cancer. It proposes that deregulation of SET-domain function has an important role in carcinogenesis.
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Scientific Article
Bannister, A.J. ; Schneider, R. ; Kouzarides, T.
Cell 109, 801-806 (2002)
Methylation of histones mediates transcriptional silencing at heterochromatin sites and affects regulated transcription at euchromatic loci. So is the methyl group a permanent mark on histones, or can it be removed by an active process necessary for regulated gene expression?
Review
Review
Santos-Rosa, H. ; Schneider, R. ; Bannister, A.J. ; Sherriff, J. ; Bernstein, B.E. ; Emre, N.C. ; Schreiber, S.L. ; Mellor, J. ; Kouzarides, T.
Nature 419, 407-411 (2002)
Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.
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Scientific Article
2001
Nielsen, S.J. ; Schneider, R. ; Bauer, U.M. ; Bannister, A.J. ; Morrison, A. ; O'Carroll, D. ; Firestein, R. ; Cleary, M. ; Jenuwein, T. ; Herrera, R.E. ; Kouzarides, T.
Nature 412, 561-565 (2001)
In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.
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Scientific Article
Travers, A. ; Schneider, R. ; Muskhelishvili, G.
Biochimie 83, 213-217 (2001)
The nucleoid-associated protein FIS modulates the topology of DNA in a growth-phase dependent manner functioning homeostatically to counteract excessive levels of negative superhelicity. We propose that this is achieved by at least two mechanisms: the physical constraint of low levels of negative superhelicity by FIS binding to DNA and by a reduction in the expression and effectiveness of DNA gyrase. In addition, high levels of expression of the fis gene do themselves require a high negative superhelical density. On DNA substrates containing phased high affinity binding sites, as exemplified by the upstream activating sequence of the tyrT promoter, FIS forms tightly bent DNA structures, or microloops, that are necessary for the optimal expression of the promoter. We suggest that these microloops compensate in part for the FIS-induced lowering of the superhelical density.
Wissenschaftlicher Artikel
Scientific Article
Schneider, R. ; Lurz, R. ; Lüder, G. ; Tolksdorf, C. ; Travers, A. ; Muskhelishvili, G.
Nucleic Acids Res. 29, 5107-5114 (2001)
The Escherichia coli chromatin protein FIS modulates the topology of DNA in a growth phase-dependent manner. In this study we have investigated the global effect of FIS binding on DNA architecture in vitro. We show that in supercoiled DNA molecules FIS binds at multiple sites in a non-random fashion and increases DNA branching. This global DNA reshaping effect is independent of the helical phasing of FIS binding sites. We propose, in addition to the previously inferred stabilisation of tightly bent DNA microloops in the upstream regions of certain promoters, that FIS may perform the distinct architectural function of organising branched plectonemes in the E.coli nucleoid.
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Scientific Article
Nasser, W. ; Schneider, R. ; Travers, A. ; Muskhelishvili, G.
J. Biol. Chem. 276, 17878-17886 (2001)
The DNA architectural proteins FIS and CRP are global regulators of transcription in Escherichia coli involved in the adjustment of cellular metabolism to varying growth conditions. We have previously demonstrated that FIS modulates the expression of the crp gene by functioning as its transcriptional repressor. Here we show that in turn, CRP is required to maintain the growth phase pattern of fis expression. We demonstrate the existence of a divergent promoter in the fis regulatory region, which reduces transcription of the fis promoter. In the absence of FIS, CRP activates fis transcription, thereby displacing the polymerase from the divergent promoter, whereas together FIS and CRP synergistically repress fis gene expression. These results provide evidence for a direct cross-talk between global regulators of cellular transcription during the growth phase. This cross-talk is manifested in alternate formation of functional nucleoprotein complexes exerting either activating or repressing effects on transcription.
Wissenschaftlicher Artikel
Scientific Article
2000
Zechiedrich, E.L. ; Khodursky, A.B. ; Bachellier, S. ; Schneider, R. ; Chen, D. ; Lilley, D.M. ; Cozzarelli, N.R.
J. Biol. Chem. 275, 8103-8113 (2000)
DNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli. Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (sigma) of -0.015 at an initial rate of 0.8 links min(-1). Topoisomerase I relaxed DNA at a faster rate, 5 links min(-1), but only to a sigma of -0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to sigma = -0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (sigma = -0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by lambda integrase site-specific recombination.
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Scientific Article
Schneider, R. ; Travers, A. ; Muskhelishvili, G.
Mol. Microbiol. 38, 167-175 (2000)
The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E. coli.
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Scientific Article
1999
Schneider, R. ; Travers, A. ; Kutateladze, T. ; Muskhelishvili, G.
Mol. Microbiol. 34, 953-964 (1999)
In Escherichia coli, the transcriptional activity of many promoters is strongly dependent on the negative superhelical density of chromosomal DNA. This, in turn, varies with the growth phase, and is correlated with the overall activity of DNA gyrase, the major topoisomerase involved in the elevation of negative superhelicity. The DNA architectural protein FIS is a regulator of the metabolic reorganization of the cell during early exponential growth phase. We have previously shown that FIS modulates the superhelical density of plasmid DNA in vivo, and on binding reshapes the supercoiled DNA in vitro. Here, we show that, in addition, FIS represses the gyrA and gyrB promoters and reduces DNA gyrase activity. Our results indicate that FIS determines DNA topology both by regulation of topoisomerase activity and, as previously inferred, by directly reshaping DNA. We propose that FIS is involved in coupling cellular physiology to the topology of the bacterial chromosome.
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Scientific Article
1997
Schneider, R. ; Travers, A. ; Muskhelishvili, G.
Mol. Microbiol. 26, 519-530 (1997)
The Escherichia coli DNA-binding protein FIS serves as a DNA architectural factor in two unrelated enzymatic reactions, the site-specific inversion of DNA and transcriptional activation of stable RNA promoters. In both these processes, FIS facilitates the assembly and dynamic transitions of two structurally distinct nucleoprotein complexes. We have proposed previously that, in these systems, FIS stabilizes writhed DNA microloops by binding at multiple helically phased sites in DNA. However, FIS also binds and bends DNA at many non-specific sites and, at its maximum levels in the early exponential phase, FIS could potentially occupy a considerable part of the E. coli chromosome. Here, we show that fis affects growth phase-specific alterations in the supercoiling level of DNA. Expression of fis accelerates the accumulation of moderately supercoiled plasmids in stationary phase, which are stabilized by FIS after nutritional shift-up. In accordance with such a function, FIS modulates the relaxing and supercoiling activities of topoisomerases in vitro in a way that keeps DNA in a moderately supercoiled state. Our results suggest that the primary role of FIS is to modulate chromosomal dynamics during bacterial growth.
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