Molecular Biology

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What is the composition of chromatin and how is it assembled?

(Marco Borso, Rupam Choudhury, Ignasi Forne Vera Kleene, Pierre Schilcher)

The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. All these factors help to establish and maintain a largely DNA sequence independent but surprisingly stable structure. The conformation of chromatin is continuously challenged through processes that use DNA as a substrate such as DNA replication, repair, recombination or transcription. During all those cases chromatin is extensively disassembled and reassembled to allow the necessary factors to gain access to their substrate resulting in a very high histone turnover rates at a given genomic location. This dynamic nature of chromatin makes it even more important that the machinery mediating this continuous restructuring is well coordinated at the molecular level to maintain the epigenetic information stored in the structure.
Vera, Rupam, Ignasi and Marco with the help of Pierre study the proteomic composition of distinct chromatin domains using high resolution mass spectrometry.

Further Reading:

Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination. (2021) Nakamura K, Kustatscher G, Alabert C, Hödl M, Forne I, Völker-Albert M, Satpathy S, Beyer TE, Mailand N, Choudhary C, Imhof A, Rappsilber J, Groth A. Mol Cell 20: 30946-1.

Alabert C, Loos C, Voelker-Albert M, Graziano S, Forne I, Reverón-Gómez N, Schuh L, Hasenauer J, Marr C, Imhof A & Groth A (2020) Domain Model Explains Propagation Dynamics and Stability of Histone H3K27 and H3K36 Methylation Landscapes. CellReports 30: 1223–1234.e8
Schuh,L., Loos,C., Pokrovsky,D., Imhof,A., Rupp,R.A.W. and Marr,C. (2020) H4K20 Methylation Is Differently Regulated by Dilution and Demethylation in Proliferating and Cell-Cycle-Arrested Xenopus Embryos. Cell Syst, 11, 653-662.e8.
Harpprecht L, Baldi S, Schauer T, Schmidt A, Bange T, Robles MS, Kremmer E, Imhof A & Becker PB (2019) A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling. Nucleic Acids Res 47: 7444–7459
Völker-Albert MC, Pusch MC, Fedisch A, Schilcher P, Schmidt A & Imhof A (2016) A Quantitative Proteomic Analysis of In VitroAssembled Chromatin. Mol Cell Proteomics 15: 945–959
Saredi G, Huang H, Hammond CM, Alabert C, Bekker-Jensen S, Forne I, Reverón-Gómez N, Foster BM, Mlejnkova L, Bartke T, Cejka P, Mailand N, Imhof A, Patel DJ & Groth A (2016) H4K20me0 marks post-replicative chromatin and recruits the TONSL–MMS22L DNA repair complex. Nature 534: 714–718
Feller C, Forne I, Imhof A & Becker PB (2015) Global and specific responses of the histone acetylome to systematic perturbation. Mol. Cell 57: 559–571
Scharf AND, Barth TK & Imhof A (2009a) Establishment of histone modifications after chromatin assembly. Nucleic Acids Res 37: 5032–5040
Scharf AND, Meier K, Seitz V, Kremmer E, Brehm A & Imhof A (2009b) Monomethylation of lysine 20 on histone H4 facilitates chromatin maturation. Mol Cell Biol 29: 57–67


How does the environment affect epigenetic marks?

(Marco Borso, Shiboyothi Lahiri, Frederike Schäfer, Anuroop Venkatasubramani)

Chromatin-modifying enzymes are thought to be the authors of an epigenetic language, but the origin and meaning of the messages they write in chromatin are still mysterious. Recent studies suggesting that the effects of diet can be passed on epigenetically to offspring add weight to the idea that these enzymes act as metabolic sensors, converting changes in metabolism into stable patterns of gene expression and mediate downstream signals.
Frederike, Anuroop, Marco and Shibo investigate how the activity of these enzymes and the modification patterns of histones are regulated by key metabolites and physiological changes such as memory formation or ageing. They use various MS based methods to study this.

Further Reading:

Lauterbach MA, Hanke JE, Serefidou M, Mangan MSJ, Kolbe C-C, Hess T, Rothe M, Kaiser R, Hoss F, Gehlen J, Engels G, Kreutzenbeck M, Schmidt SV, Christ A, Imhof A, Hiller K & Latz E (2019) Toll-like Receptor Signaling Rewires Macrophage Metabolism and Promotes Histone Acetylation via ATP-Citrate Lyase. Immunity 51: 997–1011.e7
Greco,C.M., Cervantes,M., Fustin,J.-M., Ito,K., Ceglia,N., Samad,M., Shi,J., Koronowski,K.B., Forne,I., Ranjit,S., et al. (2020) S-adenosyl-l-homocysteine hydrolase links methionine metabolism to the circadian clock and chromatin remodeling. Sci Adv, 6, eabc5629.
Serefidou,M., Venkatasubramani,A.V. and Imhof,A. (2019) The Impact of One Carbon Metabolism on Histone Methylation. Frontiers in Genetics, 10, 919–7.
Gaucher,J., Kinouchi,K., Ceglia,N., Montellier,E., Peleg,S., Greco,C.M., Schmidt,A., Forne,I., Masri,S., Baldi,P., et al. (2019) Distinct metabolic adaptation of liver circadian pathways to acute and chronic patterns of alcohol intake. Proceedings of the National Academy of Sciences of the United States of America, 116, 25250–25259
Peleg,S., Feller,C., Ladurner,A.G. and Imhof,A. (2016) The Metabolic Impact on Histone Acetylation and Transcription in Ageing. Trends in Biochemical Sciences, 41, 700–711.
Masri,S., Patel,V.R., Eckel-Mahan,K.L., Peleg,S., Forne,I., Ladurner,A.G., Baldi,P., Imhof,A. and Sassone-Corsi,P. (2013) Circadian acetylome reveals regulation of mitochondrial metabolic pathways. PNAS, 110, 3339–3344


How do species form and what keeps them apart?

(Rupam Choudhury, Eylül Ozgü, Anuroop Venkatasubramani)

Speciation involves the reproductive isolation of natural populations due to the sterility or lethality of their hybrids. The development of such an obviously maladaptive trait under the influence of natural selection is one of the main unsolved questions in evolutionary biology. In order to resolve this apparent paradox we biochemically characterized a protein complex that contains the gene products of the two speciation genes Hmr and Lhr. The two proteins are components of a larger protein complex that localizes at and close to the centromere where it represses transcription of transposable elements. In pure species this centromeric localization is important for chromosome segregation, which provides an explanation of the main driving force for the divergent evolution of their expression levels.
Eylül, Rupam biochemically analyze the complexes and the quantitative proteomic differences and their function by combining biochemical and cell biological techniques with quantitative proteomics and large-scale DNA sequencing.

Further Reading:

Kochanova,N.Y., Schauer,T., Mathias,G.P., Lukacs,A., Schmidt,A., Flatley,A., Schepers,A., Thomae,A.W. and Imhof,A. (2020) A multi-layered structure of the interphase chromocenter revealed by proximity-based biotinylation. Nucleic Acids Research, 225, 912.
Cooper,J.C., Lukacs,A., Reich,S., Schauer,T., Imhof,A. and Phadnis,N. (2019) Altered Localization of Hybrid Incompatibility Proteins in Drosophila. Molecular Biology and Evolution, 36, 1783–1792
Barth,T.K., Schade,G.O.M., Schmidt,A., Vetter,I., Wirth,M., Heun,P., Thomae,A.W. and Imhof,A. (2014) Identification of novel Drosophila centromere-associated proteins. PROTEOMICS, 14, 2167–2178.
Thomae,A.W., Schade,G.O.M., Padeken,J., Borath,M., Vetter,I., Kremmer,E., Heun,P. and Imhof,A. (2013) A Pair of Centromeric Proteins Mediates Reproductive Isolation in Drosophila Species. Developmental Cell, 27, 412–424
Ross,B.D., Rosin,L., Thomae,A.W., Hiatt,M.A., Vermaak,D., Cruz,A.F.A. de la, Imhof,A., Mellone,B.G. and Malik,H.S. (2013) Stepwise Evolution of Essential Centromere Function in a Drosophila Neogene. Science (New York, NY), 340, 1211–1214.

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Can chromatin factors be used as markers for pathological states?

(Teresa Barth, Ignasi Forne, Marc Wirth)

The discovery and use of novel molecular markers from patient tissues is a central goal for the development of more precise and personalized treatments. As chromatin components are frequently cell type specific and often altered during pathological alterations, they have a great potential to serve as protein biomarkers. That is particularly true for histones and nucleosomes, which are modified in response to various intrinsic and extrinsic signals and are abundant enough to be detected in easily accessible clinical samples such as plasma or peripheral blood monocytes.
Teresa and Ignasi with the help of Marc and in collaboration with EpiQMAx ( try to establish novel and robust mass spectrometry-based methods to eventually bring proteomic methods to clinical routine.

Further Reading:

Lahiri,S., Aftab,W., Walenta,L., Strauss,L., Poutanen,M., Mayerhofer,A. and Imhof,A. (2021) MALDI-IMS combined with shotgun proteomics identify and localize new factors in male infertility. Life Sci Alliance, 4, e202000672
Völker-Albert,M., Bronkhorst,A., Holdenrieder,S. and Imhof,A. (2020) Histone Modifications in Stem Cell Development and Their Clinical Implications. Stem Cell Rep, 15, 1196–1205.
Bux,E.M., Solis-Mezarino,V., Kuhm,C., Northoff,B.H., Karin,I., Klopstock,T., Holdt,L.M., Völker-Albert,M., Imhof,A. and Peleg,S. (2019) Determining histone H4 acetylation patterns in human peripheral blood mononuclear cells using mass spectrometry. Clinical Mass Spectrometry, 10.1016/j.clinms.2019.08.001.
Metzger,E., Wang,S., Urban,S., Willmann,D., Schmidt,A., Offermann,A., Allen,A., Sum,M., Obier,N., Cottard,F., et al. (2019) KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells. Nature Structural & Molecular Biology, 26, 361–371.
Tzika,E., Dreker,T. and Imhof,A. (2018) Epigenetics and Metabolism in Health and Disease. Frontiers in Genetics, 9, 403–8.
Lahiri,S., Sun,N., Buck,A., Imhof,A. and Walch,A. (2016) MALDI imaging mass spectrometry as a novel tool for detecting histone modifications in clinical tissue samples. Expert review of proteomics, 13, 275–284.