Podcasts about polycomb

In this episode of the Epigenetics Podcast, we talked with Giacomo Cavalli from the Institute of Human Genetics in Montpellier about his work on critical aspects of epigenetic regulation, particularly the role of Polycomb proteins and chromatin architecture. We start the Interview by talking about Dr. Cavalli's work on Polycomb function in maintaining chromatin states and how it relates to gene regulation. He shares insights from his early lab experiences, where he aimed to understand the inheritance mechanisms of chromatin states through various models, including the FAB7 cellular memory module. The discussion uncovers how Polycomb proteins can silence gene expression and the complex interplay between different epigenetic factors that govern this process. Dr. Cavalli also addresses how he has investigated the recruitment mechanisms of Polycomb complexes, highlighting the roles of several DNA-binding proteins, including DSP-1 and GAGA factor, in this intricate regulatory landscape. He emphasizes the evolution of our understanding of Polycomb recruitment, illustrating the multifactorial nature of this biological puzzle. As the conversation progresses, we explore Dr. Cavalli's fascinating research into the three-dimensional organization of the genome. He explains his contributions to mapping chromosomal interactions within Drosophila and the distinctions observed when performing similar studies in mammalian systems. Key findings regarding topologically associated domains (TADs) and their association with gene expression are presented, alongside the implications for our understanding of gene regulation in development and disease.   References Déjardin, J., Rappailles, A., Cuvier, O., Grimaud, C., Decoville, M., Locker, D., & Cavalli, G. (2005). Recruitment of Drosophila Polycomb group proteins to chromatin by DSP1. Nature, 434(7032), 533–538. https://doi.org/10.1038/nature03386 Sexton, T., Yaffe, E., Kenigsberg, E., Bantignies, F., Leblanc, B., Hoichman, M., Parrinello, H., Tanay, A., & Cavalli, G. (2012). Three-dimensional folding and functional organization principles of the Drosophila genome. Cell, 148(3), 458–472. https://doi.org/10.1016/j.cell.2012.01.010 Bonev, B., Mendelson Cohen, N., Szabo, Q., Fritsch, L., Papadopoulos, G. L., Lubling, Y., Xu, X., Lv, X., Hugnot, J. P., Tanay, A., & Cavalli, G. (2017). Multiscale 3D Genome Rewiring during Mouse Neural Development. Cell, 171(3), 557–572.e24. https://doi.org/10.1016/j.cell.2017.09.043 Szabo, Q., Donjon, A., Jerković, I., Papadopoulos, G. L., Cheutin, T., Bonev, B., Nora, E. P., Bruneau, B. G., Bantignies, F., & Cavalli, G. (2020). Regulation of single-cell genome organization into TADs and chromatin nanodomains. Nature genetics, 52(11), 1151–1157. https://doi.org/10.1038/s41588-020-00716-8   Related Episodes BET Proteins and Their Role in Chromosome Folding and Compartmentalization (Kyle Eagen) Long-Range Transcriptional Control by 3D Chromosome Structure (Luca Giorgetti) Epigenetic Landscapes During Cancer (Luciano Di Croce)   Contact Epigenetics Podcast on Mastodon Epigenetics Podcast on Bluesky Dr. Stefan Dillinger on LinkedIn Active Motif on LinkedIn Active Motif on Bluesky Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we talked with Maxim Greenberg from the Institute Jacob Monot about his work on epigenetic consequences of DNA methylation in development. In this interview we explore how Dr. Greenbergs work at UCLA involved pioneering experiments on DNA methylation mechanisms and how this period was marked by significant collaborative efforts within a highly competitive yet supportive lab environment that ultimately lead to publications in high impact journals. His transition to a postdoctoral position at the Institut Curie with Deborah Bourc'his harnessed his newfound expertise in mammalian systems, examining chromatin changes and the implications for embryonic development. Dr. Greenberg explained the nuances of his research, particularly how chromatin modifications during early development can influence gene regulatory mechanisms later in life, providing a compelling narrative about the potential long-term impacts of epigenetic changes that occur in utero. Throughout our conversation, we examined the intricate relationship between DNA methylation and Polycomb repression, discussing how these epigenetic mechanisms interact and the functional outcomes of their regulation. Dr. Greenberg's insights into his recent studies reveal a commitment to unraveling the complexities of enhancer-promoter interactions in the context of epigenetic regulation.   References Greenberg, M. V., Ausin, I., Chan, S. W., Cokus, S. J., Cuperus, J. T., Feng, S., Law, J. A., Chu, C., Pellegrini, M., Carrington, J. C., & Jacobsen, S. E. (2011). Identification of genes required for de novo DNA methylation in Arabidopsis. Epigenetics, 6(3), 344–354. https://doi.org/10.4161/epi.6.3.14242 Greenberg, M. V., Glaser, J., Borsos, M., Marjou, F. E., Walter, M., Teissandier, A., & Bourc'his, D. (2017). Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth. Nature genetics, 49(1), 110–118. https://doi.org/10.1038/ng.3718 Greenberg, M., Teissandier, A., Walter, M., Noordermeer, D., & Bourc'his, D. (2019). Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency. eLife, 8, e44057. https://doi.org/10.7554/eLife.44057 Monteagudo-Sánchez, A., Richard Albert, J., Scarpa, M., Noordermeer, D., & Greenberg, M. V. C. (2024). The impact of the embryonic DNA methylation program on CTCF-mediated genome regulation. Nucleic acids research, 52(18), 10934–10950. https://doi.org/10.1093/nar/gkae724 Richard Albert, J., Urli, T., Monteagudo-Sánchez, A., Le Breton, A., Sultanova, A., David, A., Scarpa, M., Schulz, M., & Greenberg, M. V. C. (2024). DNA methylation shapes the Polycomb landscape during the exit from naive pluripotency. Nature structural & molecular biology, 10.1038/s41594-024-01405-4. Advance online publication. https://doi.org/10.1038/s41594-024-01405-4   Related Episodes DNA Methylation and Mammalian Development (Déborah Bourc'his) Circulating Epigenetic Biomarkers in Cancer (Charlotte Proudhon) Epigenetic Mechanisms in Genome Regulation and Developmental Programming (James Hackett)   Contact Epigenetics Podcast on Mastodon Epigenetics Podcast on Bluesky Dr. Stefan Dillinger on LinkedIn Active Motif on LinkedIn Active Motif on Bluesky Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Luciano Di Croce from the Center of Genomic Regulation in Barcelona to talk about his work on epigenetic landscapes in cancer. The Di Croce Lab focuses on the Polycomb Complex and its influence on diseases like cancer. Luciano Di Croce started out his research career investigating the oncogenic transcription factor PML-RAR. They could show that in leukemic cells knockdown of SUZ12, a key component of Polycomb repressive complex 2 (PRC2), reverts not only histone modification but also induces DNA de-methylation of PML-RAR target genes. More recently the team focused on two other Polycomb related proteins Zrf1 and PHF19 and were able to characterize some of their functions in gene targeting in different disease and developmental contexts.   References Di Croce, L., Raker, V. A., Corsaro, M., Fazi, F., Fanelli, M., Faretta, M., Fuks, F., Lo Coco, F., Kouzarides, T., Nervi, C., Minucci, S., & Pelicci, P. G. (2002). Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science (New York, N.Y.), 295(5557), 1079–1082. https://doi.org/10.1126/science.1065173 Richly, H., Rocha-Viegas, L., Ribeiro, J. D., Demajo, S., Gundem, G., Lopez-Bigas, N., Nakagawa, T., Rospert, S., Ito, T., & Di Croce, L. (2010). Transcriptional activation of polycomb-repressed genes by ZRF1. Nature, 468(7327), 1124–1128. https://doi.org/10.1038/nature09574 Jain, P., Ballare, C., Blanco, E., Vizan, P., & Di Croce, L. (2020). PHF19 mediated regulation of proliferation and invasiveness in prostate cancer cells. eLife, 9, e51373. https://doi.org/10.7554/eLife.51373   Related Episodes Oncohistones as Drivers of Pediatric Brain Tumors (Nada Jabado) Transcription and Polycomb in Inheritance and Disease (Danny Reinberg) Targeting COMPASS to Cure Childhood Leukemia (Ali Shilatifard)   Contact Epigenetics Podcast on Twitter Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Active Motif on Twitter Active Motif on LinkedIn Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Karim-Jean Armache from New York University - Grossman School of Medicine to talk about his work on the structural analysis of Polycomb Complex Proteins. Karim-Jean Armache started his research career with the structural characterization of the 12-subunit RNA Polymerase II. After starting his own lab he used this knowledge in x-ray crystallography and electron microscopy to study how gene silencing complexes like the PRC complex act on chromatin and influence transcription. Further work in the Armache Lab focused on Dot, a  histone H3K79 methyltransferase, and how it acts on chromatin, as well as how it is regulated by Histone-Histone crosstalk. References Armache, K. J., Garlick, J. D., Canzio, D., Narlikar, G. J., & Kingston, R. E. (2011). Structural basis of silencing: Sir3 BAH domain in complex with a nucleosome at 3.0 Å resolution. Science (New York, N.Y.), 334(6058), 977–982. https://doi.org/10.1126/science.1210915 Lee, C. H., Holder, M., Grau, D., Saldaña-Meyer, R., Yu, J. R., Ganai, R. A., Zhang, J., Wang, M., LeRoy, G., Dobenecker, M. W., Reinberg, D., & Armache, K. J. (2018). Distinct Stimulatory Mechanisms Regulate the Catalytic Activity of Polycomb Repressive Complex 2. Molecular cell, 70(3), 435–448.e5. https://doi.org/10.1016/j.molcel.2018.03.019 De Ioannes, P., Leon, V. A., Kuang, Z., Wang, M., Boeke, J. D., Hochwagen, A., & Armache, K. J. (2019). Structure and function of the Orc1 BAH-nucleosome complex. Nature communications, 10(1), 2894. https://doi.org/10.1038/s41467-019-10609-y Valencia-Sánchez, M. I., De Ioannes, P., Wang, M., Truong, D. M., Lee, R., Armache, J. P., Boeke, J. D., & Armache, K. J. (2021). Regulation of the Dot1 histone H3K79 methyltransferase by histone H4K16 acetylation. Science (New York, N.Y.), 371(6527), eabc6663. https://doi.org/10.1126/science.abc6663   Related Episodes Transcription and Polycomb in Inheritance and Disease (Danny Reinberg) From Nucleosome Structure to Function (Karolin Luger) Oncohistones as Drivers of Pediatric Brain Tumors (Nada Jabado)   Contact Epigenetics Podcast on Twitter Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Active Motif on Twitter Active Motif on LinkedIn eMail: podcast@activemotif.com

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.29.522251v1?rss=1 Authors: Matsui, S., Granitto, M., Buckley, M., Shiley, J., Zacharias, W., Mayhew, C., Lim, H.-W., Iwafuchi, M. Abstract: Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF Forkhead box A (FOXA), required for endoderm lineage commitment, coordinates with the PR domain zinc finger 1 (PRDM1) TF to recruit Polycomb repressive complexes, which establish bivalent enhancers and repress alternative lineage programs. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to repress cell differentiation programs in pluripotent stem cells, suggesting this is a common feature of pioneer TFs. We propose that pioneer and PRDM TFs coordinate recruitment of Polycomb complexes to safeguard cell fate. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.17.516971v1?rss=1 Authors: Thambyrajah, R., Fadlullah, Z., Proffitt, M., Neo, W. H., Guillen, Y., Casado-Pelaez, M., Herrero-Molinero, P., Brujas, C., Castelluccio, N., Gonzalez, J., Iglesias, A., Marruecos, L., Ruiz-Herguido, C., Esteller, M., Mereu, E., Lacaud, G., Espinosa, L., Bigas, A. Abstract: Recent findings are challenging the classical hematopoietic model in which long-term hematopoietic stem cells (LT-HSC) are the base of the hematopoietic system. Clonal dynamics analysis of the hematopoietic system indicate that LT-HSC are not the main contributors of normal hemapoiesis in physiological conditions and the hematopoietic system is mainly maintained by multipotent progenitors (MPPs, hereafter HPC) and LT-HSCs are mostly in a non-active state. The first HSCs emerge from the aorta-gonad and mesonephros (AGM) region along with hematopoietic progenitors (HPC) within hematopoietic clusters. Molecular pathways that determine the HSC fate instead of HPC are still unknown, although inflammatory signaling, including NF-KB has been implicated in the development of HSCs. Here, we identify a chromatin binding function for IKB (also known as the inhibitor of NF-KB) that is Polycomb repression complex 2 (PRC2)- dependent and specifically determines dormant vs proliferating HSCs from the onset of their emergence in the AGM. We find a specific reduction of LT-HSCs in the IKB knockout new-born pups. This defect is manifested at the FL stage already, and traceable to the first emerging HSCs in the E11.5 AGM, without affecting the general HPC population. IKB-deficient LT-HSCs express dormancy signature genes, are less proliferative and can robustly respond to activation stimuli such as in vitro culture and serial transplantation. At the molecular level, we find decreased PRC2-dependent H3K27me3 at the promoters of several retinoic acid signaling elements in the IKB- deficient aortic endothelium and E14.5 FL LT-HSCs. Additionally, IKB binding itself is found in the promoters of retinoic acid receptors rar in the AGM, and rar{gamma} in the LT-HSC of FL. Overall, we demonstrate that the retinoic acid pathway is over-activated in the hematopoietic clusters of IKB-deficient AGMs leading to premature dormancy of LT- HSCs that persists in the FL LT-HSCs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In this episode of the Epigenetics Podcast, we caught up with Marcela Sjöberg from the University of Chile to talk about her work on the hydroxymethylation landscape in immune cells. At the beginning of her career Marcela Sjöberg worked on Polycomb and how modifications placed by this complex modulate the binding of RNA Pol II. Later, her focus shifted to hydroxymethylated cytosine and how it is involved in the inheritance of Metastable Epialleles in mouse. More recently, the laboratory is interested in transcription factor binding motifs and how hydroxymethylation of those binding motifs modulates the binding and activity of the respective transcription factors.   References Sabbattini, P., Sjoberg, M., Nikic, S., Frangini, A., Holmqvist, P.-H., Kunowska, N., Carroll, T., Brookes, E., Arthur, S. J., Pombo, A., & Dillon, N. (2014). An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation. Molecular Biology of the Cell, 25(6), 904–915. https://doi.org/10.1091/mbc.e13-10-0628 Kazachenka, A., Bertozzi, T. M., Sjoberg-Herrera, M. K., Walker, N., Gardner, J., Gunning, R., Pahita, E., Adams, S., Adams, D., & Ferguson-Smith, A. C. (2018). Identification, Characterization, and Heritability of Murine Metastable Epialleles: Implications for Non-genetic Inheritance. Cell, 175(5), 1259-1271.e13. https://doi.org/10.1016/j.cell.2018.09.043 Westoby, J., Herrera, M.S., Ferguson-Smith, A.C. et al. Simulation-based benchmarking of isoform quantification in single-cell RNA-seq. Genome Biol 19, 191 (2018). https://doi.org/10.1186/s13059-018-1571-5 Viner, C., Johnson, J., Walker, N., Shi, H., Sjöberg, M., Adams, D. J., Ferguson-Smith, A. C., Bailey, T. L., & Hoffman, M. M. (2016). Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet [Preprint]. Bioinformatics. https://doi.org/10.1101/043794   Related Episodes The Role of DNA Methylation in Epilepsy (Katja Kobow) DNA Methylation and Mammalian Development (Déborah Bourc'his) Effects of DNA Methylation on Chromatin Structure and Transcription (Dirk Schübeler)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Jan Żylicz from the Novo Nordisk Foundation Center for Stem Cell Medicine to talk about his work on epigenetic and metabolic regulation of early development. The focus of the Żylicz Lab is studying early development and how this process is influenced by epigenetic factors. In more detail, the Team focuses on the function of chromatin modifiers in this process. Primed pluripotent epiblasts in vivo show a distinct chromatin landscape that is characterized by high levels of histone H3 lysine 9 dimethylation (H3K9me2) and rearranged Polycomb-associated histone H3 lysine 27 trimethylation (H3K27me3) at thousands of genes along the genome. However, the function of only about 100 loci is impaired. The Żylicz Lab tries to understand this process behind and also the cause of this discrepancy.   References Żylicz, J. J., Bousard, A., Žumer, K., Dossin, F., Mohammad, E., da Rocha, S. T., Schwalb, B., Syx, L., Dingli, F., Loew, D., Cramer, P., & Heard, E. (2019). The Implication of Early Chromatin Changes in X Chromosome Inactivation. Cell, 176(1–2), 182-197.e23. https://doi.org/10.1016/j.cell.2018.11.041 Dossin, F., Pinheiro, I., Żylicz, J. J., Roensch, J., Collombet, S., Le Saux, A., Chelmicki, T., Attia, M., Kapoor, V., Zhan, Y., Dingli, F., Loew, D., Mercher, T., Dekker, J., & Heard, E. (2020). SPEN integrates transcriptional and epigenetic control of X-inactivation. Nature, 578(7795), 455–460. https://doi.org/10.1038/s41586-020-1974-9   Related Episodes Epigenetics and X-Inactivation (Edith Heard) The Effects of Early Life Stress on Mammalian Development (Catherine J. Peña) DNA Methylation and Mammalian Development (Déborah Bourc'his)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Sara Wickström, Director at the Max Planck Institute for Molecular Biomedicine in Münster, to talk about her work on the effect of mechanotransduction on chromatin structure and transcription in stem cells. Sara Wickström and her team focus on the stem cell niche and how that niche affects stem cell function. In order to study the native niche and to even be able to manipulate it, the Wickström Lab was able to develop a ex vivo culture system, allowing systematic identification of factors driving stem cell dynamics and plasticity. Stem cells in the stem cell niche are exposed to external stimuli such as physical forces which control their growth, fate and self renewal. Recent work in the Wickström lab showed how mechanical signals influence transcriptional regulation, chromatin organization, and nuclear architecture and how this affects aging or lineage commitment. In this Episode we also discuss how chromatin can act as a sensor of mechanical signals taking advantage of the different physical properties of eu- and heterochromatin.   References Le, H. Q., Ghatak, S., Yeung, C. Y., Tellkamp, F., Günschmann, C., Dieterich, C., Yeroslaviz, A., Habermann, B., Pombo, A., Niessen, C. M., & Wickström, S. A. (2016). Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment. Nature cell biology, 18(8), 864–875. https://doi.org/10.1038/ncb3387 Nava, M. M., Miroshnikova, Y. A., Biggs, L. C., Whitefield, D. B., Metge, F., Boucas, J., Vihinen, H., Jokitalo, E., Li, X., García Arcos, J. M., Hoffmann, B., Merkel, R., Niessen, C. M., Dahl, K. N., & Wickström, S. A. (2020). Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage. Cell, 181(4), 800–817.e22. https://doi.org/10.1016/j.cell.2020.03.052 Koester, J., Miroshnikova, Y. A., Ghatak, S., Chacón-Martínez, C. A., Morgner, J., Li, X., Atanassov, I., Altmüller, J., Birk, D. E., Koch, M., Bloch, W., Bartusel, M., Niessen, C. M., Rada-Iglesias, A., & Wickström, S. A. (2021). Niche stiffening compromises hair follicle stem cell potential during ageing by reducing bivalent promoter accessibility. Nature cell biology, 23(7), 771–781. https://doi.org/10.1038/s41556-021-00705-x Maki, K., Nava, M. M., Villeneuve, C., Chang, M., Furukawa, K. S., Ushida, T., & Wickström, S. A. (2021). Hydrostatic pressure prevents chondrocyte differentiation through heterochromatin remodeling. Journal of cell science, 134(2), jcs247643. https://doi.org/10.1242/jcs.247643   Related Episodes Nutriepigenetics: The Effects of Diet on Behavior (Monica Dus) Epigenetic Regulation of Stem Cell Self-Renewal and Differentiation (Peggy Goodell) The Effect of Vitamin D on the Epigenome (Folami Ideraabdullah)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Emily Bernstein from Icahn Schoon of Medicine at Mount Sinai to talk about her work on MacroH2A function and the role of Polycomb proteins in its epigenetic regulation, and how this affects in stem cell development and disease. The Bernstein Lab focuses on histone variants, in particular the variants of macroH2A. Chromatin architecture is influenced by the composition of the nucleosome and, hence, exchanging the core histones for histone variants can have a major impact on chromatin structure. MacroH2A is the histone with the most variants, due to a 30kDa non-histone domain (macro domain) at their C-termini. This variation leads to many macroH2A variants, which have been found to have regulatory roles in the cell. Among other things the Bernstein Lab has shown that macroH2A is enriched at a critical set of Utx target genes whose expression is critical for the early stages of induced pluripotency.   References Kapoor, A., Goldberg, M. S., Cumberland, L. K., Ratnakumar, K., Segura, M. F., Emanuel, P. O., Menendez, S., Vardabasso, C., LeRoy, G., Vidal, C. I., Polsky, D., Osman, I., Garcia, B. A., Hernando, E., & Bernstein, E. (2010). The histone variant macroH2A suppresses melanoma progression through regulation of CDK8. Nature, 468(7327), 1105–1109. https://doi.org/10.1038/nature09590 Vardabasso, C., Gaspar-Maia, A., Hasson, D., Pünzeler, S., Valle-Garcia, D., Straub, T., Keilhauer, E. C., Strub, T., Dong, J., Panda, T., Chung, C.-Y., Yao, J. L., Singh, R., Segura, M. F., Fontanals-Cirera, B., Verma, A., Mann, M., Hernando, E., Hake, S. B., & Bernstein, E. (2015). Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma. Molecular Cell, 59(1), 75–88. https://doi.org/10.1016/j.molcel.2015.05.009 Sun, Zhen, Dan Filipescu, Joshua Andrade, Alexandre Gaspar-Maia, Beatrix Ueberheide, and Emily Bernstein. 2018. “Transcription-Associated Histone Pruning Demarcates MacroH2A Chromatin Domains.” Nature Structural & Molecular Biology 25(10):958–70. doi: 10.1038/s41594-018-0134-5.   Related Episodes Influence of Histone Variants on Chromatin Structure and Metabolism (Marcus Buschbeck) Regulation of Chromatin Organization by Histone Chaperones (Geneviève Almouzni) Variants of Core Histones: Modulators of Chromatin Structure and Function (Sandra Hake)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Serena Sanulli from Stanford University to talk about her work on Heterochromatin Protein 1 (HP1), the structure of chromatin on the atomic-scale and the meso-scale, and phase separation. The Laboratory of Serena Sanulli is interested in finding connections between changes that happen on the nucleosomal level and the resulting impact on chromatin conformation on the meso-scale. They combine methods like NMR and Hydrogen-Deuterium Exchange-MS with Cell Biology and Genetics. This enables them to dissect how cells use the diverse biophysical properties of chromatin to regulate gene expression across length and time scales. A second focus of the lab is HP1, which interacts with the nucleosome and changes its conformation, enabling the compaction of the genome into heterochromatin, effectively silencing genes in that region. A high concentration of HP1 leads to the phenomenon of phase separation in the nucleus, which the Sanulli lab is now investigating.   References Sanulli, S., Justin, N., Teissandier, A., Ancelin, K., Portoso, M., Caron, M., Michaud, A., Lombard, B., da Rocha, S. T., Offer, J., Loew, D., Servant, N., Wassef, M., Burlina, F., Gamblin, S. J., Heard, E., & Margueron, R. (2015). Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation. Molecular Cell, 57(5), 769–783. https://doi.org/10.1016/j.molcel.2014.12.020 Sanulli, S., Trnka, M. J., Dharmarajan, V., Tibble, R. W., Pascal, B. D., Burlingame, A. L., Griffin, P. R., Gross, J. D., & Narlikar, G. J. (2019). HP1 reshapes nucleosome core to promote phase separation of heterochromatin. Nature, 575(7782), 390–394. https://doi.org/10.1038/s41586-019-1669-2 Sanulli, S., & Narlikar, G. J. (2021). Generation and Biochemical Characterization of Phase‐Separated Droplets Formed by Nucleic Acid Binding Proteins: Using HP1 as a Model System. Current Protocols, 1(5). https://doi.org/10.1002/cpz1.109   Related Episodes Transcription and Polycomb in Inheritance and Disease (Danny Reinberg) Heterochromatin and Phase Separation (Gary Karpen)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Ali Shilatifard from Northwestern University to talk about his work on targeting COMPASS to cure childhood leukemia. The Shilatifard Lab studies childhood leukemia and how it can potentially be treated using epigenetic targets. The team focuses on is SET1/COMPASS, a histone H3 lysine4 methylase. Ali Shilatifard was able to purify and identify its activity, with results published in 2001 in PNAS. This protein complex is conserved from yeast to drosophila to humans. Surprisingly, the Shilatifard Team could show that the catalytic activity of COMPASS is not necessary for viability of drosophila. Furthermore, they found that catalytic activity was not the decisive feature of the complex, but rather its role in the context of chromatin structure, in particular a protein domain that only spans 80 amino acids within the 4000 amino acid protein.   References Miller, T., Krogan, N. J., Dover, J., Erdjument-Bromage, H., Tempst, P., Johnston, M., Greenblatt, J. F., & Shilatifard, A. (2001). COMPASS: A complex of proteins associated with a trithorax-related SET domain protein. Proceedings of the National Academy of Sciences, 98(23), 12902–12907. https://doi.org/10.1073/pnas.231473398 Lin, C., Garruss, A. S., Luo, Z., Guo, F., & Shilatifard, A. (2013). The RNA Pol II Elongation Factor Ell3 Marks Enhancers in ES Cells and Primes Future Gene Activation. Cell, 152(1–2), 144–156. https://doi.org/10.1016/j.cell.2012.12.015 Wang, L., Zhao, Z., Ozark, P. A., Fantini, D., Marshall, S. A., Rendleman, E. J., Cozzolino, K. A., Louis, N., He, X., Morgan, M. A., Takahashi, Y., Collings, C. K., Smith, E. R., Ntziachristos, P., Savas, J. N., Zou, L., Hashizume, R., Meeks, J. J., & Shilatifard, A. (2018). Resetting the epigenetic balance of Polycomb and COMPASS function at enhancers for cancer therapy. Nature Medicine, 24(6), 758–769. https://doi.org/10.1038/s41591-018-0034-6 Morgan, M. A. J., & Shilatifard, A. (2020). Reevaluating the roles of histone-modifying enzymes and their associated chromatin modifications in transcriptional regulation. Nature Genetics, 52(12), 1271–1281. https://doi.org/10.1038/s41588-020-00736-4   Related Episodes Cancer and Epigenetics (David Jones) Transcription and Polycomb in Inheritance and Disease (Danny Reinberg) Epigenetic Mechanisms of Aging and Longevity (Shelley Berger)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Karmella Haynes from Emory University to talk about her work on synthetic chromatin epigenetics. The Haynes lab focuses on the design of synthetic chromatin sensor proteins. The first one of this kind, the Polycomb Transcription Factor (PcTF), was published in 2011. It senses H3K27me3 and recruits effector proteins to the sites of this modification. This sensor can be brought into cancer cells to activate hundreds of silenced genes. The lab now focuses on characterizing the effects of these sensor proteins genome wide, and seeks to find a way to deliver those sensor into cancer cells, without affecting healthy cells. In this Episode we discuss how Karmella Haynes got into the field of Epigenetics, how she designed the PcTF sensor proteins, and the way she came to learn how important the right control experiments are. In the end we also discuss her activities to promote diversity and inclusion in science.   References Haynes, K. A., & Silver, P. A. (2011). Synthetic Reversal of Epigenetic Silencing. Journal of Biological Chemistry, 286(31), 27176–27182. https://doi.org/10.1074/jbc.C111.229567 Haynes, K. A., Ceroni, F., Flicker, D., Younger, A., & Silver, P. A. (2012). A Sensitive Switch for Visualizing Natural Gene Silencing in Single Cells. ACS Synthetic Biology, 1(3), 99–106. https://doi.org/10.1021/sb3000035 Daer, R. M., Cutts, J. P., Brafman, D. A., & Haynes, K. A. (2017). The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells. ACS Synthetic Biology, 6(3), 428–438. https://doi.org/10.1021/acssynbio.5b00299 Tekel, S. J., & Haynes, K. A. (2017). Molecular structures guide the engineering of chromatin. Nucleic Acids Research, 45(13), 7555–7570. https://doi.org/10.1093/nar/gkx531 Tekel, S. J., Vargas, D. A., Song, L., LaBaer, J., Caplan, M. R., & Haynes, K. A. (2018). Tandem Histone-Binding Domains Enhance the Activity of a Synthetic Chromatin Effector. ACS Synthetic Biology, 7(3), 842–852. https://doi.org/10.1021/acssynbio.7b00281   Related Episodes Transcription and Polycomb in Inheritance and Disease (Danny Reinberg) Cancer and Epigenetics (David Jones)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Axel Imhof from the Ludwig Maximilian University of Munich in Germany to talk about his work on the identification of chromatin associated proteins using mass spectrometry. As the head of the Proteomics Core Facility Axel Imhof collaborates with research groups around the world. In addition, in his own lab, he focuses on the assembly and composition of chromatin, how environmental metabolites influence epigenetic marks, and how chromatin factors can be used as markers for pathological states. In this episode we discuss what has changed in the field of mass spectrometry over the years, how Axel Imhof takes advantage of collaborations, how metabolites influence chromatin, and how he is helping to bring epigenetic profiling via mass spectrometry to the clinic.   References Bonaldi, T., Regula, J. T., & Imhof, A. (2003). The Use of Mass Spectrometry for the Analysis of Histone Modifications. In Methods in Enzymology (Vol. 377, pp. 111–130). Elsevier. https://doi.org/10.1016/S0076-6879(03)77006-2 Völker-Albert, M. C., Pusch, M. C., Fedisch, A., Schilcher, P., Schmidt, A., & Imhof, A. (2016). A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin. Molecular & Cellular Proteomics, 15(3), 945–959. https://doi.org/10.1074/mcp.M115.053553 Scharf, A. N. D., Meier, K., Seitz, V., Kremmer, E., Brehm, A., & Imhof, A. (2009). Monomethylation of Lysine 20 on Histone H4 Facilitates Chromatin Maturation. Molecular and Cellular Biology, 29(1), 57–67. https://doi.org/10.1128/MCB.00989-08 Van den Ackerveken, P., Lobbens, A., Turatsinze, J.-V., Solis-Mezarino, V., Völker-Albert, M., Imhof, A., & Herzog, M. (2021). A novel proteomics approach to epigenetic profiling of circulating nucleosomes. Scientific Reports, 11(1), 7256. https://doi.org/10.1038/s41598-021-86630-3   Related Episodes Regulation of Chromatin Organization by Histone Chaperones (Geneviève Almouzni) Transcription and Polycomb in Inheritance and Disease (Danny Reinberg)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

On this weeks episode we are discussing Cancer and developmental biology in genomics with Dr Myron Evans! Myron is currently a post doctoral research fellow at St Judes Childrens research hospital in Memphis, Tennesse! Myron discusses with us his journey into cancer biology and shares a unique perspective of life working at a research hospital. He tells us all about his work into Ybx1 and how it fine tunes Polycomb repressive complex 2 activity to direct embryonic brain development. Additionally, Myron fills us in on all the cool techniques and methods he has used during his research! Access Dr.Evans Nature communications paper here: https://www.nature.com/articles/s41467-020-17878-y Connect with Myron on twitter here: https://twitter.com/myron_evansPhD

In this episode of the Epigenetics Podcast, we caught up with Monica Dus from the University of Michigan to talk about her work on nutriepigenetics and the effects of diet on behavior. The focus of Monica Dus and her team is to study the effect of sugar on the brain and how diet has an effect on behavior. The Dus lab takes a multidisciplinary approach to answer questions like "What causes animals to overeat if they consume foods rich in sugar, salt, and fat?" and "How does such a diet alter the basic physiology and biochemistry of the brain to promote food intake and weight gain?" By doing this, they showed recently that the Polycomb Repressive Complex 2 (PRC2) plays a role in reprogramming the sensory neurons of Drosophila Melanogaster, reducing sweet sensation and hence promoting obesity when flies are fed a high sugar diet. In response to that diet the binding of PRC2 to chromatin in sweet gustatory neurons is altered and reshapes the developmental transcriptional network. In this episode we discuss how flies taste food and sugar, how sugar modulates taste, and how a high sugar diet influences the taste and amount of food flies eat.   References Monica Dus, SooHong Min, … Greg S. B. Suh (2011) Taste-independent detection of the caloric content of sugar in Drosophila (Proceedings of the National Academy of Sciences of the United States of America) DOI: 10.1073/pnas.1017096108 Christina E. May, Anoumid Vaziri, … Monica Dus (2019) High Dietary Sugar Reshapes Sweet Taste to Promote Feeding Behavior in Drosophila melanogaster (Cell Reports) DOI: 10.1016/j.celrep.2019.04.027 Daniel Wilinski, Jasmine Winzeler, … Monica Dus (2019) Rapid metabolic shifts occur during the transition between hunger and satiety in Drosophila melanogaster (Nature Communications) DOI: 10.1038/s41467-019-11933-z Anoumid Vaziri, Morteza Khabiri, … Monica Dus (2020) Persistent epigenetic reprogramming of sweet taste by diet (Science Advances) DOI: 10.1126/sciadv.abc8492 How to Science Podcast NeuroEpic Podcast   Related Episodes Transcription and Polycomb in Inheritance and Disease (Danny Reinberg) The Role of Small RNAs in Transgenerational Inheritance in C. elegans (Oded Rechavi) Epigenetic Origins Of Heterogeneity And Disease (Andrew Pospisilik)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Dr. Danny Reinberg from the New York University School of Medicine to talk about his work on transcription and polycomb in inheritance and disease. Dr. Danny Reinberg is a pioneer in the characterization of transcription factors for human RNA polymerase II. In his groundbreaking work in the 1990s, he purified the essential transcription factors and reconstituted the polymerase in vitro on both naked DNA and chromatin.  Dr. Reinberg next started focusing on the polycomb repressive complex 2 (PRC2), which is the only known methyltransferase for lysine 27 on histone H3. He biochemically characterized the PRC2 subunits EZH1 and EZH2. More recently, Dr. Reinberg has been investigating the role of PRC2 in neurons.  This interview discusses the story behind how Dr. Danny Reinberg started his research career by identifying the essential RNA polymerase transcription factors, how he discovered and characterized the polycomb repressive complex 2 (PRC2), and what his research holds for the future.   References H. Lu, L. Zawel, … D. Reinberg (1992) Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II (Nature) DOI: 10.1038/358641a0 A. Merino, K. R. Madden, … D. Reinberg (1993) DNA topoisomerase I is involved in both repression and activation of transcription (Nature) DOI: 10.1038/365227a0 G. Orphanides, W. H. Wu, … D. Reinberg (1999) The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins (Nature) DOI: 10.1038/22350 Andrei Kuzmichev, Kenichi Nishioka, … Danny Reinberg (2002) Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein (Genes & Development) DOI: 10.1101/gad.1035902 Andrei Kuzmichev, Raphael Margueron, … Danny Reinberg (2005) Composition and histone substrates of polycomb repressive group complexes change during cellular differentiation (Proceedings of the National Academy of Sciences of the United States of America) DOI: 10.1073/pnas.0409875102 Ozgur Oksuz, Varun Narendra, … Danny Reinberg (2018) Capturing the Onset of PRC2-Mediated Repressive Domain Formation (Molecular Cell) DOI: 10.1016/j.molcel.2018.05.023 Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.14.297135v1?rss=1 Authors: Wright, H., Aylwin, C. F., Toro, C. A., Ojeda, S. R., Lomniczi, A. Abstract: Female puberty is subject to Polycomb Group (PcG)-dependent transcriptional repression. Kiss1, a puberty-activating gene, is a key target of this silencing mechanism. Using a gain-of-function approach and a systems biology strategy we now show that EED, an essential PcG component, acts in the arcuate nucleus of the hypothalamus to alter the functional organization of a gene network involved in the stimulatory control of puberty. A central node of this network is Kdm6b, which encodes an enzyme that erases the PcG-dependent histone modification H3K27me3. Kiss1 is a first neighbor in the network; genes encoding glutamatergic receptors and potassium channels are second neighbors. By repressing Kdm6b expression, EED increases H3K27me3 abundance at these gene promoters, reducing gene expression throughout a gene network controlling puberty activation. These results indicate that Kdm6b repression is a basic mechanism used by PcG to modulate the biological output of puberty-activating gene networks. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.20.259994v1?rss=1 Authors: Seif, E., Kang, J. J., Sasseville, C., Senkovitch, O., Kaltashov, A., Boulier, E. L., Kapur, I., Kim, C. A., Francis, N. Abstract: Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) is important for chromatin compaction and large-scale chromatin organization. Like many SAMs, Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand mechanistically how this occurs, we analyzed the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Condensate formation depends on Ph SAM, and is enhanced by but not strictly dependent on, its polymerization activity. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. Overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, phase separation is an activity of the SAM, which, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.19.254706v1?rss=1 Authors: Eeftens, J. M., Kapoor, M., Brangwynne, C. P. Abstract: Structural organization of the genome into transcriptionally active euchromatin and silenced heterochromatin is essential for eukaryotic cell function. Heterochromatin is a more compact form of chromatin, and is associated with characteristic post- translational histone modifications and chromatin binding proteins. Phase-separation has recently been suggested as a mechanism for heterochromatin formation, through condensation of heterochromatin associated proteins. However, it is unclear how phase-separated condensates can contribute to stable and robust repression, particularly for heritable epigenetic changes. The Polycomb complex PRC1 is known to be key for heterochromatin formation, but the multitude of Polycomb proteins has hindered our understanding of their collective contribution to chromatin repression. Here, we take a quantitative live cell imaging approach to show that PRC1 proteins form multicomponent condensates through hetero-oligomerization. They preferentially seed at H3K27me3 marks, and subsequently write H2AK119Ub marks. Using optogenetics to nucleate local Polycomb condensates, we show that Polycomb phase separation can induce chromatin compaction, but phase separation is dispensable for maintenance of the compacted state. Our data are consistent with a model in which the time integral of historical Polycomb phase separation is progressively recorded in repressive histone marks, which subsequently drive chromatin compaction. These findings link the equilibrium thermodynamics of phase separation with the fundamentally non-equilibrium concept of epigenetic memory. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.27.223438v1?rss=1 Authors: Kraft, K., Yost, K. E., Murphy, S., Magg, A., Long, Y., Corces, R. M., Granja, J. M., Mundlos, S., Cech, T. R., Boettiger, A., Chang, H. Y. Abstract: Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction. This process is essential for embryonic stem cell (ESCs) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spread of H3K27me3 on the linear genome. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remains unclear. Here, we apply H3K27me3 HiChIP, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes, such as Hmx1, Wnt6 and Hoxa. Genetic deletion of H3K27me3 loop anchors revealed a coupling of Polycomb-associated genome architecture and H3K27me3 deposition evidenced by disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. Further, we find that global alterations in PRC2 occupancy resulting from an EZH2 mutant selectively deficient in RNA binding is accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 acts as a "genomic wormhole", using RNA binding to enhance long range chromosome folding and H3K27me3 spreading. Additionally, developmental gene loci have novel roles in Polycomb spreading, emerging as important architectural elements of the epigenome. Copy rights belong to original authors. Visit the link for more info

Christos Velanis works at the University of Edinburgh and discusses work published in PloS Genetics entitled ‘The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2)‘. http://blog.garnetcommunity.org.uk/wp-content/uploads/2020/07/Velanis_edit-13072020-09.32.mp3 Pumi Perera is co-first author on this work from the Goodrich lab[...] The post Christos Velanis discusses the polycomb group complex, domesticated transposons and the future of scientific funding. appeared first on Weeding the Gems.

Rea Antoniou-Kourounioti works with Martin Howard and Caroline Dean at the John Innes Centre and we discuss a recent paper in Genes and Development entitled ‘Noncoding SNPs influence a distinct phase of Polycomb silencing to destabilize long-term epigenetic memory at Arabidopsis FLC‘. It’s the latest episode in exciting saga that seeks to explain the regulation[...] The post GARNet Community podcast w Rea Antoniou-Kourounioti appeared first on Weeding the Gems.

Dr van Lohuizen speaks with ecancertv at EACR 2016 about polycomb repressors, determinants of cell fate, and their significance in genetic modification of cancer cells. He describes how they exert a stabilising effect on tumourigenic stem cells, and disease states that arise through their aberrance. Dr van Lohuizen reports on results from lung cancer patients, and outlines his goals for deeper understanding of the genomic implications in future research.

The genetic basis underlying adaptive evolution is still largely unknown. Adaptive evolution is facilitated by natural selection that acts on the genetic variation present in a population. Favoring some genetic variants over others, natural selection eventually produces adaptations that allow populations to survive in changing or new environments. Populations colonizing new habitats that differ from their original habitat are often confronted with a multitude of novel ecological constraints to which they need to adapt. A well-annotated genome and a diverse genetic toolkit make the fruit fly Drosophila melanogaster an ideal model system for studying the genetics underlying adaptation. As a cosmopolitan species, D. melanogaster has adapted to a wide range of thermal environments. Despite having a tropical origin in southern-central Africa, it has successfully settled in temperate environments around the world. Thermal adaptations that have helped to deal with the greater range and variability in temperature as well as low-temperature extremes have been required to prosper in temperate environments. Chromatin-based gene regulation is known to be disrupted by varying temperatures. Variation in the temperature, at which flies live, result in varying expression levels of Polycomb group (PcG) regulated genes with higher expression at lower temperatures. Chapter 1 and 2 of this thesis aim to answer the question whether this thermosensitivity of PcG regulation has been detrimental for colonizing temperate environments and thus needed to be buffered by natural selection. Thermosensitivity of PcG regulation was observed in different natural populations of D. melanogaster. A lower degree of thermosensitive expression was consistently found for populations from temperate climates when compared to those from the tropics. In Chapter 1, evidence is presented for positive selection acting on the polyhomeotic (ph) gene region to reduce thermosensitivity of PcG regulation in temperate populations from Europe. The targets of selection appear to be single nucleotide polymorphisms (SNPs) in a relatively small cis-regulatory region between the two PcG target genes polyhomeotic proximal (ph-p) and CG3835 that are highly differentiated between European and African populations. Using reporter gene assays, it was demonstrated that these SNPs influence gene expression and that the European alleles confer reduced thermosensitivity of expression in contrast to the African alleles. In Chapter 2, thermosensitivity of another PcG target gene, vestigial (vg), was investigated in six natural populations including four temperate populations from high-altitude Africa and central to high-latitude Europe, and two tropical populations from the ancestral species range. All four temperate populations exhibited a lower degree of thermosensitive expression than the two tropical populations. The underlying mechanisms of increased buffering, however, seem to differ between these temperate populations. Thermal adaptation to temperate environments also includes dealing with low-temperature extremes. Severe cold stress is a main limiting factor imposed on D. melanogaster by temperate climates. Increased cold tolerance in temperate populations is thought to have evolved by natural selection. Cold tolerance is a quantitative trait that appears to be highly polygenic and has been mapped to different quantitative trait loci (QTL) in the genome. In Chapter 3, such a QTL region was fine-mapped to localize causal genes for increased cold tolerance in temperate flies. As a result, brinker (brk) was identified as a new candidate gene putatively involved in cold stress adaptation.

Cellular gene regulation depends on fundamental epigenetic mechanisms, but epigenetic modifications also govern the regulation of the life cycle of Epstein-Barr virus (EBV). Promoter usage during latency depends on DNA methylation of the viral genome and CpG-methylation of certain promoters with meZREs is an indispensable prerequisite to switch from the latent to the lytic phase. In my thesis, I wanted to assess the underlying epigenetic principles of EBV’s gene regulation during the establishment of latency and upon lytic reactivation. My results suggested a new classification of viral promoters and phases of gene regulation that depend on the epigenetic state of the viral chromatin. According to this new model, EBV’s infectious cycle consists of an initial abortive lytic, a latent, and a productive lytic phase, and the viral promoters can be classified into default-on, poised-on, and poised-off promoters. Default-on promoters are immediately active upon infection of primary B cells leading to the so-called abortive lytic phase of an EBV infection. Default-on promoters encounter the cell in an environment that supports binding of the basal transcription machinery to activate viral gene transcription. Default-on promoters include the promoters of BALF1, BHRF1, BZLF1, BRLF1, BNLF2a, BCRF1, and Wp. The protein products are indispensible for the permanent establishment of EBV’s genome in latently infected cells supporting growth transformation, immune evasion, and anti-apoptotic cellular pathways. Default-on promoters are epigenetically silenced upon occupancy of EBV’s DNA with nucleosomes very early after infection and a switch to poised-on promoters is initiated. Poised-on promoters embody an epigenetically active state upon infection, but their activation requires an additional, virus-encoded factor to allow initiation of transcription. Wp-induced expression of EBNA1 promotes the switch to the poised-on promoter Cp to sustain long-term EBNA expression. Other poised-on genes including the viral structural proteins are not provided with their cofactor initially. During latency, these promoters are repressed through compaction of chromatin by high nucleosome occupancy, trimethylation of H3K27, and a stable transmission of repressive modifications by Polycomb-mediated long-term silencing. The establishment of a defined DNA methylation pattern on EBV’s DNA further represses poised-on promoters. DNA methylation is a prerequisite for the activation of a third promoter class, termed default-off promoters. Default-off promoters are bound and transactivated by BZLF1 in a methylation-dependent manner. Upon infection of primary B cells, EBV’s DNA is completely unmethylated, impeding an early expression of default-off genes. Only two to three weeks post infection the viral genome has acquired a proper epigenetic configuration that supports transcription of default-off genes. Binding of BZLF1 alone does not suffice to recruit the cellular transcription machinery including RNA polymerase II, but the chromatin requires remodeling, including a loss of nucleosomes and repressive modifications at default-off promoter sites. Default-off genes encode the viral lytic DNA replication machinery. The newly synthesized DNA templates lack epigenetic modifications because lytic DNA amplification is uncoupled from cellular DNA replication, eliminating the epigenetic maintenance mechanisms during the synthesis of viral progeny. As a consequence, default-on promoters and silenced poised-on promoters, which rely on unmethylated, epigenetically naïve templates, become also activated in the onset of the lytic phase. Silenced poised-on promoters require additionally a viral cofactor for their activation. This so-far unknown factor is probably provided upon lytic DNA amplification allowing the transcription of genes encoding for structural proteins that are necessary for the packaging of viral progeny. The released EBV progeny is epigenetically unmodified and ready to infect other cells. In essence, the regulation of EBV’s life cycle by epigenetic mechanisms is a paradigm for viral coevolution with its host. Repressive epigenetic modifications are common cellular defense mechanism to fight invading pathogens. EBV has hijacked this system for the regulation of promoter usage during its own life cycle, which has become a key principle of EBV’s success in infecting and persisting in its host.

In der vorliegenden Arbeit wurden die beiden Histon-Methyltransferasen Su(var)3-9 und E(Z) aus Drosophila melanogaster charakterisiert. Die Histonmethylierung als Modifikation war schon länger bekannt gewesen, bis zum Jahr 2000 war jedoch vor allem die Acetylierung etwas genauer untersucht worden. Su(var)3-9 war die einzige bekannte Histon-Lysin-Methyltransferase, als diese Arbeit begonnen wurde. Zur Charakterisierung wurde das myc-getagte Enzym aus Drosophila-Kernextrakt durch Affinitätschromatographie aufgereinigt und zunächst die Substratspezifität festgestellt. Wie das humane Enzym Suv39H1 methyliert es ebenfalls spezifisch H3-K9 (Lysin 9 im Histon H3). Das aus den Kernextrakten aufgereinigte Enzym besitzt aber auch die Fähigkeit, ein an H3-K9 präacetyliertes Substrat zu methylieren. Die Vermutung, dass Su(var)3-9 mit einer Histondeacetylase assoziiert ist, konnte durch Verwendung von TSA als HDAC-Inhibitor bestätigt werden. Es stellte sich heraus, dass HDAC1 (Rpd3) mit Su(var)3-9 assoziiert ist. Um das Enzym besser untersuchen zu können, wurde es als Volllängenprotein und als Deletionsmutante in E. coli exprimiert. Die Aufreinigung des rekombinanten Enzyms sowie seine Lagerbedingungen wurden optimiert. Das Volllängenprotein Su(var)3-9 liegt – wie durch Gelfiltration festgestellt - als Dimer vor, die Interaktion mit sich selbst ist über den N-Terminus vermittelt. Su(var)3-9 bindet an sein eigenes, bereits methyliertes Substrat. Dies wurde an Peptiden untersucht, die den ersten 20 Aminosäuren des Histons H3 entsprechen, und entweder an Lysin 9 dimethyliert oder unmodifiziert waren. Die Interaktion mit dem methylierten Substrat ist auf die Chromodomäne von Su(var)3-9 zurückzuführen, ist jedoch schwächer als die Wechselwirkung von HP1 mit methyliertem H3-K9. Des weiteren wurde eine Drosophila-Zelllinie stabil mit Su(var)3-9 transfiziert. Das überexprimierte Protein ist jedoch nur schwach aktiv. Die Tatsachen, dass Su(var)3-9 mit HDAC1 interagiert sowie mit seinem eigenen Substrat assoziiert, ermöglichen die Aufstellung von Hypothesen über die bis jetzt kaum erhellte Ausbreitung von Heterochromatin in euchromatische Bereiche. Durch die Wechselwirkung mit der Deacetylase könnte Su(var)3-9 auch in aktiv transkribierte Bereiche vordringen und diese methylieren. Die Acetylierung, Zeichen für aktive Transkription, würde durch die Methylierung ersetzt werden. Die Interaktion mit seinem umgesetzten Substrat könnte verhindern, dass das Enzym sich nach der Reaktion entfernt, vielmehr könnte Su(var)3-9 entlang eines DNA-Stranges sukzessive alle Nukleosomen methylieren. Die darauffolgende Bindung von HP1 an methyliertes H3-K9 könnte den heterochromatischen Charakter des Chromatins verstärken und für längere Zeit festlegen. Aus Drosophila-Kernextrakten gelang es weiterhin, den E(Z)/ESC-Komplex über Säulenchromatographie aufzureinigen. Dieser enthält neben E(Z), ESC, p55 und Rpd3 auch Su(z)12. E(Z), ESC und Su(z)12 gehören der Polycomb-Gruppe an. Deren Funktion ist die dauerhafte Repression der homöotischen Gene. Sie spielen daher eine wichtige Rolle im „Zellgedächtnis“ während der frühen Entwicklung von Drosophila. Es konnte gezeigt werden, dass der E(Z)/ESC-Komplex Lysin 9 sowie Lysin 27 im Histon H3 methyliert. Außerdem wurde in vitro ein Teilkomplex aus rekombinantem E(Z), p55 und ESC rekonstituiert, der das Histon H3 methylieren kann. Ein Teilkomplex, der E(Z) mit mutierter SET-Domäne enthält, ist nicht in der Lage, H3 zu methylieren. Die Vorhersage, dass E(Z) aufgrund seiner SET-Domäne eine Methyltransferase sein müsse, konnte durch vorliegende Untersuchungen bestätigt werden. Polycomb ist ein weiteres Protein aus der Polycomb-Gruppe. In dieser Arbeit konnte gezeigt werden, dass dieses Protein spezifisch an das Histon H3 bindet, das an K27 trimethyliert ist. Polycomb besitzt wie HP1 eine Chromodomäne. Aus den vorliegenden Daten kann folgendes Modell aufgestellt werden: Nach der Methylierung von H3-K9 sowie H3-K27 durch den E(Z)/ESC-Komplex in homöotischen Genen, die schon abgeschaltet sind und weiterhin reprimiert werden müssen, bindet Polycomb an dieses Methylierungsmuster. Polycomb befindet sich in einem großen Komplex mit weiteren Polycomb-Gruppen-Proteinen. Die Bindung dieses Komplexes an Chromatin könnte ein denkbarer Mechanismus sein, wie die dauerhafte Repression der homöotischen Gene vermittelt wird. Um den E(Z)/ESC-Komplex genauer untersuchen zu können, wurden Viren für das Baculosystem hergestellt, so dass eine Einzel- oder auch Coexpression der Proteine möglich ist. Die Aktivität von E(Z), das im Baculosystem exprimiert wurde, ist nicht besonders hoch. Es bindet unter den in dieser Arbeit verwendeten Bedingungen weder an DNA, noch an Histone noch an H3-Peptide, die methyliert sind. Innerhalb des E(Z)/ESC-Komplexes bindet E(Z) an p55, Rpd3, ESC sowie Su(z)12. Su(z)12 interagiert mit p55, Rpd3 und E(Z). Die weiteren Interaktionen werden am besten durch eine bildliche Darstellung (siehe Abb. 86) vermittelt. In einem Luciferase-Assay wurde eine repressive Wirkung von E(Z) festgestellt. Dieses Experiment bedarf allerdings eines aktivierten Systems. Ferner muss durch Mutationsanalysen sichergestellt werden, dass die repressive Wirkung auf die Methyltransferase-Aktivität von E(Z) zurückzuführen ist. Kürzlich wurde entdeckt, dass E(Z) sowie Su(z)12 in verschiedenen Tumoren überexprimiert sind. Noch ist weder deren Funktion in den Tumorzellen klar, noch weiss man, ob die Überexpression der Grund oder eine Folge der Tumorbildung ist, noch kennt man alle Zielgene, die durch eine Überexpression von E(Z) und Su(z)12 beeinflusst werden. In nächster Zeit sind hier Einsichten in die Wirkungsweise von E(Z), Su(z)12 und anderen Polycomb-Gruppen-Proteinen zu erwarten.