Podcasts about h3k27me3

A new editorial paper was published in Oncotarget's Volume 14 on May 12, 2023, entitled, “Targeting H3K27me3 loss in pediatric brain tumors - a perspective on epigenetically guided cancer therapy.” High-grade tumors of the central nervous system, including medulloblastoma, ependymoma and DMG (diffuse midline glioma, formerly known as DIPG (diffuse intrinsic pontine glioma)), constitute a major challenge in pediatric oncology. They are characterized by an aggressive growth and high relapse rates and claim the lives of many pediatric cancer patients. Both medulloblastoma and ependymoma are treated with surgical resection followed by adjuvant radiation therapy. DMG, on the other hand, diffusely infiltrates the brain stem making a resection virtually impossible. Thus, radiotherapy is the primary treatment modality for this tumor. While radiation temporarily attenuates the progression of DMG this brain cancer remains incurable and most children succumb to their disease. In his new editorial, Dr. Michael Goldstein from Johns Hopkins University School of Medicine discusses the extensively investigated molecular profiles of the aforementioned pediatric brain tumors demonstrating distinct epigenetic traits. “Strikingly, a global loss of H3K27 tri-methylation (H3K27me3) as a result of the dominant-negative histone H3K27M mutation was found to be a hallmark of DMG occurring in the majority of the tumors.” H3K27me3 is a product of the EZH2 histone methyltransferase affecting multiple cellular processes including transcription, chromatin structure and DNA damage response. Similarly, the aggressive PFA ependymoma subgroup is characterized by a lack of H3K27me3 due to an overexpression of the EZHIP protein that acts as an EZH2 inhibitor whereas less aggressive PFB tumors retain normal H3K27me3 levels. However, no comprehensive analysis of H3K27me3 expression patterns in medulloblastoma has been performed and the significance of this epigenetic mark in pediatric brain tumors has remained unknown. “To address this, we have investigated the levels of the H3K27me3 histone mark and its role in treatment response of non-WNT/SHH medulloblastoma comprising group 3 and group 4 tumors. We demonstrated that about 50% of the tumors in patients with group 3 and group 4 medulloblastoma are H3K27me3 deficient. Strikingly, loss of H3K27me3 was associated with high relapse rates and poor survival.” DOI - https://doi.org/10.18632/oncotarget.28427 Correspondence to - Michael Goldstein - mgolds33@jhu.edu Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28427 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, epigenetics, brain tumor, EZH2, H3K27me3, radiation therapy About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.545208v1?rss=1 Authors: Oikawa, T., Hasegawa, J., Ohnishi, N., Onodera, Y., Hashimoto, A., Sasaki, J., Sasaki, T., Ueda, K., Sabe, H. Abstract: H3.1 is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, although the underlying mechanism remains unknown. Here we show that p53 is involved in this process. CTDNEP1 converts phosphatidic acid (PA) into diacylglycerol, and EZH2 generates H3K27me3. p53 increased CTDNEP1 and decreased EZH2 in the nuclear H3.1 interactome of the G1/S phase. Moreover, H3.1 bound robustly to PA but not to diacylglycerol. p53 deficiency caused perinuclear accumulation of EZH2-modified H3K27me3 of non-nucleosomal histones during the G1/S phase. p53 induced the expression of TMEM255A, which reduced nuclear PA levels by increasing CTDNEP1 levels. Therefore, H3.1 entering the nucleus in the absence of p53 may be trapped near the nuclear envelope (NE) and epigenetically marked as repressive without forming nucleosomes. Our study identified the NE as a novel target of p53, in which p53 downregulates nuclear PA levels to normalize H3.1 nuclear entry and epigenetic modification. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

A new editorial paper was published in Oncotarget's Volume 14 on February 11, 2023, entitled, “Unveiling the non-canonical functions of EZH2 in prostate cancer.” Prostate cancer (PCa) is ranked as the second leading cause of cancer-related death among American men excluding skin cancer. In this new editorial, researchers Yang Yi, Yanqiang Li, Kaifu Chen, and Qi Cao from Northwestern University's Feinberg School of Medicine discuss a well-known oncogenic driver in PCa: enhancer of zeste homolog 2 (EZH2)—canonically known for the functions as the catalytic subunit of Polycomb Repressive Complex 2 (PRC2) that deposes histone H3 lysine 27 mono, di-, and tri-methylation (H3K27me1-3) and represses transcription. “Although the oncogenic role of EZH2 mainly relies on its enzymatic activity and the PRC2, accumulating evidence suggests that targeting the lysine methyltransferase activity of EZH2 alone is ineffective in treating EZH2-dependent malignancies including PCa [4, 5].” Hence, deeply investigating the multifaceted tumorigenic functions of EZH2 will shed new light on understanding the etiology of PCa. It is noteworthy that two recent studies published in Nature Cell Biology and Oncogene by Yi et al. described previously unrecognized roles of EZH2 in regulation of translation and coactivation of transcription, respectively. In both cases, EZH2 exerts oncogenic functions independently of PRC2 and H3K27me3 to promote tumorigenesis and aggressiveness in PCa. “In summary, both articles by Yi et al. emphasized the significance of non-canonical functions of EZH2 during PCa development, which may provide novel insights into the advancement of EZH2-targeting strategies to treat PCa patients. In fact, a new wave has been ushed for the discovery of EZH2 inhibitors to eliminate both the catalytic and non-catalytic activities of EZH2 [12–14]. Will these newly developed EZH2 degraders be successfully applied in PCa therapy? Will additional noncanonical functions of EZH2 be characterized in the PCa model? Let's eagerly wait and see.” Full editorial: DOI: https://doi.org/10.18632/oncotarget.28357 Correspondence to: Qi Cao - qi.cao@northwestern.edu Keywords: EZH2, prostate cancer, FBL, CDCA8, E2F1 Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28357 Keywords - EZH2, prostate cancer, FBL, CDCA8, E2F1 About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.02.526881v1?rss=1 Authors: Mätlik, K., Govek, E.-E., Paul, M. R., Allis, C. D., Hatten, M. E. Abstract: Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature, multipolar neurons. Here we use RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron, the mouse cerebellar granule cell (GC). We find that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent H3K4me3 and H3K27me3 domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes and induced the downregulation of migration-related genes and upregulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons. 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/2023.01.31.526222v1?rss=1 Authors: Dror, E., Fagnocchi, L., Wegert, V., Apostle, S., Grimaldi, B., Gruber, T., Panzeri, I., Heyne, S., Hoeffler, K. D., Kreiner, V., Ching, R., Lu, T. T.-H., Semwal, A., Johnson, B., Senapati, P., Lempradl, A. M., Schones, D., Imhof, A., Shen, H., Pospisilik, J. A. Abstract: Despite the recent explosion in surveys of cell-type heterogeneity, the mechanisms that specify and stabilize highly related cell subtypes remain poorly understood. Here, focusing initially on exploring quantitative histone mark heterogeneity, we identify two major sub-types of pancreatic {beta}-cells ({beta}HI and {beta}LO). {beta}HI and {beta}LO cells differ in their size, morphology, cytosolic and nuclear ultrastructure, transcriptional output, epigenomes, cell surface marker, and function. Importantly, {beta}HI and {beta}LO cells can be FACS separated live into CD24+ ({beta}HI) and CD24- ({beta}LO) fractions. From an epigenetic viewpoint, {beta}HI-cells exhibit ~4-fold higher levels of H3K27me3, more compacted chromatin, and distinct chromatin organization that associates with a specific pattern of transcriptional output. Functionally, {beta}HI cells have increased mitochondrial mass, activity, and insulin secretion both in vivo and ex vivo. Critically, Eed and Jmjd3 loss-of-function studies demonstrate that H3K27me3 dosage is a significant regulator of {beta}HI / {beta}LO cell ratio in vivo, yielding some of the first-ever specific models of {beta}-cell sub-type distortion. {beta}HI and {beta}LO sub-types are conserved in humans with {beta}HI-cells enriched in human Type-2 diabetes. These data identify two novel and fundamentally distinct {beta}-cell subtypes and identify epigenetic dosage as a novel regulator of {beta}-cell subtype specification and heterogeneity. 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/2023.01.13.523596v1?rss=1 Authors: Mannion, A. J., Zhao, H., Zhang, Y., von Wright, Y., Bergman, O., Saharinen, P., Holmgren, L. Abstract: Endothelial cells (ECs) are constantly exposed to mechanical forces in the form of fluid shear stress, extracellular stiffness, and cyclic strain. How these forces are sensed by ECs remains an understudied aspect in the homeostatic regulation of the circulatory system. Angiomotin-like 2 (AmotL2) is localised to EC junctions and is required for alignment and actin reorganisation under conditions of high shear stress. Here we show that AmotL2 crucially regulates transcription and promotor activity of the YAP gene. Functionally, density-dependent proliferation of ECs in vitro and proliferation of a subpopulation of ECs within the inner aortic arch, were both reliant on AmotL2 and Yap/Taz endothelial expression in vivo. Mechanistically, depletion of AmotL2 led to altered nuclear morphology, chromatin accessibility and suppression of YAP-promotor activity through increased H3K27me3 mediated by the polycromb repressive complex component EZH2. Our data describe a previously unknown role for junctional mechanotransduction in shaping the epigenetic landscape and transcriptional regulation of YAP in vascular homeostasis. 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 Tim Petros from the Eunice Kennedy Shriver National Institute of Child Health and Human Development at the NIH to talk about his work on Single Cell Epigenomics in Neuronal Development.  The Petros lab focuses on “interneurons”, their diversity and how environmental signals interact to generate this diversity. This subgroup of neurons comprise about 20% of neutrons in the brain, however, they are the primary source of inhibition. Furthermore, interneurons are critical components in modulating information flow throughout the nervous system. The Petros lab seeks to uncover the genetic programs that lead to the incredible diversity in interneurons, as well as how the local environment influences this process.  To lay a foundation for this and to provide a data-base for other researchers the Petros lab generated an epigenome atlas of neural progenitor cells of the mouse brain. This data includes scRNA-Seq, snATAC-Seq, CUT&Tag (H3K4me3, H3K27me3), CUT&RUN (H3K27ac), Hi-C and Capture-C. This data can be downloaded at the link below:   https://www.nichd.nih.gov/research/atNICHD/Investigators/petros/data-sharing    References Datasets: https://www.nichd.nih.gov/research/atNICHD/Investigators/petros/data-sharing Quattrocolo G, Fishell G, Petros TJ. Heterotopic Transplantations Reveal Environmental Influences on Interneuron Diversity and Maturation. Cell Rep. 2017 Oct 17;21(3):721-731. doi: 10.1016/j.celrep.2017.09.075. PMID: 29045839; PMCID: PMC5662128. Dongjin R Lee, Christopher Rhodes, Apratim Mitra, Yajun Zhang, Dragan Maric, Ryan K Dale, Timothy J Petros (2022) Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain eLife 11:e71864 https://doi.org/10.7554/eLife.71864 Rhodes, C. T., Thompson, J. J., Mitra, A., Asokumar, D., Lee, D. R., Lee, D. J., Zhang, Y., Jason, E., Dale, R. K., Rocha, P. P., & Petros, T. J. (2022). An epigenome atlas of neural progenitors within the embryonic mouse forebrain. Nature communications, 13(1), 4196. https://doi.org/10.1038/s41467-022-31793-4   Related Episodes The Role of Histone Dopaminylation and Serotinylation in Neuronal Plasticity (Ian Maze) Single-Cell Technologies using Microfluidics (Ben Hindson, CSO of 10x Genomics) The Role of DNA Methylation in Epilepsy (Katja Kobow)   Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com

A new research paper was published in Aging (Aging-US) on the cover of Volume 14, Issue 12, entitled, “Time makes histone H3 modifications drift in mouse liver.” Aging is known to involve epigenetic histone modifications, which are associated with transcriptional changes, occurring throughout the entire lifespan of an individual. “So far, no study discloses any drift of histone marks in mammals which is time-dependent or influenced by pro-longevity caloric restriction treatment.” To detect the epigenetic drift of time passing, researchers—from Istituto di Ricovero e Cura a Carattere Scientifico, University of Urbino ‘Carlo Bo', University of Milan, and University of Padua—determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. “In this study, we used chromatin immunoprecipitation sequencing technology to acquire 108 high-resolution profiles of H3K4me3, H3K4me1, H3K27me3 and H3K27ac from the livers of mice aged between 3 months and 12 months and fed 30% caloric restriction diet (CR) or standard diet (SD).” The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions. “In conclusion, while our data do not establish that the observed changes in H3 modification are causally involved in aging, they indicate age, buffered by caloric restriction, releases the histone H3 marking process of transcriptional suppression in gene desert regions of mouse liver genome most of which remain to be functionally understood.” DOI: https://doi.org/10.18632/aging.204107 Corresponding Author: Marco Giorgio - marco.giorgio@unipd.it Keywords: epigenetics, aging, histones, ChIP-seq, diet Sign up for free Altmetric alerts about this article: https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.204107 About Aging-US: Launched in 2009, Aging (Aging-US) publishes papers of general interest and biological significance in all fields of aging research and age-related diseases, including cancer—and now, with a special focus on COVID-19 vulnerability as an age-dependent syndrome. Topics in Aging go beyond traditional gerontology, including, but not limited to, cellular and molecular biology, human age-related diseases, pathology in model organisms, signal transduction pathways (e.g., p53, sirtuins, and PI-3K/AKT/mTOR, among others), and approaches to modulating these signaling pathways. Follow Aging on social media: SoundCloud – https://soundcloud.com/Aging-Us Facebook – https://www.facebook.com/AgingUS/ Twitter – https://twitter.com/AgingJrnl Instagram – https://www.instagram.com/agingjrnl/ YouTube – https://www.youtube.com/agingus​ LinkedIn – https://www.linkedin.com/company/aging/ Pinterest – https://www.pinterest.com/AgingUS/ For media inquiries, please contact media@impactjournals.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

Part. 1に続きDANDRITE研究所で来夏独立予定の北沢太郎さん(@kitazawa_taro)にお越しいただき、直近のNature Genetics論文の裏話と、今後行うEngram関連のプロジェクトについて伺いました（11/4収録） Shownotes: 1st著者Bipartite 論文 Kitazawa et al., Nat Genet 2021 バレレットの発生について1st 著者としての総説 Kitazawa et al., Curr Opin Neurobiol 2018 最初期遺伝子、Immediate early gene (IEG) ニューロ、エピジェネティクス、クロマチンについて総説たち 1 2 ChIPseq、抗体を使って特定のタンパク質と相互作用のあるゲノム領域を同定 H3K4me2= パーミッシブにアクティブなマーク H3K27ac=強く転写アクティブ H3K27me3=ポリコーム (Polycomb) 依存の抑制性マーク ATACseq、トランスポゼースを使ってオープンなクロマチンの存在するゲノム領域を同定 今までよく言われていたポリコーム・バイバレント(bivalent)→H3K27me3とH3K4meの共存 今回発見したポリコーム・バイパタイト(bipartite)→IEGのプロモーターがH3K27acでアクティブ、ジーンボディーがH3K27me3抑制性 クラウンチングスタートのFMIプレスリリース JST創発的研究支援事業、破壊的イノベーション？ エングラム細胞のシステムスコンソリデーション中のステートシフトの論文、レビュー 1 2 リアルエングラム仮説についてレビュー、ジョセリン＆利根川 再活性化されてこそエングラムという前提でのレビュー、ジェノミクスの観点から BICCN回はこちら 投射細胞発生に関してジャボドンBioRxiv、最近Natureに出ましたね。リバイス3年半。。。 ムルシックフローゲルのV1投射論文 キャロッサル：対側投射 IEGのヘテロ性に関して、例えばこれ ヌタウナギのエンブリオの研究ができるのは世界中でRIKEN倉谷研究室だけ。参考文献 1 2 3 4 エピジェネティックな記憶とかDavid Allisのレビュー グリーンバーグのYap et al., Neuron 2018 レビュー Li Huei Tsaiのところのリーセント記憶エングラム・エピジェネティック論文 HDAC2論文 エンハンサーのモチーフ解析から転写因子を同定するアプローチ。FMIで開発したパイプライン Editorial notes: うちでピザ食べながら録ったんですが、今後もin-person収録定期的にやっていきたいと思いました。ただ欠点もあって、近くにマイク２本立てた事によるカブりの処理に一苦労。指向性高めのマイクじゃないと厳しいな...買うか(萩原) 休憩でピザ食べ過ぎて苦しかったぜ。しゃべり続けるのって大変なのね。 (北沢) スイスピザ美味しそう。。Part 1聞いてる時はまさかここまでエングラムの話題になるとは想像していませんでした（１リスナー並感）。in-person収録やってみたいですね〜　(宮脇)

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

Dr. Evan Noch interviews Drs. Farshad Nassiri and Justin Wang from the team of Dr. Gelareh Zadeh about their recently published paper entitled “Loss of H3K27me3 in meningiomas” published online in Neuro-Oncology in May 2021.

Dr. Maya Graham interviews Drs. Ghazaleh Tabatabai and Felix Behling about their paper entitled “H3K27me3 loss indicates an increased risk of recurrence in the Tübingen meningioma cohort” published online in Neuro-Oncology in December 2020.

In this episode of the Epigenetics Podcast, we caught up with Céline Vallot from L'Institut Curie in Paris to discuss her work on investigating the dynamics of epigenetic plasticity in cancer with single cell technologies. During her Post-Doc years Céline Vallot worked on the inactive X chromosome. Using RNA-Seq she discovered a novel long noncoding RNA (lncRNA) called XACT. This lncRNA is expressed from and coats the active X chromosome in human pluripotent cells. Céline Vallot also showed that XACT is specific to humans and cannot be found in mice. After starting her own lab, Céline Vallot began to focus on Single Cell Epigenomics in Cancer. She and her team developed a high-throughput single-cell ChIP-seq approach which relies on a droplet microfluidics platform to profile the chromatin landscape of thousands of cells. By doing so they could show that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature. This would have been impossible with common bulk approaches. These cells are characterized by the loss of H3K27me3, which leads to stable transcriptional repression, influencing genes that are known to promote resistance to treatment. In this episode we discuss how Céline Vallot had her once-in-a-lifetime scientific eureka-moment, when, during her postdoc, she first saw XACT coating the whole X-Chromosome in humans and then how she pivoted when starting her own lab and focuses now on single-cell epigenomics in cancer.   References Céline Vallot, Christophe Huret, … Claire Rougeulle (2013) XACT , a long noncoding transcript coating the active X chromosome in human pluripotent cells (Nature Genetics) DOI: 10.1038/ng.2530 Kevin Grosselin, Adeline Durand, … Annabelle Gérard (2019) High-throughput single-cell ChIP-seq     identifies heterogeneity of chromatin states in breast cancer (Nature Genetics) DOI: 10.1038/s41588-019-0424-9 Pacôme Prompsy, Pia Kirchmeier, … Céline Vallot (2020) Interactive analysis of single-cell epigenomic landscapes with ChromSCape (Nature Communications) DOI: 10.1038/s41467-020-19542-x Justine Marsolier, Pacôme Prompsy, … Céline Vallot (2021) H3K27me3 is a determinant of chemotolerance in triple-negative breast cancer (bioRxiv) DOI: 10.1101/2021.01.04.423386   Related Episodes Dosage Compensation in Drosophila (Asifa Akhtar) Epigenetics and X-Inactivation (Edith Heard) 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

Dr. Iyad Alnahhas talks with Dr. Felix Sahm about his recently published article in Neuro-Oncology entitled: "A subset of pediatric-type thalamic gliomas share a distinct DNA methylation profile, H3K27me3 loss and frequent alteration of EGFR," published online on November 1, 2020.

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/2020.09.02.279703v1?rss=1 Authors: Bartosovic, M., Kabbe, M., Castelo-Branco, G. Abstract: The development of the mouse central nervous system (CNS) involves coordinated execution of transcriptional and epigenetic programs. These programs have been extensively studied through single-cell technologies in a pursuit to characterize the underlying cell heterogeneity. However, histone modifications pose additional layers of both positive and negative regulation that defines cellular identity. Here we show that the Cut&Tag technology can be coupled with a droplet-based single cell library preparation platform to produce high quality chromatin modifications data at a single cell resolution in tens of thousands of cells. We apply single-cell Cut&Tag (scC&T) to probe histone modifications characteristic of active promoters (H3K4me3), active promoters and enhancers (H3K27ac), active gene bodies (H3K36me3) and inactive regions (H3K27me3) and generate scC&T profiles for almost 50,000 cells. scC&T profiles of each of these histone modifications were sufficient to determine cell identity and deconvolute at single cell level regulatory principles such as promoter bivalency, spreading of H3K4me3 and promoter-enhancer connectivity. Moreover, we used scC&T to investigate the single-cell chromatin occupancy of transcription factor Olig2 and the cohesin complex component Rad21. Our results indicate that analysis of histone modifications and transcription factor occupancy at a single cell resolution can provide unique insights of epigenomic landscapes in the CNS. We also provide an online resource that can be used to interactively explore the data at https://castelobranco.shinyapps.io/BrainCutAndTag2020/. 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/2020.08.12.247361v1?rss=1 Authors: Wang, G., Xia, B., Zhou, M., Lv, J., Zhao, D., Li, Y., Bu, Y., Wang, X., Cooke, J. P., Cao, Q., Lee, M., Zhang, L., Chen, K. Abstract: Numerous studies of relationship between epigenomic features have focused on their strong correlation across the genome, likely because such relationship can be easily identified by many established methods for correlation analysis. However, two features with little correlation may still colocalize at many genomic sites to implement important functions. There is no bioinformatic tool for researchers to specifically identify such feature pair. Here, we develop a method to identify feature pair in which two features have maximal colocalization but minimal correlation (MACMIC) across the genome. By MACMIC analysis of 3,385 feature pairs in 15 cell types, we reveal a dual role of CTCF in epigenetic regulation of cell identity genes. Although super-enhancers are associated with activation of target genes, only a subset of super-enhancers colocalized with CTCF regulate cell identity genes. At super-enhancers colocalized with CTCF, the CTCF is required for the active marker H3K27ac in cell type requiring the activation, and also required for the repressive marker H3K27me3 in other cell types requiring the repression. Our work demonstrates the biological utility of the MACMIC analysis and reveals a key role for CTCF in epigenetic regulation of cell identity. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.10.236562v1?rss=1 Authors: Roule, T., Ariel, F., Hartmann, C., Crespi, M., Blein, T. Abstract: Clustered organization of biosynthetic non-homologous genes is emerging as a characteristic feature of plant genomes. The co-regulation of clustered genes seems to largely depend on epigenetic reprogramming and three-dimensional chromatin conformation. Here we identified the long noncoding RNA (lncRNA) MARneral Silencing (MARS), localized inside the Arabidopsis marneral cluster, and which controls the local epigenetic activation of its surrounding region in response to ABA. MARS modulates the POLYCOMB REPRESSIVE COMPLEX 1 (PRC1) component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1) binding throughout the cluster in a dose-dependent manner, determining H3K27me3 deposition and chromatin condensation. In response to ABA, MARS decoys LHP1 away from the cluster and promotes the formation of a chromatin loop bringing together the MARNERAL SYNTHASE 1 (MRN1) locus and a distal ABA-responsive enhancer. The enrichment of co-regulated lncRNAs in clustered metabolic genes in Arabidopsis suggests that the acquisition of novel noncoding transcriptional units may constitute an additional regulatory layer driving the evolution of biosynthetic pathways. 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

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.23.218552v1?rss=1 Authors: van der Velde, A. G., Fan, K., Tsuji, J., Moore, J. E., Purcaro, M., Pratt, H., Weng, Z. Abstract: The morphologically and functionally distinct cell types of a multicellular organism are maintained by epigenomes and gene expression programs. Phase III of the ENCODE Project profiled 66 mouse epigenomes across twelve tissues at daily intervals from embryonic day 10.5 to birth. Applying the ChromHMM algorithm to these epigenomes, we annotated eighteen chromatin states with characteristics of promoters, enhancers, transcribed regions, repressed regions, and quiescent regions throughout the developmental time course. Our integrative analyses delineate the tissue specificity and developmental trajectory of the loci in these chromatin states. Approximately 0.3% of each epigenome is assigned to a bivalent chromatin state, which harbors both active marks and the repressive mark H3K27me3. Highly evolutionarily conserved, these loci are enriched in silencers bound by Polycomb Repressive Complex proteins and the transcription start sites of their silenced target genes. This collection of chromatin state assignments provides a useful resource for studying mammalian development. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.21.213876v1?rss=1 Authors: Meijer, M., Agirre, E., Kabbe, M., van Tuijn, C., Heskol, A., Falcao, A. M., Corces, M. R., Montine, T., Chen, X., Chang, H. Y., Castelo-Branco, G. Abstract: Multiple sclerosis (MS) is a disease characterized by a targeted immune attack on myelin in the central nervous system (CNS). We have previously shown that oligodendrocytes (OLs), myelin producing cells in the CNS, and their precursors (OPCs), acquire disease-specific transcriptional states in MS1,2. To understand how these alternative transcriptional programs are activated in disease, we performed single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) on the OL lineage in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We identified regulatory regions with increased accessibility in oligodendroglia (OLG) in EAE, some of which in the proximity of immune genes. A similar remodeling of chromatin accessibility was observed upon treatment of postnatal OPCs with interferon-gamma (IFN-gamma), but not with dexamethasone. These changes in accessibility were not exclusive to distal enhancers, but also occurred at promoter regions, suggesting a role for promoters in mediating cell-state transitions. Notably, we found that a subset of immune genes already exhibited chromatin accessibility in OPCs ex vivo and in vivo, suggesting a primed chromatin state in OLG compatible with rapid transitions to an immune-competent state. Several primed genes presented bivalency of H3K4me3 and H3K27me3 at promoters in OPCs, with loss of H3K27me3 upon IFN-gamma treatment. Inhibition of JMJD3/Kdm6b, mediating removal of H3K27me3, led to the inability to activate these genes upon IFN-gamma treatment. Importantly, OLGs from the adult human brain showed chromatin accessibility at immune gene loci, particularly at MHC-I pathway genes. A subset of single-nucleotide polymorphisms (SNPs) associated with MS susceptibility overlapped with these primed regulatory regions in OLG from both mouse and human CNS. Our data suggest that susceptibility for MS may involve activation of immune gene programs in OLG. These programs are under tight control at the chromatin level in OLG and may therefore constitute novel targets for immunological-based therapies for MS. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.04.132571v1?rss=1 Authors: Pearl, J. R., Shetty, A. C., Cantle, J. P., Bergey, D. E., Bragg, R. M., Coffey, S. R., Kordasiewicz, H. B., Hood, L. E., Price, N. D., Ament, S. A., Carroll, J. B. Abstract: Progressive striatal gene expression changes and epigenetic alterations are a prominent feature of Huntingtons disease (HD), but direct relationships between the huntingtin (HTT) protein and chromatin remain poorly described. Here, using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that HTT reproducibly occupies specific locations in the mouse genome, including thousands of genomic loci that are differentially occupied in striatal tissue from a knock-in mouse model of HD (B6.HttQ111/+) versus wildtype controls. ChIP-seq of histone modifications, generated in parallel, revealed genotype-specific colocalization of HTT with trimethylation of histone 3 lysine 27 (H3K27me3), a repressive chromatin mark. Close to genes that are differentially regulated in HD, greater HTT occupancy in HttQ111/+ vs. wildtype mice predicted increased H3K27me3, reduced histone 3 lysine 4 (H3K4me3, a marker of poised and active promoters), and down-regulated gene expression. Altered huntingtin-chromatin interactions may therefore play a direct role in driving transcriptional dysregulation in HD. Copy rights belong to original authors. Visit the link for more info

Hepatoblastoma is a malignant disease of the liver. It accounts for about 1 % of all childhood cancers and is the most common malignant liver tumor in infancy. Hepatoblastoma is assumed to arise from immature liver progenitor cells by aberrant activation of genes important in the embryonic development. Based on its early manifestation it is generally assumed that hepatoblastoma displays a relatively normal genomic background. Whole-exome sequencing performed in our group identified hepatoblastoma as one of the genetically simplest tumors ever described, with recurrent mutations in beta-catenin (CTNNB1) and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2). Based on this finding we performed targeted genotyping of a large cohort of primary hepatoblastomas, hepatoblastoma cell lines and transitional liver cell tumors and identified CTNNB1 and NFE2L2 to be mutated in 72.5 % and 9.8 % of cases, respectively. CTNNB1 is a key effector molecule of canonical WNT signaling pathway, a pathway that is essential in organogenesis and cellular processes such as cell proliferation, differentiation, survival and apoptosis. However, NFE2L2 is involved in the activation of the cellular antioxidant response to combat the harmful effects such as xenobiotics and oxidative stress. Interestingly, all NFE2L2 mutations were located in or adjacent to the DLG and ETGE motifs of the NFE2L2 protein that are needed to get recognized by the KEAP1/CUL3 complex for proteasomal degradation. Functional analysis showed that cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signaling and that depletion of the NFE2L2 via siRNA downregulates the NAD(P)H dehydrogenase (quinine) 1 (NQO1), a target gene of NFE2L2, and inhibits proliferation. In the clinical setting, NQO1 overexpression in tumors was significantly associated with metastasis, vascular invasion, the adverse prognostic C2 gene signature as well as poor outcome. RNA sequencing in our group identified the ubiquitin-like with PHD and ring finger domains 1 (UHRF1), a protein known to preferentially bind to hemi-methylated DNA, to be highly overexpressed in hepatoblastoma tumors. UHRF1 is as a key regulator in the epigenetic crosstalk, by controlling DNA methylation and histone modification. Using immunoprecipitation, we were able to show that UHRF1 binds in concert with DNA methyltransferase 1 (DNMT1) and ubiquitin specific peptidase 7 (USP7) as a trimeric complex to promoter regions of tumor suppressor genes (TSG) relevant in hepatoblastoma, such as hedgehog interacting protein (HHIP), insulin-like growth factor binding protein 3 (IGFBP3), and secreted frizzled-related protein 1 (SFRP1). These genes are epigenetically silenced in hepatoblastoma, as evidenced by heavy DNA methylation and enrichment of the repressive H3K27me3 and H3K9me2 chromatin mark. Interestingly, knockdown of UHRF1 expression via RNA interference resulted in promoter demethylation, but no reactivation of TSG gene expression. Additionally, no effect on tumor cell proliferation was observed after UHRF1 knockdown. Chromatin immunoprecipitation experiments revealed a decrease of the repressive chromatin marks H3K27me3 and H3K9me2 after UHRF1 depletion, but neither a clear shift towards the active H3K4me2 chromatin mark nor enrichment of RNA Polymerase at the TSG loci was observed. Statistical analyses revealed that a high expression of UHRF1 was associated with advanced disease state and a worse overall survival. Taken together our study demonstrates that activation of WNT signaling in concert with activation of the NFE2L2-KEAP1 pathway might be the driving force in the development of liver cancers. Moreover, we defined aberrant NQO1 expression as a marker for adverse course of disease and poor outcome. In addition, we showed that an aberrant expression of the epigenetic regulator UHRF1 and its excessive binding on promoter regions results in methylation of TSGs. This may represent an important mechanism in the initial phases of embryonal tumorigenesis. However, UHRF1 depletion alone was not sufficient to re-induce TSG expression. Therefore, UHRF1 might be more useful as a biomarker for the prognosis of hepatoblastoma than a direct anti-cancer target for hepatoblastoma therapy.

The CHO-K1 cell line is the most common expression system for therapeutic proteins in the pharmaceutical industry. Due to the nature of economics, the cell lines and the vector design are subject to constant change to increase product quality and quantity. During the cultivation, the production cell lines are susceptible to decreasing productivity over time. Often the loss of production can be associated with a reduction of copy number and the silencing of transgenes. During cell line development, the most promising cell lines are cultivated in large batch culture. Consequently, the loss of a stable production cell line can be very cost-intensive. For this reason I developed different strategies to avoid a reduced productivity. Instability of production cell lines can be predicted by the degree of CpG methylation of the driving promoter. Considering that the DNA methylation is at the end of an epigenetic cascade and associated with the maintenance of the repressive state, I investigated the upstream signals of histone modifications with the assumption to obtain a higher predictive power of production instability. For this reason I performed a chromatin immunoprecipitation of the histone modifications H3K9me3 and H3K27me3 as repressive signals and H3ac as well as H3K4me3 as active marks. The accumulations of those signals were measured close to the hCMV-MIE at the beginning of the cultivation and were then compared with the loss of productivity over two month. I found that the degree of the H3 acetylation (H3ac) correlated best with the production stability. Furthermore I was able to identify an H3ac threshold to exclude most of the unstable producers. In the second project I aimed to improve the vector design by considering epigenetic mechanisms. To this end I designed on the one hand a target-oriented histone acetyltransferase to enforce an open and active chromatin status at the transgene. On the other hand I point-mutated methylation-susceptible CpGs within the hCMV-MIE to impede the maintenance of inactive heterochromatin formation. Remarkably, the C to G mutation located 179 bp upstream of transcription start site resulted in very stable antibody producing cell lines. In addition, the examination of cell pools expressing eGFP showed that G-179 promoter variants were less prone to a general methylation and gene amplification, which illustrates the dominating effect in epigenetic mechanisms of one single CpG. The last project was performed to localize stable integration sites within the CHO-K1 genome. In so doing I could show that the transfection leads predominantly to integration into inactive regions. Furthermore I identified promising integration sites with a high potential to induce stable expression. However, those results are preliminary and must be viewed with caution. Further examination needs to be done to confirm these results. Considering the results of all three projects, I propose that the interplay of metabolic burden and selection pressure at an early time point of cultivation plays an important role in cell line development. Small alterations of selection pressure can lead to a decisive change of cell properties. Therefore, stable cells are less susceptible than weak producers. The increase of selection pressure leads to compensatory effect by gene amplification in the instable cell lines. The resulting adjustment of productivity masks the truly stable cells, which precludes the selection of the right cell lines. For this reason the selection pressure, the copy number as well as the growth rate should be considered to minimize repressive effects.

X chromosome inactivation (XCI) in female mammalian cells is an ideal model system to study the relationship of epigenetic regulation and higher-order chromatin structure. However, light microscopic studies of chromosomal organization have long been limited by the diffraction barrier of optical resolution. Super-resolution 3D-structured illumination microscopy (3D-SIM) – one of several recent techniques that circumvent this limitation – enables multicolor optical sectioning of entire cells with eightfold-improved volumetric resolution compared to conventional fluorescence imaging methods. In the present work, 3D-SIM has been applied to analyze higher-order chromatin structure of the Barr body in mammalian nuclei, a characteristic hallmark of XCI, with yet unprecedented detail. First, the increased resolution prompted to reappraise the potential detrimental effect of the DNA-FISH procedure on chromatin structure. Comparative analyses revealed slight deteriorations at the resolution level of 3D-SIM, especially within more decondensed euchromatin sites within the nuclear interior. In contrast, overall nuclear morphology and the nuclear envelope as well as heterochromatic sites in general maintained well preserved. The results suggest that DNA-FISH studies can benefit from a combination with super-resolution microscopy. In particular, when keeping in mind the current developments of the FISH technique with increasingly small and higher-complexity probes. The compact shape of the Barr body led to the assumption of a contribution of this special higher-order chromatin structure to the establishment and maintenance of the silenced state in the inactive X chromosome (Xi). However, a confirmation of this view has always been hampered by the restrictions of conventional light microscopy. In this work, the 3D chromosomal organization of the Xi and autosomes has been investigated with 3D-SIM in various human and mouse somatic cells and in mouse embryonic stem cell (ESC) lines. The precise subchromosomal localization of a variety of factors involved in XCI in different developmental states was qualitatively and quantitatively assessed utilizing combined immunofluorescence, EdU- pulse and RNA-/DNA-FISH labeling protocols and novel data analysis tools customized for the special requirements of 3D-SIM. The results demonstrate that all autosomes are made of a three-dimensional interconnected network of chromatin domains (CDs, or topology associated domains, TADs) of highly-variable shape and dynamics. CDs/TADs are comprised of a compacted chromatin core enriched with repressive marks, which is collectively proposed to be the functionally passive chromatin compartment (PNC). This PNC is surrounded by a 50 – 150 nm locally defined, less compacted perichromatin region (PR) that is enriched with active histone modifications and pervaded by a three-dimensional interchromatin (IC) network. The PR and the IC are collectively referred to as being the functionally relevant active nuclear compartment (ANC) that harbors all major nuclear processes, including transcription and replication. 3D-SIM data revealed that the Barr body maintains this principle compartmentalization and that it is still pervaded by a narrow ANC network, which is able to fulfill its functional role as a hub for replication or rarely occurring expression of XCI-escape genes. Live-cell super-resolution imaging on HeLa H2B-GFP cells confirmed that the observed chromatin features do not reflect fixation artifacts. Xist RNA, the key factor of XCI, has been found to be preferentially located as distinct discernible foci within the ANC throughout the entire volume of the Barr body. Here, it is tightly associated with a Xi-specific form of the nuclear matrix protein SAF-A, which confirms a previously suggested role for this Xi-enriched protein in Xist RNA spreading. In contrast, Xist RNA shows no spatial correlation with repressive Xi-enriched histone marks that are found within compacted chromatin sites. This specific localization of Xist RNA reflects an intrinsic feature as it is already present during early spreading in differentiating female ESCs, where it precedes chromatin compaction concomitant with RNA Polymerase II exclusion. Its localization is further confirmed in a male ESC line carrying an inducible Xist transgene on an autosome, but where Xist RNA fails to form a true autosomal Barr body, which is less compacted and maintains transcriptional activity. Last, Xist RNA shows no direct association with PRC2, the mediator of H3K27me3, which is in contrast to the generally believed direct recruitment model of PRC2 to the Xi by Xist RNA. The data collected in this work reflects further support and a refinement of the not unequivocally accepted CT-IC (chromosome territory - interchromatin compartment) model of higher-order chromosome architecture. In addition, a first attempt has been made to integrate these findings with a recently growing number of studies using chromosome conformation capturing (3C)-based techniques and to complement them on the single-cell level. Finally, a novel model for Xist RNA function in XCI is presented, which proposes a sequence-independent structural role for gene silencing and the formation of a repressive chromatin compartment.

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state.