Podcasts about Nucleosome

TWiV reviews new committee of experts to counter vaccine misinformation, resignation of NSF Director, dependence of immune response to rabies virus on the gut microbiome, and a nucleosome switch primes hepatitis B virus infection. Hosts: Vincent Racaniello, Rich Condit, and Angela Mingarelli Subscribe (free): Apple Podcasts, RSS, email Become a patron of TWiV! Links for this episode Support science education at MicrobeTV ASV 2025 Committee of experts to counter vaccine misinformation (Science) NSF Director resigns (Science) Dependence of vaccine immune response on gut microbiome (Cell Host Microbe) Nucleosome switch primes HepB replication (Cell) Letters read on TWiV 1212 Timestamps by Jolene Ramsey. Thanks! Weekly Picks Angela – Wild chimpanzees filmed by scientists bonding over alcoholic fruit Rich – Malcolm Gladwell Revisionist History “The Joe Rogan Intervention” Vincent – Measles Misinformation Is on the Rise — and Americans Are Hearing It, Survey Finds Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv Content in this podcast should not be construed as medical advice.

In this episode of the Epigenetics Podcast, we talked with Vijay Ramani from the Gladstone Institute to talk about his work on Single-Molecule Adenine Methylated Oligonucleosome Sequencing Assay (SAMOSA). Our discussion starts with Vijay Ramani's impactful contributions to the field during his time in Jay Shendure's lab, where he worked on several innovative methods, including RNA proximity ligation. This project was conceived during his graduate studies, aiming to adapt techniques from DNA research to investigate RNA structures—a largely unexplored area at the time. We delved into the nuances of his experiences in graduate school, emphasizing how critical it was to have mentors who provided room for creativity and autonomy in experimental design. Dr. Ramani then shares insights about his foray into developing more refined methodologies, such as in-situ DNA Hi-C, a revolutionary protocol tailored for three-dimensional genomic mapping. He explained the rationale behind his projects, comparing the outcomes with contemporaneous advancements in methods like Micro-C. The discussion highlighted the importance of understanding enzyme bias in chromatin studies and the need for meticulous experimental design to ensure the validity of biological interpretations. We further explored exciting advancements in single-cell genomics, specifically Ramani's work on developing sci-Hi-C. This innovative technique leverages combinatorial indexing to allow high-resolution mapping of chromatin architecture at the single-cell level, a significant leap forward in understanding the complexities of gene regulation. As we progress, Ramani detailed his transition from graduate student to independent investigator starting his own lab. He elaborated on the challenges and excitements associated with establishing his research focus in chromatin structure and function using advanced sequencing technologies. Employing various strategies, including the innovative SAMOSA assay, his research seeks to elucidate the mechanisms by which chromatin structure influences transcriptional regulation. We also discussed the heterogeneity of chromatin and its implications for gene expression. Ramani provided a fascinating perspective on how variations in chromatin structure could affect gene activity, highlighting potential avenues for future research that aims to untangle the complex dynamics at play in both healthy and diseased states.   References Ramani, V., Cusanovich, D., Hause, R. et al. Mapping 3D genome architecture through in situ DNase Hi-C. Nat Protoc 11, 2104–2121 (2016). https://doi.org/10.1038/nprot.2016.126 Nour J Abdulhay, Colin P McNally, Laura J Hsieh, Sivakanthan Kasinathan, Aidan Keith, Laurel S Estes, Mehran Karimzadeh, Jason G Underwood, Hani Goodarzi, Geeta J Narlikar, Vijay Ramani (2020) Massively multiplex single-molecule oligonucleosome footprinting eLife 9:e59404. https://doi.org/10.7554/eLife.59404 Abdulhay, N.J., Hsieh, L.J., McNally, C.P. et al. Nucleosome density shapes kilobase-scale regulation by a mammalian chromatin remodeler. Nat Struct Mol Biol 30, 1571–1581 (2023). https://doi.org/10.1038/s41594-023-01093-6 Nanda, A.S., Wu, K., Irkliyenko, I. et al. Direct transposition of native DNA for sensitive multimodal single-molecule sequencing. Nat Genet 56, 1300–1309 (2024). https://doi.org/10.1038/s41588-024-01748-0   Related Episodes Epigenetic Mechanisms in Genome Regulation and Developmental Programming (James Hackett) Chromatin Profiling: From ChIP to CUT&RUN, CUT&Tag and CUTAC (Steven Henikoff) Split-Pool Recognition of Interactions by Tag Extension (SPRITE) (Mitch Guttman)   Contact Epigenetics Podcast on X Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Epigenetics Podcast on Bluesky Epigenetics Podcast on Threads Active Motif on X Active Motif on LinkedIn Email: podcast@activemotif.com

TWiV notes the passing of virologist Diane Griffin, first H5N1 influenza virus in US pigs, Innate immune control of influenza virus interspecies adaptation via IFITM3, and antiviral trained innate immunity in alveolar macrophages after SARS-CoV-2 infection reduces secondary influenza A virus disease. Hosts: Vincent Racaniello, Alan Dove, Rich Condit, Kathy Spindler, and Brianne Barker Subscribe (free): Apple Podcasts, RSS, email Become a patron of TWiV! Links for this episode MicrobeTV Discord Server MicrobeTV Fundraiser Diane Griffin passes (Johns Hopkins) Diane Griffin on TWiV 453 First H5N1 influenza virus in US pigs (CIDRAP) IFITM3 controls interspecies influenza virus infection (Nat Comm) Trained innate immunity by alveolar macrophages (Immunity) Timestamps by Jolene. Thanks! Weekly Picks Angela – See a giant ‘ghost particle' detector and more — October's best science images Brianne – October 27 APoD: Bat nebula Dickson – Nikon Small World Contest 2024 winners Kathy – AAAS 150th anniversary video, celebrating scientists and Pew's 2024 annual Trust in Science survey findings Rich – Cats Basically Are a Liquid After All, Study Confirms Alan – HHMI's Beautiful Biology site Vincent – EcoHealth Alliance Fights Back Listener Picks Hunter – Don't stop me now: Queen's Brian May on saving badgers — and the scientific method Anne – Reasons to be cheerful Intro music is by Ronald Jenkees Send your virology questions and comments to twiv@microbe.tv Content in this podcast should not be construed as medical advice.

In this episode of the Epigenetics Podcast, we talked with Karine Le Roch from the University of California at Riverside about her work on malaria chromatin structure and its transcriptional regulation. In this Interview Dr. Le Roch discusses her investigation of post-transcriptional controls and nucleosome positioning in Plasmodium falciparum, employing next-generation sequencing and chromatin profiling methods. Karin emphasizes how these methodologies contribute to a comprehensive understanding of gene regulation beyond mere transcription initiation, emphasizing the significance of mRNA binding proteins and their role in stabilizing gene transcripts for translation. This exploration of the interaction between chromatin structure, transcriptional dynamics, and post-transcriptional regulation reveals a multidimensional perspective of gene expression. Transitioning to her lab's focus on high-throughput genomic technologies, we discuss how Karin and her team are uncovering conserved and species-specific genomic organization principles within various Plasmodium species. By generating 3D genomic models through Hi-C experiments, she describes how they have identified patterns that underline the parasite's immune evasion strategies. In particular, we learn how genes involved in antigenic variation are controlled through intricate epigenetic mechanisms, illuminating the pathways that allow these parasites to elude host immune responses.   References Le Roch, K. G., Zhou, Y., Blair, P. L., Grainger, M., Moch, J. K., Haynes, J. D., De La Vega, P., Holder, A. A., Batalov, S., Carucci, D. J., & Winzeler, E. A. (2003). Discovery of gene function by expression profiling of the malaria parasite life cycle. Science (New York, N.Y.), 301(5639), 1503–1508. https://doi.org/10.1126/science.1087025 Ponts, N., Harris, E. Y., Prudhomme, J., Wick, I., Eckhardt-Ludka, C., Hicks, G. R., Hardiman, G., Lonardi, S., & Le Roch, K. G. (2010). Nucleosome landscape and control of transcription in the human malaria parasite. Genome research, 20(2), 228–238. https://doi.org/10.1101/gr.101063.109 Bunnik, E. M., Cook, K. B., Varoquaux, N., Batugedara, G., Prudhomme, J., Cort, A., Shi, L., Andolina, C., Ross, L. S., Brady, D., Fidock, D. A., Nosten, F., Tewari, R., Sinnis, P., Ay, F., Vert, J. P., Noble, W. S., & Le Roch, K. G. (2018). Changes in genome organization of parasite-specific gene families during the Plasmodium transmission stages. Nature communications, 9(1), 1910. https://doi.org/10.1038/s41467-018-04295-5   Related Episodes Epigenetics in Human Malaria Parasites (Elena Gómez-Diaz)   Contact Epigenetics Podcast on X Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Epigenetics Podcast on Bluesky Epigenetics Podcast on Threads Active Motif on X Active Motif on LinkedIn Email: podcast@activemotif.com

In this episode of the Epigenetics Podcast, we caught up with Vladimir Teif from the University of Essex to talk about his work on nucleosome positioning in development and disease. Vladimir's research has been pivotal in understanding nucleosome positioning and its implications for cell differentiation, particularly in embryonic stem cells and cancer. We discuss his groundbreaking studies that first mapped nucleosome positions in various cell types and how these findings led to uncovering the intricate relationships between nucleosome stability, transcription factors, and DNA modifications such as methylation. This understanding has immense significance for cancer diagnostics, where knowing the spatial arrangement of nucleosomes could influence how aggressive a cancer type might be, or how a patient might respond to treatment. Transitioning from foundational research to clinical applications, Vladimir elaborates on his exciting work with liquid biopsies. By analyzing cell-free DNA from blood plasma, researchers can infer the nucleosome positioning and, ultimately, the presence of cancer without the need for invasive tissue biopsies. We explore how this new approach holds potential for earlier detection of cancers and more effective patient stratification, demonstrating a profound shift in how we leverage epigenetic data in clinical settings.   References Vladimir B. Teif, Karsten Rippe, Predicting nucleosome positions on the DNA: combining intrinsic sequence preferences and remodeler activities, Nucleic Acids Research, Volume 37, Issue 17, 1 September 2009, Pages 5641–5655, https://doi.org/10.1093/nar/gkp610 Teif, V., Vainshtein, Y., Caudron-Herger, M. et al. Genome-wide nucleosome positioning during embryonic stem cell development. Nat Struct Mol Biol 19, 1185–1192 (2012). https://doi.org/10.1038/nsmb.2419 Beshnova DA, Cherstvy AG, Vainshtein Y, Teif VB (2014) Regulation of the Nucleosome Repeat Length In Vivo by the DNA Sequence, Protein Concentrations and Long-Range Interactions. PLoS Comput Biol 10(7): e1003698. https://doi.org/10.1371/journal.pcbi.1003698 Shtumpf, M., Piroeva, K.V., Agrawal, S.P. et al. NucPosDB: a database of nucleosome positioning in vivo and nucleosomics of cell-free DNA. Chromosoma 131, 19–28 (2022). https://doi.org/10.1007/s00412-021-00766-9   Related Episodes Circulating Epigenetic Biomarkers in Cancer (Charlotte Proudhon) Epigenome-based Precision Medicine (Eleni Tomazou)   Contact Epigenetics Podcast on X Epigenetics Podcast on Instagram Epigenetics Podcast on Mastodon Epigenetics Podcast on Bluesky Epigenetics Podcast on Threads Active Motif on X Active Motif on LinkedIn Email: podcast@activemotif.com

Nels and Vincent discuss the genome sequence of an ancient wooly mammoth, which shows that the three-dimensional architecture of the DNA can persist after 50,000 years. Hosts: Nels Elde and Vincent Racaniello Subscribe (free): Apple Podcasts, Google Podcasts, RSS, email Become a patron of TWiEVO Links for this episode Join the MicrobeTV Discord server Three dimensional architecture of 50,000 year old wooly mammoth genome (Cell) Timestamps by Jolene Science Picks Nels – Mysterious SARS-CoV-2 variants showing up in sewer samples Vincent – ‘Cocaine sharks' found in waters off Brazil Music on TWiEVO is performed by Trampled by Turtles Send your evolution questions and comments to twievo@microbe.tv

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.25.546476v1?rss=1 Authors: Xie, Z., Chai, Y., Zhu, Z., Shen, Z., Zhao, Z., Xiao, L., Du, Z., Ou, G., Li, W. Abstract: Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates. 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/2022.12.09.519750v1?rss=1 Authors: Cho, M.-G., Kumar, R. J., Lin, C.-C., Boyer, J. A., Shahir, J. A., Fagan-Solis, K., Simpson, D. A., Fan, C., Foster, C. E., Goddard, A. M., Wang, Q., Wang, Y., Ho, A. Y., Liu, P., perou, c. J., Zhang, Q., McGinty, R. K., Purvis, J. E., Gupta, G. P. Abstract: Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS/STING-mediated innate immune signaling and tumor suppression1-3. However, the mechanism for cGAS activation by endogenous DNA damage remains enigmatic, particularly given the constitutive inhibition of cGAS by high-affinity histone acidic patch (AP) binding4-10. Here we report an in vivo CRISPR screen that identified the DNA double strand break sensor Mre11 as a suppressor of mammary tumorigenesis induced by Myc overexpression and p53 deficiency. Mre11 antagonizes Myc-induced proliferation through cGAS/STING activation. Direct binding of the Mre11-Rad50-Nbn (MRN) complex to nucleosomes displaces cGAS from AP sequestration, which is required for DNA damage-induced cGAS mobilization and activation by cytosolic DNA. Mre11 is thereby essential for cGAS activation in response to oncogenic stress, cytosolic DNA transfection, and ionizing radiation. Furthermore, we show Mre11-dependent cGAS activation suppresses Myc-induced proliferation through ZBP1/RIPK3/MLKL-mediated necroptosis. In human triple-negative breast cancer, ZBP1 downregulation correlates with increased genome instability, decreased immune infiltration, and poor patient prognosis. These findings establish Mre11 as a critical link between DNA damage and cGAS activation that regulates tumorigenesis through ZBP1-dependent necroptosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In today's episode, we are excited to be talking to Dr Christopher Clarkson, currently based at UCL, where he has just started a post-doctoral research position. In 2020, Dr. Clarkson completed his PhD at the University of Essex where he investigated changes of nucleosome positioning and 3D chromatin organization! In today's episode, we discuss how CTCF binding strength correlates with nucleosome repeat length and what the implications are of this on gene regulation. We further discuss how regions in the genome where CTCF is able to bind are extremely important for cellular differentiation and boundary formation within the genome. Email: c.clarkson@ucl.ac.uk Chris' Nucleic Acid Research article: CTCF-dependent chromatin boundaries formed by asymmetric nucleosome arrays with decreased linker length Twitter: find Chris on twitter here. Check out Biobox Analytics here, the ideal platform for working with NGS data, running and designing bioinformatic pipelines and generating the perfect plots: https://biobox.io/

In this episode of the Epigenetics Podcast, we caught up with Dr. Keji Zhao from the National Heart, Lung, and Blood Institute at the National Institutes of Health in Bethesda, MD, to talk about his work on the genome-wide investigation of epigenetic marks and nucleosome positioning. Dr. Keji Zhao pioneered in the development of cutting-edge techniques in the field of epigenetics. Current methods at that time relied on DNA microarrays, however, Dr. Zhao wanted a more comprehensive and unbiased approach that would avoid the shortfalls of these array-based methods. Hence, he set out to develop new sequencing-based methods like ChIP-Seq and MNase-Seq with accompanying computational methods to analyze the huge amount of sequencing data that would be generated. Using the above-mentioned techniques, Dr. Zhao was able to show that histone deacetylases (HDACs) and histone acetyltransferases (HATs) were found at inactive and active genes, respectively, as previously thought. Surprisingly, he was also able to show that HDACs were also located at active genes. Furthermore, both, HATs and HDACs can be found at low levels at silenced genes. In this episode we discuss the story behind how Dr. Keji Zhao was one of the pioneers of the chromatin immunoprecipitation technology, how he discovered the genomic locations of HATs and HDACs, and in the end he shares some tips and tricks on how to get the best results in ChIP-Seq assays.   References Artem Barski, Suresh Cuddapah, … Keji Zhao (2007) High-resolution profiling of histone methylations in the human genome (Cell) DOI: 10.1016/j.cell.2007.05.009 Dustin E. Schones, Kairong Cui, … Keji Zhao (2008) Dynamic regulation of nucleosome positioning in the human genome (Cell) DOI: 10.1016/j.cell.2008.02.022 Zhibin Wang, Chongzhi Zang, … Keji Zhao (2009) Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes (Cell) DOI: 10.1016/j.cell.2009.06.049 Wenfei Jin, Qingsong Tang, … Keji Zhao (2015) Genome-wide detection of DNase I hypersensitive sites in single cells and FFPE tissue samples (Nature) DOI: 10.1038/nature15740 Binbin Lai, Weiwu Gao, … Keji Zhao (2018) Principles of nucleosome organization revealed by single-cell micrococcal nuclease sequencing (Nature) DOI: 10.1038/s41586-018-0567-3 Related Episodes In Vivo Nucleosome Structure and Dynamics (Srinivas Ramachandran) Development of Site-Specific ChIP Technologies (Hodaka Fujii) Multiple Challenges in ChIP (Adam Blattler)   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/2020.11.19.389429v1?rss=1 Authors: Lee, E., Kang, C., Purhonen, P., Hebert, H., Bouazoune, K., Hohng, S., Song, J.-J. Abstract: Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed integrative biophysical analysis on CHD7 chromatin remodeler using crosslinking-mass spectrometry (XL-MS), cryo-Electron Microscopy (cryo-EM) and single-molecule Forster Resonance Energy Transfer (smFRET). We uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. The cryo-EM analysis on the N-CRD_Chromodomain bound to nucleosome reveals that the N-CRD interacts with the acidic patch of nucleosome. Furthermore, smFRET analysis shows the mutations in the N-CRD result in slow or highly-fluctuating remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities. Copy rights belong to original authors. Visit the link for more info

In this episode of the Epigenetics Podcast, we caught up with Dr. Srinivas Ramachandran, Assistant Professor at the University of Colorado, Anschutz Medical Campus, to talk about his work on ​in vivo nucleosome structure and dynamics. Dr. Srinivas Ramachandran studies the structure and dynamics of nucleosomes during cellular processes like transcription and DNA replication. During transcription, as the RNA polymerase transcribes along the DNA, it needs to pass nucleosomes. Dr. Ramachandran investigated the effect of nucleosomes on transcription and also studied how different histone variants affect this process. He found that the first nucleosome within a gene body is a barrier for the progression of RNA polymerase, and that presence of the histone variant H2A.Z in this first nucleosome lowers this barrier. Furthermore, Dr. Ramachandran developed a method called mapping in vivo nascent chromatin using EdU and sequencing (MINCE-Seq), enabling the study of chromatin landscapes right after DNA replication. In MINCE-Seq, newly replicated DNA is labeled right after the replication fork has passed by with the nucleotide analog ethynyl deoxyuridine (EdU), which can then be coupled with biotin using click chemistry. After the purification of newly replicated DNA and MNase digestion, the chromatin landscape can be analyzed. In this interview, we discuss the story behind how Dr. Ramachandran found his way into chromatin research, what it was like to start a wet lab postdoc with a bioinformatics background, and what he is working on now to unravel nucleosomal structure and dynamics in his own lab.   References Christopher M. Weber, Srinivas Ramachandran, Steven Henikoff (2014) Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase (Molecular Cell) DOI: 10.1016/j.molcel.2014.02.014 Srinivas Ramachandran, Steven Henikoff (2016) Transcriptional Regulators Compete with Nucleosomes Post-replication (Cell) DOI: 10.1016/j.cell.2016.02.062 Srinivas Ramachandran, Kami Ahmad, Steven Henikoff (2017) Transcription and Remodeling Produce Asymmetrically Unwrapped Nucleosomal Intermediates (Molecular Cell) DOI: 10.1016/j.molcel.2017.11.015 Satyanarayan Rao, Kami Ahmad, Srinivas Ramachandran (2020) Cooperative Binding of Transcription Factors is a Hallmark of Active Enhancers (bioRxiv) DOI: 10.1101/2020.08.17.253146 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/2020.11.13.382465v1?rss=1 Authors: Knight, P., Gauthier, M.-P. L., Pardo, C. E., Darst, R. P., Riva, A., Kladde, M. P., Bacher, R. Abstract: Differential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we developed methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin landscapes. Methylscaper implements a weighted principle component analysis that orders sequencing reads, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscaper's utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets. Methylscaper, is available on GitHub at https://github.com/rhondabacher/methylscaper. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.21.348664v1?rss=1 Authors: Reddy, G., Thirumalai, D. Abstract: Nucleosomes, the building blocks of chromosomes, are also transcription regulators. Single molecule pulling experiments have shown that nucleosomes unwrap in two major stages, releasing nearly equal length of DNA in each stage. The first stage, attributed to the rupture of the outer turn is reversible, occurs at low forces ({approx} (3 - 5) pNs) whereas in the second stage the inner turn ruptures irreversibly at high forces (between {approx} (9 - 15) or higher) pNs. We show that Brownian dynamics simulations using the Self-Organized Polymer model of the nucleosome capture the experimental findings, thus permitting us to discern the molecular details of the structural changes not only in DNA but also in the Histone Protein Core (HPC). Upon unwrapping of the outer turn, which is independent of the pulling direction, there is a transition from 1.6 turns to 1.0 turn DNA wound around the HPC. In contrast, the rupture of the inner turn, leading to less than 0.5 turn DNA around the HPC, depends on the pulling direction, and is controlled by energetic and kinetic barriers. The latter arises because the mechanical force has to produce sufficient torque to rotate (in an almost directed manner) the HPC by 180{degrees}. In contrast, during the rewrapping process, HPC rotation is stochastic, with the quenched force fQ playing no role. Interestingly, if fQ = 0 the HPC rotation is not required for rewrapping because the DNA ends are unconstrained. The assembly of the outer wrap upon force quench, as assessed by the decrease in the end-to-end distance (Ree) of the DNA, nearly coincides with the increase in Ree as force is increased, confirming the reversible nature of the 1.6 turns to 1.0 turn transition. The asymmetry in HPC rotation during unwrapping and rewrapping accounts for the observed hysteresis in the stretch-release cycles in single molecule pulling experiments. Experiments that could validate the prediction that HPC rotation, which gives rise to the kinetic barrier in the unwrapping process, are proposed. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.20.305581v1?rss=1 Authors: Woods, D. C., Rodríguez-Ropero, F., Wereszczynski, J. Abstract: Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction. Copy rights belong to original authors. Visit the link for more info

In this episode of the Epigenetics Podcast, we caught up with Dr. Christine Cucinotta and Dr. Melvin Noe Gonzalez to talk about how they brought the #fragilenucleosome seminar series and Discord channel to life.   Christine Cucinotta and Melvin Noe Gonzales are part of the organizing committee of the independent scientific community "Fragile Nucleosome." This community consists of a Discord channel with more than 1,000 members, a biweekly seminar series, a mentoring program, and a journal club series. The Fragile Nucleosome is organized exclusively by early-career scientists, without external sponsors or under the roof of a single graduate program or university.   In this interview, Christine and Melvin share the story on how the Fragile Nucleosome community got started, what has happened so far, and what the future plans are for the #fragilenucleosome.     References #fragilenucleosome on Twitter Fragile Nucleosome Discord Channel Fragile Nucleosome on generegulation.org Christine Cucinotta on Twitter Melvin Noe Gonzalez on Twitter   Contact   Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on Linked-In Active Motif on Facebook eMail: podcast@activemotif.com

The nucleosome is the central organizing structure of the eukaryotic genome. It consists of DNA wrapped around histone proteins. Dr. Karolin Luger shares her discovery of the three-dimensional structure of the nucleosome using X-ray crystallography, which provided a deeper understanding of chromatin organization.

The nucleosome is the central organizing structure of the eukaryotic genome. It consists of DNA wrapped around histone proteins. Dr. Karolin Luger shares her discovery of the three-dimensional structure of the nucleosome using X-ray crystallography, which provided a deeper understanding of chromatin organization.

In this episode of the Epigenetics Podcast, we caught up with Karolin Luger, Ph.D., from the University of Colorado in Boulder to talk about her work on solving the crystal structure of the nucleosome and on how histone chaperones like FACT act on chromatin. During her postdoc with Timothy Richmond at the Swiss Federal Institute of Technology in Zürich, Karolin Luger was the first author on an all-time classic paper called "Crystal structure of the nucleosome core particle at 2.8 A resolution" which was published in Nature. This article was published more than 20 years ago now and it has been cited about 9000 times. After completing her postdoc, she moved to Colorado to set up her own lab where she continued to work on the structure of the nucleosome and the factors that influence their structure. The most recent Nature paper published by her lab investigated how the FACT complex promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication, and DNA repair.   In this interview, we discuss the efforts that went into solving the crystal structure of the nucleosome back in 1997, her work on histone chaperones, and her recent work on how FACT keeps nucleosomes intact after gene transcription.   References  K. Luger, A. W. Mäder, … T. J. Richmond (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution (Nature) DOI: 10.1038/38444 Yang Liu, Keda Zhou, … Karolin Luger (2020) FACT caught in the act of manipulating the nucleosome (Nature) DOI: 10.1038/s41586-019-1820-0   Contact   Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on Linked-In Active Motif on Facebook eMail: podcast@activemotif.com

This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from recent issues of Circulation Research and talks with Steve Lim and James M. Murphy about their article on nuclear FAK regulation of smooth muscle cell proliferation. Article highlights: Li et al: Histone Turnover in Adult Heart Kurosawa et al: Celastramycin Ameliorates Pulmonary Hypertension Urban et al: NOS3 Gene Polymorphism and Coronary Heart Disease   Transcript Cindy S.:                               Hi, welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host Cindy St. Hilaire, and I'm an assistant professor of medicine and bioengineering at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the July 5th and July 19th issues of the Circulation Research Journal. We'll also have an in-depth conversation with Drs. Steve Limb and James Murphy, from the University of South Alabama College of Medicine, who are the lead authors in one of the exciting discoveries presented in the July 5th issue. Cindy S.:                               The first article I want to share with you is titled, Replication-Independent Histone Turnover Underlines the Epigenetic Homeostasis in the Adult Heart. The co-first authors are Yumei Li, Shanshan Ai, Xianhong Yu, and the corresponding author is Aibin He. This research was conducted at the Institute of Molecular Medicine Beijing Key Laboratory of Cardiometabolic Molecular Medicine and the Peking-Tsinghua Center for Life Sciences. Both of which are part of the Peking University in Beijing, China. Cindy S.:                               In the nucleus of cells, DNA is packaged into a structure called chromatin. Chromatin can reside in an open state that is permissive to gene transcription, or closed state where transcription is inhibited. The core units of chromatin are called nucleosomes. A nucleosome consists of DNA that is wrapped around proteins called histones. It's the position of these nucleosomes that determines whether the chromatin allows for DNA transcription or not. There is a large body of research that is focused on understanding the epigenetic processes that promote or repress transcription. Most of this research focuses on the processes that read, write, and erase covalent histone modifications. But, histones are proteins, and proteins, as we all know, have finite half-lives. Cindy S.:                               Far less research has been conducted to understand the dynamics of histone assembly and disassembly on specific regions of DNA. In this study, the authors took a novel approach of using a GFP-tagged histone H2B protein to track in vivo the rate at which nucleosomes are replaced in cardiac chromatin, and to what extent this rate varies across the genome of those cells. This is particularly interesting, and a particularly good cell type to study, as cardiomyocytes rarely divide or proliferate in the adult heart. What they found was intriguing. Nucleosome recycling is not even across the epigenome of cardiac cells. Instead, gene promoters, enhancer, and other regulatory regions that are known to promote gene transcription all exhibited a higher histone turnover rate than regions of the epigenome that are not occupied by these permissive remarks. Cindy S.:                               Further, they found greater histone turnover at loci for cardiac specific transcription factors as compared to loci for pluripotency transcription factors. This implies preferential access to these regions. Digging further into the mechanism, they discovered that the repressive chromatin regulator, EED, promoted this histone turnover. The epigenetic signature is what helps to define the identity and function of a fully differentiated cell. This study suggests that loss of histone turnover may promote loss of the proper epigenetic signature of a fully differentiated cell. These exciting findings suggest replication independent histone turnover is a requirement in maintaining both epigenetic and functional homeostasis in the adult heart. From this, one may hypothesize that perhaps aberrations in histone turnover contribute to age related diseases in the cardiac tissue, as well as possibly other tissues. Cindy S.:                               The next article I'd like to highlight is titled, Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension-High-throughput Screening of 5,562 Compounds. The first author is Ryo Kurosawa, and the corresponding author is Hiroaki Shimokawa, both from the department of cardiovascular medicine at Tohoku University Graduate School of Medicine in Sendai, Japan. This article is focusing on the disease pulmonary arterial hypertension. Cindy S.:                               Pulmonary arterial hypertension, or PAH, is a disease that stems from the increased proliferation of arterial smooth muscle cells in the lungs. This proliferation leads to a progressive occlusion of the pulmonary arteries. This occlusion also causes increased pressure in the right heart ventricle. That can lead to heart failure, and ultimately death. Basal dilatory drugs are currently used as therapy in PAH, as they help to open the blood vessels, which can alleviate some of the symptoms. However, these drugs do not target the underlying cause of the symptoms, which is the hyperproliferation of the smooth muscle cell. Cindy S.:                               To identify novel compounds that inhibit smooth muscle cell proliferation, Kurosawa and colleagues used a high-throughput approach. They isolated cells from patients with pulmonary arterial hypertension and used these cells in a high-throughput approach to test 5,562 novel molecules on their ability to inhibit the proliferation of these cells. This unbiased approach yielded several potential compounds that potentially reduced smooth muscle cell proliferation from these patients, and also had very minimal deleterious effects on healthy control smooth muscle cells. From there, the team tinkered with the structure of the drug Celastramycin to try to increase its efficacy, and with that tinkering they found in vitro, that their new molecule could reduce both the inflammatory signal that helps to drive the proliferation of the smooth muscle cells, as well as reactive oxygen species, which helps to drive the inflammatory signaling. Cindy S.:                               Moving forward to in vivo studies, the team found that their new treatment also reduced right ventricle systolic pressure and hypertrophy in three different rodent models of pulmonary arterial hypertension. This treatment improved exercise capacity in one of the models. Together, these exciting results indicate that Celastramycin could be developed as a potential therapy for pulmonary arterial hypertension. Cindy S.:                               The last paper we're going to talk about before switching to our interview with Drs. Steve Limb and James Murphy, is a paper titled, 15-Deoxy-Δ12,14-Prostaglandin J2 Reinforces the Anti-Inflammatory Capacity of Endothelial Cells With a Genetically Determined Nitric Oxide Deficit. The co-first authors are Ivelina Urban, Martin Turinsky, Sviatlana Gehrmann, and the corresponding author is Marcus Hecker, all from the department of cardiovascular physiology at Heidelberg University in Heidelberg, Germany. Cindy S.:                               Nitric oxide is a vasodilatory and anti-inflammatory molecule, and thus, beneficial to cardiovascular health. Homozygosity of a single nucleotide polymorphism, or SNP, is a gene nitric oxide synthase results in reduced ability of endothelial cells to produce nitric oxide, specifically in response to fluid share stress. Decreased bioavailability of nitric oxide in the vessel wall helps to promote atherosclerosis. The SNP that we're referring to in this paper is called T-786C, where TT homozygosity is considered the control, or healthy genotype, and CC homozygosity is the disease associated. CC homozygosity of this SNP is predictive of atherosclerotic related diseases, and consequently, individuals with CC homozygosity have an increased risk for coronary heart disease. Cindy S.:                               Now, despite this detrimental evidence, homozygous patients do not develop atherosclerosis at an accelerated rate. This suggests that there's a compensatory mechanism at play. To identify how CC homozygous cells compensate for reduced nitric oxide synthase activity, the authors utilized human umbilical vein endothelial cells, that are also called huvecs, that harbored either the TT or the CC version of this SNP. They also used these in combination with a monocytic cell line. Cindy S.:                               Urban and colleagues found that under fluid share stress conditions, human endothelial cells homozygous with for the CC variant, had increased production of an anti-inflammatory prostaglandin called 15d-PGJ2. Signaling, via this prostaglandin, helps to compensate in part for the reduced endo production. This prostaglandin suppressed monocyte activation by reducing expression of pro-inflammatory genes such as aisle 1 beta, and decreased monocyte transmigration through endothelial cells. The team also found that patients with coronary heart disease were more likely to have the CC homozygous variant than age match controls. Thus, not only did they identify a partial compensatory mechanism, the authors suggest that 15d-PGJ2 could be a useful biomarker for the diagnosis of coronary heart disease. Cindy S.:                               So that's it for the highlights of the July issues of Circulation Research. Thank you very much to Ruth Williams, who writes the In This Issue copy for the journal, as well as the editorial team at the journal and at the podcast. Cindy S.:                               Okay, so now we're going to talk to our team of first author and last author. Today's paper that we're talking about is Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription. The first authors of this papers are Kyuho Jeong, Jung-Hyun Kim, and James M. Murphy, and the corresponding author is Steve Lim. Today, we're going to be speaking with James and Steve about this paper. So thank you, both of you, for joining us today. Steve Lim:                           Thanks for having us today. Cindy S.:                               Great. Congratulations on your beautiful paper. I was wondering if maybe we could just start by both of you introducing yourselves, telling us your current position, and maybe about how you came into this field. Steve Lim:                           Hi, Cindy. We appreciate the opportunity to discuss our paper. I'm Steve Lim, an associate professor in the department of biochemistry and molecular biology and medicine at the University of South Alabama. I received my PhD from University of Alabama at Birmingham. I did my post-doctoral training studying the law of FAK in cancer biology at UC San Diego Moores Cancer Center. In 2012 I started my own lab here at South Alabama, where I decided to focus on vascular biology using some pharmacy data I generated at the end of my post-doctoral study. James Murphy:                 I'm James Murphy, I'm a post-doctoral fellow in Dr. Lim's lab at the University of South Alabama. My path to science was a little different than most. I got an undergraduate and graduate degree in mathematics before I joined the PhD program here at South Alabama. Due to a family history of cardiovascular related deaths, I decided to join Dr. Lim's lab due to his interest in studying vascular disease to find new therapeutic targets. Cindy S.:                               Interesting, a math major. James Murphy:                 Yeah. Cindy S.:                               Has that been able to help with any of your basic science studies? James Murphy:                 I'm pretty good at doing concentrations. Cindy S.:                               You're the expert in lab math. James Murphy:                 Yeah. I think the logic skills and critical thinking skills that I picked up in math really help out here in science. Cindy S.:                               Oh, I bet, that's wonderful. You're the dream PhD student who can hit the ground running with M1V1 equals M2V2. Great. Well, thank you so much. I really liked this paper because I love the mechanosensing and how does a cell read what's outside, and how does that message get brought to the inside. Really, that's what you're finding in this paper, specifically looking at how FAK is mediating transcriptional regulation. Maybe you can start by just telling us, what was your overarching question when you started this study? Steve Lim:                           Sure. It is very well-known fact that promotes cell proliferation and migration through interior receptors and gross factor receptor signaling. Both of which are key components in the smooth muscle cell hyperplasia. So naturally, we asked ourselves a simple question, "Is FAK activity important for smooth muscle cell proliferation, and leading into hyperplasia?" Cindy S.:                               So when you say FAK activity I think one thing that's interesting in your paper is, FAK really has kind of two different functions, and one is the kinase function. A kinase is when it can phosphorylate another protein, so it itself is an enzyme. But, then it has another function, so can you maybe tell us about those different functions of FAK? Steve Lim:                           Right. So FAK can function as a kinase, as well as a kind of independent scaffold, which can recruit different proteins. In the paper, we specifically described a kinase independent function as a nuclear function, nuclear FAK function. Cindy S.:                               Interesting. So what premise, or what gaps and knowledge were present before your study, that you were trying to address? Steve Lim:                           Actually, a study showed that the knocking off FAK in the smooth muscle cells prevented neointimal hyperplasia. As just you asked question, FAK has two different functions. Since FAK has both kind of dependent and independent [inaudible 00:14:48], this study lets the unanswered question, which of these two different functions of FAK plays a larger role in dealing with hyperplasia. We aimed to inhibit FAK activity to distinguish between FAK kind of dependent and independent roles in dealing with hyperplasia. Cindy S.:                               Interesting. How exactly were you able to do that? How could you take and dissect apart the two different functions of this protein? Steve Lim:                           We started off with a small pile of experiment to test if a small molecule FAK inhibitor could block neointimal hyperplasia, and we were very surprised at the degree to FAK inhibition actually prevented neointimal hyperplasia following vascular injury. Cindy S.:                               Yeah, and that's in figure one. I was looking at that, it's quite striking. Steve Lim:                           Actually, to distinguish these two different functions we generated new genetic FAK–Kinase-Dead mouse model in conjunction with a FAK inhibitor model, and that would allow us to study a lot of FAK activity in smooth muscle cells. Cindy S.:                               Great. James, could you tell us about the mouse model that you developed for this study, and the specific mutations that you created and what you were allowed to test with those models. James Murphy:                 So the FAK–Kinase-Dead knock-in model was actually generated during Dr. Lim's post-doctoral studies. Cindy S.:                               Is that the exciting data? James Murphy:                 The mutation is just a simple lysine to arginine mutation of amino acid 454. What they found was that, actually, homozygous kinase-dead embryos was lethal. So you need FAK activity to actually develop a full grown organism. We kind of had to cross a hetero wild-type kinase-dead mouse with a phlox FAK mouse, which eventually, if you cross with tissue-specific Cres, what you end up with is a phlox wild-type or a phlox kinase-dead mouse. Then, when you treat Tamoxifen in your Cre mouse, then you delete one copy of wild-type FAK and you're left with either a single copy of wild-type FAK, or a single copy of kinase-dead FAK. Cindy S.:                               Very nice. So for your study, you used, if I recall correctly, the myosin-11, Tamoxifen-inducible Cre model. Can you maybe talk about why you chose that model and why not the SM22 Cre or a non-inducible model? What was your strategy? James Murphy:                 As I mentioned, FAK activity is important for embryo genesis, so we thought we had to use an inducible model, so as to make sure we had an adult mouse at the time of the experiment. We originally actually had the SMA Cre model, however, some grant reviewers had told us that we should kind of shift to the more myosin-11 mouse to be more specific to the vascular. One downside to that, as we mentioned in the paper, is that that's actually only on the Y chromosome, so you can only use male mice. Cindy S.:                               Yes. But, at least it's in only the smooth muscle cells. Is that kind of the pros and cons of that model? James Murphy:                 Yes, and the MYH-11 Cre is kind of the most accepted model when you're doing smooth muscle studies. Cindy S.:                               Great. So can both of you go over some of the key findings of your paper? If we're going to say this in a tweet, what would we say? James Murphy:                 In a tweet. So I think, as we talked about, FAK can go to the nucleus. It's kind of constantly shuttling between the nucleus and the cytoplasm, at least what we've been able to observe in vitro. However, kind of a its localization in vivo still kind of was up in the air at the time. However, our immunostaining data actually rebuild that healthy uninjured arteries primarily showed FAK was in the nucleus. Suggesting that FAK was inactive, and maybe somehow suppressing smooth muscle cell proliferation by staying in the nucleus. But, after wire injury, FAK not only increased its activation, but also shifted to be primarily within the cytoplasm, and eventually we showed that that increase of GABA4 protein stability leading to proliferation. Cindy S.:                               Very interesting. That's great. So what was the hardest part of this whole study? James Murphy:                 Dr. Lim did the preliminary FAK inhibitor studies, but he had people when he started his own lab, he had to teach us how to do the wire injury. At first, learning gets kind of technical, you have to get used to using the microscope. Cindy S.:                               Could you describe the wire injury model for us? James Murphy:                 Yes. What you do is you anesthetize the mouse and you actually locate the femoral artery, and you want to kind of reveal the muscular branch. What you do is you add suture proximal and distal to the muscular branch of the femoral artery to stop blood flow. Then, you're going to cut a small incision in the muscular branch, and you insert a small wire through the branch up into the femoral artery towards the iliac branch. What this does is denude the endothelial layer and kind of causes an extension of the artery, damaging the smooth muscle layer. Once you remove it and suture off the muscular branch, then after a couple weeks you start to see hyperplasia. Cindy S.:                               Interesting. So what does this model clinically? James Murphy:                 This model kind of mimics angioplasty procedures that one may have if they have an occluded artery. There's multiple angioplasty procedures. There's a physical dislodging and opening of the artery. Then, there's some other methods such as using a stent to keep it open. Cindy S.:                               Great. Very interesting. What do you think would happen in maybe, I don't know, an LDLR knockout that was crossed with your FAK kinase deficient mutant? What do you think would happen in an athero model? James Murphy:                 We're actually- Cindy S.:                               Or is that the next paper? We don't have to talk about it if it's the next paper. James Murphy:                 We're actually currently testing that right now. Cindy S.:                               Oh, okay. James Murphy:                 So that's kind of our next step is to test this in atherosclerotic models to see what happens. Cindy S.:                               So, what might this mean for potential therapeutic target? How could we leverage this data to possibly translate it to the clinical setting, even if it's far off? What might we want to do moving forward? Steve Lim:                           Speaking of translational potential, currently most of the treatment options for narrow vessels rely on thrombolytic stents, that provides local delivery of anti-proliferative drugs. However, DES comes with several disadvantages, including location, work, size of these affected vessels. In fact, inhibitors are under cancer clinical development, have never been used in the vascular diseases. Our study, I think, at least to show the potential for using this type of FAK inhibitors in treating hyperplasia, which was not possible before. Cindy S.:                               That's interesting. So essentially, there's already potential, therapy's already available that would just have to be tested in this new ... in this new vascular realm, essentially. Steve Lim:                           Yeah. I was thinking about effication of these type of drugs. I think it could be, as you said, PAH could be one of the targets, because they're not really useful drugs available now. In the future, what we actually, we started already, but it's known, these moments of proliferation plays key role in the arthrosclerosis progression. Studies targeting neointimal hyperplasia and atherosclerosis, it's not existing. I think in the future probably, we would like to test whether in fact inhibition and the smooth muscle cells reduce its atherosclerity in animal models, and hopefully in humans. Cindy S.:                               Yeah, yeah, hopefully in humans, always. Yeah, and in those mouse models, there's always interesting studies where you can block things from the beginning. But, I think one of the beautiful things about the mouse model that you created, the fact that it's Tamoxifen inducible, you could essentially let that atherosclerotic plaque build up for a bit and then knock it out and see if it can reverse it. So the model you created is a really wonderful tool to use for a whole bunch of studies. So congratulations. Steve Lim:                           Thank you. Cindy S.:                               Yeah, I thought the most interesting aspect of this paper was really the fact that it could link this FAK protein, this integrin signal mediating protein to the transcription factor GABA4. So could you possibly tell us a little bit about that interaction, and exactly what GAB is doing in the smooth muscle cell? Steve Lim:                           I actually think that the identifying GABA4 factor actually was one of the difficulties, because normal cells do not express GABA4, that's what is known. I think it's because, based on our finding, the smooth muscle cells in vivo, you could package more predominantly localized in vivo, the nuclear FAK is predominant. So that nuclear FAK finds GABA4 and reduces ability through the process on degradation. But, actually, not changing ... Nuclear factor is not changed. GABA4 mRNA are the expression, so GABA4 is always expressed in smooth muscle cells. But, we never see in healthy, or very freshly isolated smooth muscle cells. We never see Gaba4. That was the most difficult part actually. Cindy S.:                               So the mRNA is always there, it's just never making it to a protein that accumulates in any measurable quantity. Steve Lim:                           So you become a protein, but FAK, nuclear FAK kills all GABA4 in the nucleus. Cindy S.:                               That's the proteasome mediated degradation? Steve Lim:                           Right. Then, GABA4 actually promotes cycling D1 transcription. So no GABA4, no cycling the new one, and smooth muscle cells do not cycle. Cindy S.:                               Interesting. So can you maybe close the loop and tell us essentially what's in figure nine, like this. Could you talk us through that? Steve Lim:                           It summarizes in figure nine, I think it would be best, we can put two different situations. In healthy R3, FAK is in the nucleus, and GABA4 is reduced, cycling D1 is not expressed, and smooth muscle cells become high acid. They don't proliferate. But, in injured, actually, FAK localization is it's the vaso injury promotes FAK localization, vaso injury shifts FAK nuclear localization to cytoplasm. Actually, FAK is activated. Now, GABA4, that increases cycling T1 expression. So that causes intimal hyperplasia. That could be a kind of summary. Cindy S.:                               No, that's perfect. Congratulations on a very nice paper. I thoroughly enjoyed reading it, and I enjoyed even more speaking with the two of you. So thank you very much. Steve Lim:                           Well, thank you so much. Cindy S.:                               Thank you for listening. I'm your host Cindy St. Hilaire, and this is Discover CircRes, your source for the most up to date and exciting discoveries in basic cardiovascular research.  

The TWiPsters solve the case of the Brazilian Immigrant With Heart Problems, and describe how genome organization controls trypanosome antigenic variation. Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiP. Links for this episode: Trypanosome genome organization and antigenic variation (Nature) Hero: Arthur Looss Letters read on TWiP 161 Case Study for TWiP 161 Daniel was asked to see 30 yo female from Bolivia, had to travel back during 3rd trimester. Was there for most of 3rd trimester. Child born in US, pericardial effusion, ascites, moderate PDA. Heart function is ok. Woman was healthy, no issues during pregnancy. Baby’s  white count elevated, diagnostic evaluation. It is a parasite. Send your case diagnosis, questions and comments to twip@microbe.tv Music by Ronald Jenkees

The Nucleosome is the basic building unit of chromatin. It consists out of 147 base pairs of double stranded DNA wrapped around the Histone core octamer that consists out of 2 copies of each dimer of H2A/H2B, and H3/H4. Nucleosomes are organized like "beads on a string" to form a modifiable regulatory basis for higher order structures of chromatin. The first images of the nucleosome as a particle was published by our guests Ada and Don Olins from the University of New England in 1974 (Olins, A. L. & Olins, D. E. Spheroid Chromatin Units (ν Bodies). Science 183, 330–332 (1974).). This observation lead the way to numerous discoveries around chromatin which ultimately culminated in the discovery of the 2.8 Angstrom high-resolution crystal structure 20 years ago in the year 1997 (Luger, K., Mäder, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389, 251–260 (1997).) References for this episode Ada L. Olins, Donald E. Olins. Spheroid Chromatin Units (ν Bodies). Science. 25 Jan 1974: Vol. 183, Issue 4122, pp. 330-332. DOI: 10.1126/science.183.4122.330 Ada L. Olins, Donald E. Olins, et al. An epichromatin epitope. Nucleus. 2011 Jan-Feb; 2(1): 47–60. DOI: 10.4161/nucl.2.1.13271 Active Motif Contact Details Follow us on Twitter Join us on LinkedIn Like us on Facebook Email us @Active Motif Europe or Active Motif North America.

Hosts: Nels Elde and Vincent Racaniello Nels and Vincent reveal how introns - the parts of pre-mRNAs that are removed by splicing - were generated by DNA transposons in two different picoeukaryotes.   Become a patron of TWiEVO Introns from DNA transposons (Nature) Reverse transcription and integration lecture Image: Elde Lab Mobile Studios on location in Santa Fe, NM Letters read on TWiEVO 16 This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get your first 3 meals free with your first purchase – WITH FREE SHIPPING – by going to blueapron.com/twie. Science Picks Nels - Did eukaryotes invent anything? (TWiM 144) Vincent - Virology course online Music on TWiEVO is performed by Trampled by Turtles Send your evolution questions and comments to twievo@microbe.tv

Eukaryotic genomes are organized inside the cell nucleus in a structured macromolecular DNA-protein polymer named chromatin, formed by single discrete unites called Nucleosomes. The packing of the genetic information into chromatin allows the efficient regulation of several nuclear processes, such as gene expression and transcription, DNA replication, cell cycle progression, chromosome segregation and DNA damage repair. Chromatin comes in two flavors: a transcriptionally active, more loosened state, called euchromatin and a transcriptionally silent or low expressed, more compact state, called heterochromatin. The assembly of silent chromatin or heterochromatin is fundamental for the regulation of every nuclear process and it is driven in most Eukaryotes by the deposition and the read-out of the histone H3 lysine 9 methylation (H3K9me) post-translational modification (PTM). H3K9me on the nucleosome is specifically bound by chromatin readers called chromodomains (CD) and this recognition is fundamental for the downstream processes that lead to the formation of heterochromatin and shut down the expression of single genes or entire gene clusters. Despite several studies have been done on different chromodomains binding to H3K9me histone tail peptides, to date there was no structural information on how chromodomains interact with their natural binding partners, the H3K9me3 Nucleosomes. In a preliminary structural study carried out in our laboratory we solved the cryo-electron microscopy (Cryo-EM) structure of the chromodomain of the fission yeast Chp1 protein (Chp1CD) in complex with an H3K9me nucleosome. The structure showed that the Chp1CD interacts not only with the histone H3 tail but also with the histone globular domains in the Nucleosome core, primarily with histone H3. Mutations in the residues of Chp1CD that form the binding interface with the Nucleosome core (two loops in the β-sheet of the domain) caused a drop of the affinity in vitro for the H3K9me Nucleosome, which was independent from the histone H3K9me tail interaction. Cells harboring the same Chp1CD loop mutations were defective in silencing centromeric transcripts and maintain the deposition of the H3K9me mark for heterochromatin formation. This indicated that Chp1CD-nucleosome core interaction is fundamental for heterochromatin formation in fission yeast and opened up to the possibility that chromodomains could read multiple histone PTMs, on both the recruiting histone tail and on the nucleosome core. This study substantially contributes to understand how chromodomains interact with chromatin, how much the nucleosome core interaction is conserved among different CDs and how different chromodomain proteins are regulated at the same loci. Understanding how chromodomain readers recognize nucleosomes is fundamental to uncover the basics of gene silencing and heterochromatin formation.

Eukaryotic genomes make use of nucleosomes to considerably reduce their packaging volumes. As a consequence, the underlying DNA is rendered inaccessible. Cells make use of ATP-dependent remodeling factors to disrupt histone-DNA contacts and bring about access to the DNA. ACF1 is the largest regulatory subunit of two nucleosome remodeling factors, namely ACF and CHRAC. These complexes assemble, slide or evenly space nucleosomes on DNA with an ability to sense the linker lengths. However, roles of ACF1 in organizing nucleosomes in vivo and their physiological consequences are largely unclear. To understand the roles of ACF1 on chromatin organization, I compared nucleosome occupancy and transcription profiles in wild-type and ACF1-deficient Drosophila embryos. To further investigate and corroborate these chromatin changes, I performed genomewide mapping of ACF1 using chromatin immunoprecipitation. Nucleosome occupancy was mapped by subjecting DNA obtained from MNase-digested chromatin to deep sequencing and the occupancies were analyzed using advanced analog signal processing methods. We found discontinuous and discrete patches of regularly positioned nucleosomes in wild-type tissue, referred to as ‘regularity regions’. These regions span actively transcribing and silent chromatin domains and show associated variation in the linker lengths across them. A subset of these regions located at sides remote from the transcriptional start sites loses regularity upon ACF1 deletion and show presence of a novel DNA sequence motif. Analyzing nucleosome periodicity by autocorrelation function revealed that nucleosome linker length is longer in ACF1-deficient embryos. Despite profound quantifiable changes in the chromatin organization the RNA expression analyses did not show any major changes. Genomewide localization of ACF1 was studied using by chromatin immunoprecipitation. We observed a strong enrichment of ACF1 along active promoter regions, coinciding strikingly well with another remodeling factor, RSF-1. However, careful analyses using mutant tissues for both proteins demonstrated that the observed enrichments were in fact false positive. We define 3100 genomic sites as false positive ‘Phantom Peaks’ that tend to enrich in the ChIP-seq experiments. By comparing publicly accessible profiles and the Phantom regions, we showed that several ChIP-seq profiles of the epigenetic regulators show strong enrichment along the Phantom Peaks. In conclusion, we identify regions of regularly organized nucleosomes across the genome and show that a subset localized in silent chromatin regions is affected by ACF1 deletion. Moreover, we identified a class of false positive ChIP-seq peaks at active promoters. This list of Phantom Peaks can be used to assess potential false positive signal in a ChIP-seq profile, especially when mutant tissue is not available as a control.

Tue, 12 Nov 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17714/ https://edoc.ub.uni-muenchen.de/17714/1/Pointner_Julia.pdf Pointner, Julia ddc:570, ddc:500,

Davide Corona, University of Palermo, Palermo - Italy speaks on "Chromatin Binding, Nucleosome Spacing and ncRNA-mediated Regulation of the Remodeling ATPase ISWI". This seminar has been recorded at University of Trieste by ICGEB Trieste

Tue, 20 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11878/ https://edoc.ub.uni-muenchen.de/11878/1/Lantermann_Alexandra.pdf Lantermann, Alexandra

The goal of this study has been to elucidate the mechanisms responsible for rebuilding nucleosomes at the PHO5 promoter upon rerepression. In this work, I could unambiguously show that histones are incorporated at the PHO5 promoter upon repression. Regarding the source of these histones, I provide evidence that a significant fraction of the deposited histones originate from a soluble histone pool, i.e. a histone source in trans. Promoter closure occurs with strikingly rapid kinetics and is independent of replication. In agreement with the finding that PHO5 repression does not require cell division, I found that histone chaperones which are associated with replication-independent nucleosome assembly are important for rapid PHO5 promoter closure. Strains deleted for histone chaperones involved in replication-dependent nucleosome assembly did not exhibit any defect in promoter closure. Other factors contributing to rapid PHO5 repression turned out to be nucleosome remodelers, whose characteristic mode of action is chromatin assembly in trans. Nucleosome remodeling mutants typically catalyzing nucleosome movements in cis are not implicated in PHO5 promoter reassembly. The phenomenon of trans-deposition of histones upon repression is not restricted to the PHO5 promoter but is also found at two other phosphate regulated promoters, PHO8 and PHO84. By its rapid mode of action, this mechanism contributes to efficiently shutting off transcription. This might also hold true for other yeast genes. In the second part of this work I present results that indicate a role for the histone chaperone Asf1p in the activation of the PHO5 gene. Interestingly, the induction of PHO5 in an asf1 mutant is dependent on the phosphate concentration of the growth medium. Full induction occurs only when the medium is completely free of phosphate. The abundance of even trace amounts of phosphate precludes PHO5 activation altogether.

The PHO5 and PHO8 genes in yeast provide typical examples for the role of chromatin in promoter regulation. Both genes are regulated by the same transcriptional activator, Pho4, which initiates nucleosome remodeling and transcriptional activation. In spite of this co-regulation, there are important differences in gene activity and in the way promoter chromatin undergoes chromatin remodeling. First, PHO5 belongs to one of the most strongly induced genes in yeast being 10-fold more active than the PHO8 gene (Oshima, 1997; Barbaric et al., 1992). Second, chromatin remodeling at the PHO5 promoter affects four nucleosomes (Almer et al., 1986), whereas only two nucleosomes are afffected at the PHO8 promoter (Barbaric et al., 1992). Third, neither the histone acetyl transferase Gcn5 nor chromatin remodeling complex Swi/Snf seem to be critically required for chromatin remodeling at the PHO5 promoter (Barbaric et al., 2001; Reinke and Hörz, 2003; Dhasarathy and Kladde, 2005; Neef and Kladde, 2003). At the PHO8 promoter, on the other hand, absence of Swi/Snf results in the complete loss of chromatin remodeling under inducing conditions. Furthermore, Gcn5 is required for full remodeling and transcriptional activation at this promoter (Gregory et al., 1999). Ever since these differences were recognized there have been speculations about the underlying reasons. This work shows that these discrepancies are not a direct consequence of the position or strength of the UASp elements driving the activation of transcription. Instead, these differences result from different stabilities of the two promoter chromatin structures. The basis for these results was the development of a competitive yeast in vitro assembly technique in which differences in nucleosome stability between promoter regions could be directly compared. This technique originated from a yeast in vitro chromatin assembly system that generated the characteristic PHO5 promoter chromatin structre (Korber and Hörz, 2004). As shown here, this system also assembles the native PHO8 promoter nucleosome pattern. Using the competitive assembly system it was shown that the PHO8 promoter has greater nucleosome positioning power, and that the properly positioned nucleosomes are more stable than at the PHO5 promoter. This provided for the first time evidence for the correlation of inherently more stable chromatin with stricter co-factor requirements. Remarkably, the positioning information for the in vitro assembly of the native PHO5 and PHO8 promoter chromatin patterns was specific to the yeast extract. Salt gradient dialysis or Drosophila embryo extract assemblies did not support the proper nucleosome positioning. However, nucleosomes in chromatin generated in these systems could be shifted to their in vivo-like positions by the addition of yeast extract. This indicates that the nucleosome positioning mechanisms in vitro are uncoupled from the nucleosome loading machinery. The nucleosome positioning at the PHO5 and PHO8 promoters was energy dependent suggesting a role of chromatin remodeling machines in generation of the repressed promoter chromatin structure. In spite of this, the chromatin remodeling machines Swi/Snf, Isw1, Isw2 and Chd1 were dispensable nucleosome positioning at both promoters.

Sat, 1 Jan 1994 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7385/1/atp_dependent_nucleosome_disruption_7385.pdf Wu, Chun-Guey; Becker, Peter B.; Tsukiyama, T.