Epigenetics Podcast

Nucleosome Positioning in Cancer Diagnostics (Vladimir Teif)

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
 
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The Effect of Histone Demethylases on Gene Expression and Cancer Cell Stability (Johnathan Whetstine)

In this episode of the Epigenetics Podcast, we talked with Johnathan Whetstine from Fox Chase Cancer Center about his work on how histone demethylases affect gene expression and cancer cell stability.
The Interview start by discussing a pivotal paper from Jonathan's lab in 2010, where they identified a role for the KDM4A histone demethylase in replication timing and cell cycle progression. They elaborate on the discoveries made regarding the link between histone marks, replication timing, and gene expression control. Jonathan explains the impact of microRNAs on regulating KDM4A and how protein turnover rates can influence cellular responses to treatments like mTOR inhibitors.
Further, they explore the causal relationship between histone marks and replication timing, demonstrating how alterations in epigenetic regulation can affect genome stability. Jonathan shares insights from his latest research on H3K9 methylation balance at the MLL-KM2A locus, elucidating how these epigenetic modifications regulate amplifications and rearrangements in cancer cells. The episode concludes with a discussion on the establishment of the Cancer Epigenetics Institute at Fox Chase Cancer Center, aiming to bridge academia and industry to accelerate translational research in cancer epigenetics.
 
References
Black, J. C., Allen, A., Van Rechem, C., Forbes, E., Longworth, M., Tschöp, K., Rinehart, C., Quiton, J., Walsh, R., Smallwood, A., Dyson, N. J., & Whetstine, J. R. (2010). Conserved antagonism between JMJD2A/KDM4A and HP1γ during cell cycle progression. Molecular cell, 40(5), 736–748. https://doi.org/10.1016/j.molcel.2010.11.008
Mishra, S., Van Rechem, C., Pal, S., Clarke, T. L., Chakraborty, D., Mahan, S. D., Black, J. C., Murphy, S. E., Lawrence, M. S., Daniels, D. L., & Whetstine, J. R. (2018). Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications. Cell, 174(4), 803–817.e16. https://doi.org/10.1016/j.cell.2018.06.018
Van Rechem, C., Ji, F., Chakraborty, D., Black, J. C., Sadreyev, R. I., & Whetstine, J. R. (2021). Collective regulation of chromatin modifications predicts replication timing during cell cycle. Cell reports, 37(1), 109799. https://doi.org/10.1016/j.celrep.2021.109799
Gray, Z. H., Chakraborty, D., Duttweiler, R. R., Alekbaeva, G. D., Murphy, S. E., Chetal, K., Ji, F., Ferman, B. I., Honer, M. A., Wang, Z., Myers, C., Sun, R., Kaniskan, H. Ü., Toma, M. M., Bondarenko, E. A., Santoro, J. N., Miranda, C., Dillingham, M. E., Tang, R., Gozani, O., … Whetstine, J. R. (2023). Epigenetic balance ensures mechanistic control of MLL amplification and rearrangement. Cell, 186(21), 4528–4545.e18. https://doi.org/10.1016/j.cell.2023.09.009
 
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Stem Cell Transcriptional Regulation in Naive vs. Primed Pluripotency (Christa Buecker)

In this episode of the Epigenetics Podcast, we talked with Christa Buecker from the Max Perutz Laboratories in Vienna about her work on transcriptional regulation during early embryonic development. Dr. Buecker unravels the differences between naive and primed pluripotency states, showcasing how OCT4 relocalization and enhancer chromatin landscapes play pivotal roles during this transition. The conversation delves into the intricate interplay of transcription factors like OCT4 and OTX2, shedding light on their collaborative efforts in regulating gene expression during differentiation.
Dr. Buecker then shares insights from her study on enhancer elements controlling FGF5 expression and discusses the surprising revelation that individual enhancers show no intrinsic activity but work together in a super additive fashion. She also touches upon her research on IRF1's connection to the gene regulatory network and its role in protecting cells against viral infections.
The conversation shifts to Dr. Buecker's current research endeavors, focusing on exploring the strength of enhancers and their impact on gene expression at different distances from promoters. She shares her vision for future experiments and the integration of enhancers to decipher their impact on transcription regulation.
 
References
Buecker, C., Srinivasan, R., Wu, Z., Calo, E., Acampora, D., Faial, T., Simeone, A., Tan, M., Swigut, T., & Wysocka, J. (2014). Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell stem cell, 14(6), 838–853. https://doi.org/10.1016/j.stem.2014.04.003
Thomas, H. F., Kotova, E., Jayaram, S., Pilz, A., Romeike, M., Lackner, A., Penz, T., Bock, C., Leeb, M., Halbritter, F., Wysocka, J., & Buecker, C. (2021). Temporal dissection of an enhancer cluster reveals distinct temporal and functional contributions of individual elements. Molecular cell, 81(5), 969–982.e13. https://doi.org/10.1016/j.molcel.2020.12.047
Romeike, M., Spach, S., Huber, M., Feng, S., Vainorius, G., Elling, U., Versteeg, G. A., & Buecker, C. (2022). Transient upregulation of IRF1 during exit from naive pluripotency confers viral protection. EMBO reports, 23(9), e55375. https://doi.org/10.15252/embr.202255375
 
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Gene Expression Control and Intricacies of X-chromosome Inactivation (Claire Rougeulle)

In this episode of the Epigenetics Podcast, we talked with Claire Rougeulle from the Epigenetics and Cell Fate Center at Université Paris City about her work on gene expression control, the intricacies of X-chromosome inactivation, and the potential of non-coding RNAs in this process.
In this episode Claire Rougeulle explains her discoveries regarding the transcription regulation of XIST by factors like YY1 and the erosion of X-chromosome inactivation in human pluripotent stem cells. She shares the complexity of distinguishing between epigenetics and transcriptional regulation, highlighting the challenges in studying allelic expression of X-chromosomes at the single-cell level.
The Episode further explores Claire's findings on the XACT locus regulation, evolution from retroviruses, and its potential role in preventing X-chromosome silencing. Claire also shares her future research focus on understanding X-inactivation establishment in humans and the transition from XIST attenuating to silencing X-chromosomes after implantation.
 
References
Makhlouf, M., Ouimette, J. F., Oldfield, A., Navarro, P., Neuillet, D., & Rougeulle, C. (2014). A prominent and conserved role for YY1 in Xist transcriptional activation. Nature communications, 5, 4878. https://doi.org/10.1038/ncomms5878
Vallot, C., Ouimette, J. F., Makhlouf, M., Féraud, O., Pontis, J., Côme, J., Martinat, C., Bennaceur-Griscelli, A., Lalande, M., & Rougeulle, C. (2015). Erosion of X Chromosome Inactivation in Human Pluripotent Cells Initiates with XACT Coating and Depends on a Specific Heterochromatin Landscape. Cell stem cell, 16(5), 533–546. https://doi.org/10.1016/j.stem.2015.03.016
Casanova, M., Moscatelli, M., Chauvière, L. É., Huret, C., Samson, J., Liyakat Ali, T. M., Rosspopoff, O., & Rougeulle, C. (2019). A primate-specific retroviral enhancer wires the XACT lncRNA into the core pluripotency network in humans. Nature communications, 10(1), 5652. https://doi.org/10.1038/s41467-019-13551-1
 
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Epigenetic Mechanisms in Genome Regulation and Developmental Programming (James Hackett)

In this episode of the Epigenetics Podcast, we talked with James Hackett from the EMBL in Rome about his work on epigenetic mechanisms in genome regulation and developmental programming.
One of James Hackett's significant studies focused on DNA methylation and genome defense mechanisms in the germline, exploring the role of chromatin modifications in mammalian gene regulation. He delves into investigating the erasure of DNA methylation in the germline, highlighting the key role of the TET-enzymes in demethylation processes.
Dr. Hackett shares insights from his research on pluripotent stem cells, where he mapped genome-wide DNA methylation and hydroxymethylation in different pluripotent states. He discusses the impact of extrinsic conditions on pluripotent states and the biases observed in lineage preferences.
Furthermore, the discussion delves into the development of a CRISPR screening tool to study cell fate transitions, particularly focusing on the genetic factors contributing to germline specification. He also talks about his work on epigenetic inheritance, highlighting the importance of precise perturbations in understanding chromatin modifications and their functional implications.
In a recent study, the Hackett lab focuses on systematic epigenome editing to investigate the context-dependent functions of chromatin modifications. We hear about this work, and the complexity of interactions between chromatin marks, DNA sequences, and transcription factors, shedding light on the nuanced effects of various chromatin modifications on gene expression.
 
References
Hackett JA, Reddington JP, Nestor CE, et al. Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline. Development (Cambridge, England). 2012 Oct;139(19):3623-3632. DOI: 10.1242/dev.081661. PMID: 22949617; PMCID: PMC3436114.
Hackett JA, Sengupta R, Zylicz JJ, et al. Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine. Science (New York, N.Y.). 2013 Jan;339(6118):448-452. DOI: 10.1126/science.1229277. PMID: 23223451; PMCID: PMC3847602.
Hackett JA, Kobayashi T, Dietmann S, Surani MA. Activation of Lineage Regulators and Transposable Elements across a Pluripotent Spectrum. Stem Cell Reports. 2017 Jun;8(6):1645-1658. DOI: 10.1016/j.stemcr.2017.05.014. PMID: 28591649; PMCID: PMC5470235.
Hackett JA, Huang Y, Günesdogan U, et al. Tracing the transitions from pluripotency to germ cell fate with CRISPR screening. Nature Communications. 2018 Oct;9(1):4292. DOI: 10.1038/s41467-018-06230-0. PMID: 30327475; PMCID: PMC6191455.
 
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Advanced Optical Imaging in 3D Nuclear Organisation (Lothar Schermelleh)

In this episode of the Epigenetics Podcast, we talked with Lothar Schermelleh from the University of Oxford about his work on advanced optical imaging in 3D nuclear organisation. 
The interview starts by Lothar Schermelleh sharing his groundbreaking work in understanding chromatin organization using super-resolution microscopy techniques. He then delves into his past experiments, including his publication on imaging chromatin domains and X chromosome inactivation. His work showcases the power of structured illumination microscopy in overcoming diffraction limits, revealing insights into nuclear organization and regulation.
Lothar also discusses refining methods for labeling chromosome territories and replication domains, as well as exploring structural and functional nuclear organization using advanced microscopy techniques. They touch on the potential of AI in microscopy, the importance of quality control in imaging, and Lothar's grant proposal for developing artifact-free, super-resolution imaging under cryo conditions with adaptive optics.
The conversation emphasizes the intersection of technology development and biological applications, highlighting the importance of addressing specific biological questions through innovative imaging approaches. 
 
References
Schermelleh, L., Carlton, P. M., Haase, S., Shao, L., Winoto, L., Kner, P., Burke, B., Cardoso, M. C., Agard, D. A., Gustafsson, M. G., Leonhardt, H., & Sedat, J. W. (2008). Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science (New York, N.Y.), 320(5881), 1332–1336. https://doi.org/10.1126/science.1156947
Schermelleh, L., Heintzmann, R., & Leonhardt, H. (2010). A guide to super-resolution fluorescence microscopy. The Journal of cell biology, 190(2), 165–175. https://doi.org/10.1083/jcb.201002018
Smeets, D., Markaki, Y., Schmid, V. J., Kraus, F., Tattermusch, A., Cerase, A., Sterr, M., Fiedler, S., Demmerle, J., Popken, J., Leonhardt, H., Brockdorff, N., Cremer, T., Schermelleh, L., & Cremer, M. (2014). Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci. Epigenetics & chromatin, 7, 8. https://doi.org/10.1186/1756-8935-7-8
Ball, G., Demmerle, J., Kaufmann, R., Davis, I., Dobbie, I. M., & Schermelleh, L. (2015). SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy. Scientific reports, 5, 15915. https://doi.org/10.1038/srep15915
 
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DNA Replication, Transcription and R-loops (Stephan Hamperl)

In this episode of the Epigenetics Podcast, we talked with Dr. Stephan Hamperl from the Helmholtz Zentrum Munich about his work on how conflicts between transcription, replication, and R-loop formation influence genome stability in human cells.
During the early stages of his career Stephan studied conflicts between transcription and replication in human cells, particularly focusing on R-loop structures. In our discussion, he explains the formation of R-loops and their impact on genome stability, emphasizing the importance of the orientation of replication forks approaching R-loops in determining DNA damage outcomes.
Stephan then delves into his work on the MATAC-Seq method, which analyzes chromatin domains at DNA replication origins to understand replication timing variability. The method involves methylating DNA linkers between nucleosomes and using nanopore sequencing for single-molecule readouts, revealing heterogeneity in chromatin structure at replication origins.
Finally, Stephan discusses his automated image analysis pipeline for quantifying transcription and replication activity overlap in mammalian genomes, addressing the challenge of visualizing these processes simultaneously. The conversation concludes with insights into Stefan's future research directions, focusing on understanding transcription-replication conflicts' molecular basis and their potential implications in cancer cell transformation.
References
Hamperl, S., Brown, C. R., Garea, A. V., Perez-Fernandez, J., Bruckmann, A., Huber, K., Wittner, M., Babl, V., Stoeckl, U., Deutzmann, R., Boeger, H., Tschochner, H., Milkereit, P., & Griesenbeck, J. (2014). Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae. Nucleic acids research, 42(1), e2. https://doi.org/10.1093/nar/gkt891
Hamperl, S., Bocek, M. J., Saldivar, J. C., Swigut, T., & Cimprich, K. A. (2017). Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses. Cell, 170(4), 774–786.e19. https://doi.org/10.1016/j.cell.2017.07.043
Chanou, A., Weiβ, M., Holler, K., Sajid, A., Straub, T., Krietsch, J., Sanchi, A., Ummethum, H., Lee, C. S. K., Kruse, E., Trauner, M., Werner, M., Lalonde, M., Lopes, M., Scialdone, A., & Hamperl, S. (2023). Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency. Nucleic acids research, 51(22), 12303–12324. https://doi.org/10.1093/nar/gkad1022
 
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Mutations of Gene Regulatory Elements in Human Disease (Nadav Ahituv)

In this episode of the Epigenetics Podcast, we talked with Nadav Ahituv from University of California, San Francisco about his work on mutations of gene regulatory elements in human disease.
Using massively parallel experiments, his lab revolutionized functional genomics by studying the impact of transcription factor binding sites on gene expression. His groundbreaking technology deciphered the regulatory language of gene expression by exploring transcription factor combinations, spacing, and orientation. By delving into the influence of DNA shape and gene topology, Nadav Ahituv's research provides a comprehensive understanding of gene regulation at the molecular level, shedding light on the complexity of genetic interactions.
The conversation delves into specific cases involving enhancers, gene sequencing, and 3D genomic structure, highlighting the impact of critical elements such as CTCF sites on gene expression. Discussions of haploid insufficiency and its implications for human health, using CRISPR technology to enhance gene expression, offer new possibilities for treating genetic diseases. Explorations of leptin-responsive regulatory elements in the hypothalamus and liver-associated transcription factors provide insights into metabolic regulation and gene expression networks in different tissues.
The episode also explores the epigenomic landscape, the evolution of methods from bulk approaches to single-cell analyses, and the role of AI and machine learning in deciphering complex genetic patterns. The conversation transitions to a unique study of bat embryonic development, dietary differences, and their implications for understanding wing development and metabolism in different bat species.
 
References
Ahituv, N., Zhu, Y., Visel, A., Holt, A., Afzal, V., Pennacchio, L. A., & Rubin, E. M. (2007). Deletion of ultraconserved elements yields viable mice. PLoS biology, 5(9), e234. https://doi.org/10.1371/journal.pbio.0050234
Matharu, N., Rattanasopha, S., Tamura, S., Maliskova, L., Wang, Y., Bernard, A., Hardin, A., Eckalbar, W. L., Vaisse, C., & Ahituv, N. (2019). CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science (New York, N.Y.), 363(6424), eaau0629. https://doi.org/10.1126/science.aau0629
Ushiki, A., Zhang, Y., Xiong, C., Zhao, J., Georgakopoulos-Soares, I., Kane, L., Jamieson, K., Bamshad, M. J., Nickerson, D. A., University of Washington Center for Mendelian Genomics, Shen, Y., Lettice, L. A., Silveira-Lucas, E. L., Petit, F., & Ahituv, N. (2021). Deletion of CTCF sites in the SHH locus alters enhancer-promoter interactions and leads to acheiropodia. Nature communications, 12(1), 2282. https://doi.org/10.1038/s41467-021-22470-z
Georgakopoulos-Soares, I., Deng, C., Agarwal, V., Chan, C. S. Y., Zhao, J., Inoue, F., & Ahituv, N. (2023). Transcription factor binding site orientation and order are major drivers of gene regulatory activity. Nature communications, 14(1), 2333. https://doi.org/10.1038/s41467-023-37960-5
Gordon, W. E., Baek, S., Nguyen, H. P., Kuo, Y. M., Bradley, R., Fong, S. L., Kim, N., Galazyuk, A., Lee, I., Ingala, M. R., Simmons, N. B., Schountz, T., Cooper, L. N., Georgakopoulos-Soares, I., Hemberg, M., & Ahituv, N. (2024). Integrative single-cell characterization of a frugivorous and an insectivorous bat kidney and pancreas. Nature communications, 15(1), 12. https://doi.org/10.1038/s41467-023-44186-y
 
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