Epigenetics Podcast

Split-Pool Recognition of Interactions by Tag Extension (SPRITE) (Mitch Guttman)

In this episode of the Epigenetics Podcast, we talked with Mitch Guttman from California Institute of Technology about his work on characterising the 3D interactions of the genome using Split-Pool Recognition of Interactions by Tag Extension (SPRITE).
Mitch Guttman discusses his exploration of the long non-coding RNA Xist, which plays a crucial role in X chromosome inactivation. He explains how they discovered that Xist is present everywhere in the nucleus, not just in specific locations on the X chromosome. Through their research, they identified critical proteins like SHARP that are involved in X chromosome silencing.
The discussion then shifts to SPRITE, a method they developed to map multi-way contacts and generalize beyond DNA to include RNA and proteins. They compare SPRITE to classical proximity ligation methods like Hi-C and discuss how cluster sizes in SPRITE can estimate 3D distances between molecules. The conversation also touches upon the potential of applying SPRITE to single-cell experiments, allowing for the mapping of higher order nucleic acid interactions and tracking the connectivity of DNA fragments in individual cells.
 
References
Jesse M. Engreitz et al., The Xist lncRNA Exploits Three-Dimensional Genome Architecture to Spread Across the X Chromosome. Science 341,1237973(2013). DOI:10.1126/science.1237973
Chun-Kan Chen et al., Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing. Science 354, 468-472(2016). DOI: 10.1126/science.aae0047
Quinodoz, S. A., Ollikainen, N., Tabak, B., Palla, A., Schmidt, J. M., Detmar, E., Lai, M. M., Shishkin, A. A., Bhat, P., Takei, Y., Trinh, V., Aznauryan, E., Russell, P., Cheng, C., Jovanovic, M., Chow, A., Cai, L., McDonel, P., Garber, M., & Guttman, M. (2018). Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus. Cell, 174(3), 744-757.e24. https://doi.org/10.1016/j.cell.2018.05.024
Goronzy, I. N., Quinodoz, S. A., Jachowicz, J. W., Ollikainen, N., Bhat, P., & Guttman, M. (2022). Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription. Cell Reports, 41(9), 111730. https://doi.org/10.1016/j.celrep.2022.111730
Perez, A. A., Goronzy, I. N., Blanco, M. R., Guo, J. K., & Guttman, M. (2023). ChIP-DIP: A multiplexed method for mapping hundreds of proteins to DNA uncovers diverse regulatory elements controlling gene expression [Preprint]. Genomics. https://doi.org/10.1101/2023.12.14.571730
 
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MLL Proteins in Mixed-Lineage Leukemia (Yali Dou)

In this episode of the Epigenetics Podcast, we talked with Yali Dou from Keck School of Medicine of USC about her work on MLL Proteins in Mixed-Lineage Leukemia.
To start off this Interview Yali describes her early work on MLL1 and its function in transcription, particularly its involvement in histone modification. She explains her successful purification of the MLL complex and the discovery of MOF as one of the proteins involved.
Next, the interview focuses on her work in reconstituting the MLL core complex and the insights gained from this process. She shares her experience of reconstituting the MLL complex and discusses her focus on the crosstalk of H3K4 and H3K79 methylation, regulated by H2BK34 ubiquitination.
The podcast then delves into the therapeutic potential of MLL1, leading to the discovery of a small molecule inhibitor. Finally, we talk about the importance of the protein WDR5 in the assembly of MLL complexes and how targeting the WDR5-ML interaction can inhibit MLL activity.
 
References
Dou, Y., Milne, T., Ruthenburg, A. et al. Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat Struct Mol Biol 13, 713–719 (2006). https://doi.org/10.1038/nsmb1128
Wu, L., Zee, B. M., Wang, Y., Garcia, B. A., & Dou, Y. (2011). The RING Finger Protein MSL2 in the MOF Complex Is an E3 Ubiquitin Ligase for H2B K34 and Is Involved in Crosstalk with H3 K4 and K79 Methylation. Molecular Cell, 43(1), 132–144. https://doi.org/10.1016/j.molcel.2011.05.015
Cao, F., Townsend, E. C., Karatas, H., Xu, J., Li, L., Lee, S., Liu, L., Chen, Y., Ouillette, P., Zhu, J., Hess, J. L., Atadja, P., Lei, M., Qin, Z. S., Malek, S., Wang, S., & Dou, Y. (2014). Targeting MLL1 H3K4 Methyltransferase Activity in Mixed-Lineage Leukemia. Molecular Cell, 53(2), 247–261. https://doi.org/10.1016/j.molcel.2013.12.001
Park, S.H., Ayoub, A., Lee, YT. et al. Cryo-EM structure of the human MLL1 core complex bound to the nucleosome. Nat Commun 10, 5540 (2019). https://doi.org/10.1038/s41467-019-13550-2
 
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Sex-biased Imprinting and DNA Regulatory Landscapes During Reprogramming (Sam Buckberry)

In this episode of the Epigenetics Podcast, we talked with Sam Buckberry from the Telethon Kids Institute about his work on gene imprinting, sex-biased gene expression, DNA regulatory landscapes, and genomics in the indigenous population of Australia.
Sam Buckberry's research career started with working on the imprinting of H19, IGF2, and IGF2R genes in the placenta. We talk about the controversy surrounding the imprinting of IGF2R and how his study used pyrosequencing to quantify gene expression. We also discuss Sam's work on sex-biased gene expression in the placenta and the identification of a cluster of genes related to placental development and pregnancy.
In addition, we talk about Sam's research on reprogramming and the characterization of DNA regulatory landscapes during the process. We discuss the challenges of working with sequencing data, the discovery of epigenetic memories, and erasing them during reprogramming. Towards the end of the conversation, Sam mentions his current work in setting up an epigenetics group focused on indigenous genomics. They are conducting a large-scale, multi-omics study on cardiometabolic conditions in samples from indigenous Australian communities, with the goal of identifying biomarkers and better understanding the molecular basis of these conditions.
 
References
Buckberry, S., Liu, X., Poppe, D. et al. Transient naive reprogramming corrects hiPS cells functionally and epigenetically. Nature 620, 863–872 (2023). https://doi.org/10.1038/s41586-023-06424-7
Knaupp AS1, Buckberry S1, Pflueger J, Lim SM, Ford E, Larcombe MR, Rossello FJ, de Mendoza A, Alaei S, Firas J, Holmes ML, Nair SS, Clark SJ, Nefzger CM, Lister R and Polo JM (2017). Transient and permanent reconfiguration of chromatin and transcription factor occupancy drive reprogramming. Cell Stem Cell 21, 1-12 1 Co-first author
 
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BET Proteins and Their Role in Chromosome Folding and Compartmentalization (Kyle Eagen)

In this episode of the Epigenetics Podcast, we talked with Kyle Eagen from Baylor College of Medicine about his work on BET Proteins and their role in chromosome folding and compartmentalization.
In the early days of his research career Dr. Eagen made use of genomics and microscopy to study chromosomes, particularly polytene chromosomes in Drosophila. The correlation between the folding patterns detected by Hi-C and polytene bands highlights the similarities between the two, bridging traditional cytology with modern NGS methods. This work formed the basis of Kyle's thesis and sparked his interest in nuclear organization and chromosome 3D structure.
In his independent lab Kyle then studied compartments in chromatin structure and focused on the relationship between histone modifications and the 3D structure of chromosomes. The discovery of BRD4-NUT, a fusion oncoprotein that reprograms chromosome 3D structure, is highlighted as a significant step forward in understanding chromatin structure.
The conversation then shifts to the use of a tool to test hypotheses about the involvement of BRD4 in a specific process, leading to consistent results and considerations for manipulating chromosome organization for therapeutic purposes. The role of BET proteins in genome folding and the need for further research on other factors involved in 3D genome structure are discussed.
 
References
Rosencrance, C. D., Ammouri, H. N., Yu, Q., Ge, T., Rendleman, E. J., Marshall, S. A., & Eagen, K. P. (2020). Chromatin Hyperacetylation Impacts Chromosome Folding by Forming a Nuclear Subcompartment. Molecular Cell, 78(1), 112-126.e12. https://doi.org/10.1016/j.molcel.2020.03.018
Huang, Y., Durall, R. T., Luong, N. M., Hertzler, H. J., Huang, J., Gokhale, P. C., Leeper, B. A., Persky, N. S., Root, D. E., Anekal, P. V., Montero Llopis, P. D. L. M., David, C. N., Kutok, J. L., Raimondi, A., Saluja, K., Luo, J., Zahnow, C. A., Adane, B., Stegmaier, K., … French, C. A. (2023). EZH2 Cooperates with BRD4-NUT to Drive NUT Carcinoma Growth by Silencing Key Tumor Suppressor Genes. Cancer Research, 83(23), 3956–3973. https://doi.org/10.1158/0008-5472.CAN-23-1475
 
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Epigenetic Underpinnings of Human Addiction (Francesca Telese & Jessica Zhou)

In this episode of the Epigenetics Podcast, we talked with Francesca Telese from UC San Diego and Jessica Zhou from Cold Spring Harbour about their work on the molecular underpinnings of human addiction.
Francesca Telese worked on neuronal enhancers and their pivotal role in governing gene activity. She sheds light on her remarkable findings concerning the epigenetic signature of neuronal enhancers that are intricately involved in synaptic plasticity.
Jessica Zhou joined Francesca Telese's lab as a PhD student where she worked on elucidating the effects of chronic cannabis use on memory and behavior in mice. She takes us through the fascinating correlation between THC and gene co-expression networks. Francesca and Jessicathen discuss the utilization of genetically diverse outbred rats in their research, along with the crucial exploration of cell type specificity in gene expression studies. They then delve into the long-term changes that occur in the brain after drug exposure and the profound implications for relapse. Additionally, they touch upon the challenges they face in analyzing single-cell data.
 
References
Zhou, J. L., de Guglielmo, G., Ho, A. J., Kallupi, M., Pokhrel, N., Li, H. R., Chitre, A. S., Munro, D., Mohammadi, P., Carrette, L. L. G., George, O., Palmer, A. A., McVicker, G., & Telese, F. (2023). Single-nucleus genomics in outbred rats with divergent cocaine addiction-like behaviors reveals changes in amygdala GABAergic inhibition. Nature neuroscience, https://doi.org/10.1038/s41593-023-01452-y
Wang, J., Telese, F., Tan, Y., Li, W., Jin, C., He, X., Basnet, H., Ma, Q., Merkurjev, D., Zhu, X., Liu, Z., Zhang, J., Ohgi, K., Taylor, H., White, R. R., Tazearslan, C., Suh, Y., Macfarlan, T. S., Pfaff, S. L., & Rosenfeld, M. G. (2015). LSD1n is an H4K20 demethylase regulating memory formation via transcriptional elongation control. Nature neuroscience, 18(9), 1256–1264. https://doi.org/10.1038/nn.4069
 
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H3K79 Methylation, DOT1L, and FOXG1 in Neural Development (Tanja Vogel)

In this episode of the Epigenetics Podcast, we talked with Tanja Vogel from the University Clinics Freiburg about her work on epigenetic modifications in stem cells during central nervous system development.
During our discussion, Dr. Vogel shared that she and her team have investigated H3K79 methylation and its functional significance, which remains a topic of debate in the scientific community. They’ve also investigated the role of DOT1L in neural development and its implications for neuronal networks, as disrupting DOT1L can lead to conditions such as epilepsy and schizophrenia. They explored the function of the SOX2 enhancer in the presence or absence of DOT1L enzymatic inhibition.
The conversation then shifts to FoxG1, a vital player in forebrain development. The team uncovered its role in chromatin accessibility and its connection to microRNA processing. Their study, utilizing ChIP-Seq, reveals FoxG1's interactions with enhancer regions and other transcription factors, like NeuroD1.
 
### References
Britanova, O., de Juan Romero, C., Cheung, A., Kwan, K. Y., Schwark, M., Gyorgy, A., Vogel, T., Akopov, S., Mitkovski, M., Agoston, D., Sestan, N., Molnár, Z., & Tarabykin, V. (2008). Satb2 is a postmitotic determinant for upper-layer neuron specification in the neocortex. Neuron, 57(3), 378–392. https://doi.org/10.1016/j.neuron.2007.12.028
Büttner, N., Johnsen, S. A., Kügler, S., & Vogel, T. (2010). Af9/Mllt3 interferes with Tbr1 expression through epigenetic modification of histone H3K79 during development of the cerebral cortex. Proceedings of the National Academy of Sciences of the United States of America, 107(15), 7042–7047. https://doi.org/10.1073/pnas.0912041107
Franz, H., Villarreal, A., Heidrich, S., Videm, P., Kilpert, F., Mestres, I., Calegari, F., Backofen, R., Manke, T., & Vogel, T. (2019). DOT1L promotes progenitor proliferation and primes neuronal layer identity in the developing cerebral cortex. Nucleic acids research, 47(1), 168–183. https://doi.org/10.1093/nar/gky953
Ferrari, F., Arrigoni, L., Franz, H., Izzo, A., Butenko, L., Trompouki, E., Vogel, T., & Manke, T. (2020). DOT1L-mediated murine neuronal differentiation associates with H3K79me2 accumulation and preserves SOX2-enhancer accessibility. Nature communications, 11(1), 5200. https://doi.org/10.1038/s41467-020-19001-7
Akol, I., Izzo, A., Gather, F., Strack, S., Heidrich, S., Ó hAilín, D., Villarreal, A., Hacker, C., Rauleac, T., Bella, C., Fischer, A., Manke, T., & Vogel, T. (2023). Multimodal epigenetic changes and altered NEUROD1 chromatin binding in the mouse hippocampus underlie FOXG1 syndrome. Proceedings of the National Academy of Sciences of the United States of America, 120(2), e2122467120. https://doi.org/10.1073/pnas.2122467120
 
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Function of Insulators in 3D Genome Folding (Maria Gambetta)

In this episode of the Epigenetics Podcast, we talked with Maria Gambetta from the University of Lausanne about her work on the function of insulators in 3D genome folding.
Maria Gambetta focuses on investigating 3D contact dynamics between enhancers and promoters, providing insights into tissue-specific gene activation. The team used capture-C to analyze dynamic looping events, emphasizing the significance of accessible chromatin peaks in enhancer-promoter interactions. Furthermore, they focused on gene insulation and CTCF's role in forming topologically associating domains in Drosophila. Hi-C analysis on CTCF mutants revealed the conservation of TAD boundary mechanisms, identifying CP-190 as a potential binding protein.
Their findings on the loss of TAD boundaries in mutants and the role of transcription in TAD boundary formation are discussed as well as the function of CP190 and insulators in preventing interactions between promoters and enhancers. Their work challenges existing models of insulator function and seeks to understand their mechanisms better. The conversation concludes with insights into long-range regulatory associations in Drosophila, emphasizing the punctual interactions between transcription factor binding sites and their effect on neural gene transcription and genome folding.
 
References
Gambetta, M. C., Oktaba, K., & Müller, J. (2009). Essential role of the glycosyltransferase sxc/Ogt in polycomb repression. Science (New York, N.Y.), 325(5936), 93–96. https://doi.org/10.1126/science.1169727
Kaushal, A., Mohana, G., Dorier, J., Özdemir, I., Omer, A., Cousin, P., Semenova, A., Taschner, M., Dergai, O., Marzetta, F., Iseli, C., Eliaz, Y., Weisz, D., Shamim, M. S., Guex, N., Lieberman Aiden, E., & Gambetta, M. C. (2021). CTCF loss has limited effects on global genome architecture in Drosophila despite critical regulatory functions. Nature communications, 12(1), 1011. https://doi.org/10.1038/s41467-021-21366-2
Hoencamp, C., Dudchenko, O., Elbatsh, A. M. O., Brahmachari, S., Raaijmakers, J. A., van Schaik, T., Sedeño Cacciatore, Á., Contessoto, V. G., van Heesbeen, R. G. H. P., van den Broek, B., Mhaskar, A. N., Teunissen, H., St Hilaire, B. G., Weisz, D., Omer, A. D., Pham, M., Colaric, Z., Yang, Z., Rao, S. S. P., Mitra, N., … Rowland, B. D. (2021). 3D genomics across the tree of life reveals condensin II as a determinant of architecture type. Science (New York, N.Y.), 372(6545), 984–989. https://doi.org/10.1126/science.abe2218
Mohana, G., Dorier, J., Li, X., Mouginot, M., Smith, R. C., Malek, H., Leleu, M., Rodriguez, D., Khadka, J., Rosa, P., Cousin, P., Iseli, C., Restrepo, S., Guex, N., McCabe, B. D., Jankowski, A., Levine, M. S., & Gambetta, M. C. (2023). Chromosome-level organization of the regulatory genome in the Drosophila nervous system. Cell, 186(18), 3826–3844.e26. https://doi.org/10.1016/j.cell.2023.07.008
 
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Contribution of the Estrogen Receptor to Breast Cancer Progression (Jason Carroll)

In this episode of the Epigenetics Podcast, we talked with Jason Carroll from the Cambridge Research Institute about his work on contribution of estrogen receptor to breast cancer progression.
The Podcast centers around the crucial role of the forkhead protein FOXA1 in breast cancer. FOXA1 acts as a pioneer transcription factor, facilitating gene regulation by recruiting nuclear receptors to chromatin, profoundly influencing gene expression in various breast cancer subtypes. The FOXA1-positive subtype of triple-negative breast cancer, despite being estrogen receptor-negative, shares gene expression profiles with estrogen receptor-positive breast cancer, shedding light on the importance of targeting the androgen receptor for treatment.
The challenges of studying transcription factor mappings from clinical samples are explored, with a focus on the ChIP-seq method's success in mapping estrogen receptor binding sites. Various techniques for transcription factor mapping, including CUT&RUN, CUT&Tag, and ChIP-exo, are discussed, as well as the potential of mass spec techniques like the RIME method in analyzing protein interactions. An intriguing experiment involving the purification of multiple proteins to identify interactions is highlighted.
 
References
Carroll, J. S., Meyer, C. A., Song, J., Li, W., Geistlinger, T. R., Eeckhoute, J., Brodsky, A. S., Keeton, E. K., Fertuck, K. C., Hall, G. F., Wang, Q., Bekiranov, S., Sementchenko, V., Fox, E. A., Silver, P. A., Gingeras, T. R., Liu, X. S., & Brown, M. (2006). Genome-wide analysis of estrogen receptor binding sites. Nature genetics, 38(11), 1289–1297. https://doi.org/10.1038/ng1901
Hurtado, A., Holmes, K. A., Geistlinger, T. R., Hutcheson, I. R., Nicholson, R. I., Brown, M., Jiang, J., Howat, W. J., Ali, S., & Carroll, J. S. (2008). Regulation of ERBB2 by oestrogen receptor-PAX2 determines response to tamoxifen. Nature, 456(7222), 663–666. https://doi.org/10.1038/nature07483
Ross-Innes, C. S., Stark, R., Teschendorff, A. E., Holmes, K. A., Ali, H. R., Dunning, M. J., Brown, G. D., Gojis, O., Ellis, I. O., Green, A. R., Ali, S., Chin, S. F., Palmieri, C., Caldas, C., & Carroll, J. S. (2012). Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature, 481(7381), 389–393. https://doi.org/10.1038/nature10730
Mohammed, H., Russell, I. A., Stark, R., Rueda, O. M., Hickey, T. E., Tarulli, G. A., Serandour, A. A., Birrell, S. N., Bruna, A., Saadi, A., Menon, S., Hadfield, J., Pugh, M., Raj, G. V., Brown, G. D., D'Santos, C., Robinson, J. L., Silva, G., Launchbury, R., Perou, C. M., … Carroll, J. S. (2015). Progesterone receptor modulates ERα action in breast cancer. Nature, 523(7560), 313–317. https://doi.org/10.1038/nature14583
 
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