Epigenetics Podcast

ATAC-Seq, scATAC-Seq and Chromatin Dynamics in Single-Cells (Jason Buenrostro)

In this episode of the Epigenetics Podcast, we caught up with Jason Buenrostro from Harvard University to talk about his work on developing biological tools to measure chromatin dynamics in single-cells. He explains how his lab uses these tools to study chromatin alterations in different cell types and disease states to uncover new mechanisms of gene regulation and their contribution to those diseases.
In his first years of his research career Jason Buenrostro took a risk and just added an enzyme called Transposase to cells in a cell culture. What he saw on a subsequent agarose gel astonished him. He was able to recreate a nucleosomal ladder that he knew from experiments using MNase or DNase-Seq, however, without the tedious steps of optimization. In the following years he optimized that method and data analyzation into a method known today as ATAC-Seq. In recent years he was also able to bring ATAC-Seq to the next level and developed single cell ATAC-Seq (scATAC-Seq), and combining it with RNA-Seq in a multi-omics approach.
In this Episode we discuss how Jason Buenrostro developed ATAC-Seq in William Greenleaf's lab, how a lack of equipment shaped the ATAC-Seq protocol, and how scATAC-Seq has enabled a whole different way of looking at biological samples.
 
References
Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., & Greenleaf, W. J. (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature Methods, 10(12), 1213–1218. https://doi.org/10.1038/nmeth.2688
Buenrostro, J. D., Wu, B., Litzenburger, U. M., Ruff, D., Gonzales, M. L., Snyder, M. P., Chang, H. Y., & Greenleaf, W. J. (2015). Single-cell chromatin accessibility reveals principles of regulatory variation. Nature, 523(7561), 486–490. https://doi.org/10.1038/nature14590
Buenrostro, J. D., Corces, M. R., Lareau, C. A., Wu, B., Schep, A. N., Aryee, M. J., Majeti, R., Chang, H. Y., & Greenleaf, W. J. (2018). Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation. Cell, 173(6), 1535-1548.e16. https://doi.org/10.1016/j.cell.2018.03.074
Lareau, C. A., Duarte, F. M., Chew, J. G., Kartha, V. K., Burkett, Z. D., Kohlway, A. S., Pokholok, D., Aryee, M. J., Steemers, F. J., Lebofsky, R., & Buenrostro, J. D. (2019). Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility. Nature Biotechnology, 37(8), 916–924. https://doi.org/10.1038/s41587-019-0147-6
 
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Chromatin Profiling: From ChIP to CUT&RUN, CUT&Tag and CUTAC (Steven Henikoff)
Hi-C and Three-Dimensional Genome Sequencing (Erez Lieberman Aiden)
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Synthetic Chromatin Epigenetics (Karmella Haynes)

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

In this episode of the Epigenetics Podcast, we caught up with Enrico Glaab from the University of Luxemburg to talk about his work on the development of integrative machine learning tools for neurodegenerative diseases.
The work of Dr. Enrico Glaab focuses on neurodegenerative disorders like Parkinson’s and Alzheimer’s disease. In his group his team works on the development of software tools to analyze molecular, clinical and neuroimaging data for those diseases that can be used and applied easily by scientists and deliver publication ready figures. More recently, Enrico Glaab's group got interested in the influence of Epigenetics in Parkinson's and Alzheimer's disease.
In this Episode we discuss how Enrico Glaab made the switch from wet-lab to becoming a bioinformatician and how he uses integrative machine learning tools to find approaches to not only cure but also be able to detect neurodegenerative diseases like Alzheimer's or Parkinson's early on.
 
References
Enrico Glaab, Reinhard Schneider (2015) RepExplore: addressing technical replicate variance in proteomics and metabolomics data analysis (Bioinformatics) DOI: 10.1093/bioinformatics/btv127
Enrico Glaab, Reinhard Schneider (2015) Comparative pathway and network analysis of brain transcriptome changes during adult aging and in Parkinson’s disease (Neurobiology of Disease) DOI: 10.1016/j.nbd.2014.11.002
Sandra Köglsberger, Maria Lorena Cordero-Maldonado, … Enrico Glaab (2017) Gender-Specific Expression of Ubiquitin-Specific Peptidase 9 Modulates Tau Expression and Phosphorylation: Possible Implications for Tauopathies (Molecular Neurobiology) DOI: 10.1007/s12035-016-0299-z
Enrico Glaab, Paul Antony, … Manuel Buttini (2019) Transcriptome profiling data reveals ubiquitin-specific peptidase 9 knockdown effects (Data in Brief) DOI: 10.1016/j.dib.2019.104130
 
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Ultraconserved Enhancers and Enhancer Redundancy (Diane Dickel)

In this episode of the Epigenetics Podcast, we caught up with Diane Dickel from Lawrence Berkeley National Laboratory to talk about her work on ultraconserved enhancers and enhancer redundancy.
Diane Dickel and her co-workers study non-coding regions of the genome that harbor distant-acting transcriptional regulatory regions, called enhancers. Enhancers have been shown to be critical for normal embryonic development, implying evolutional conservation. Diane Dickel and her team try to identify and characterize enhancers at a genomic scale. Their efforts include the use of CRISPR/CAS9 to mutate enhancer sequences in order to understand sequence dependent functional relevance.
In this episode we discuss the function of ultraconserved enhancers, what ultraconservation actually means, how enhancer redundancy works and how Diane Dickel dealt with a failed PhD project.
 
References
Dickel, D. E., Ypsilanti, A. R., Pla, R., Zhu, Y., Barozzi, I., Mannion, B. J., Khin, Y. S., Fukuda-Yuzawa, Y., Plajzer-Frick, I., Pickle, C. S., Lee, E. A., Harrington, A. N., Pham, Q. T., Garvin, T. H., Kato, M., Osterwalder, M., Akiyama, J. A., Afzal, V., Rubenstein, J. L. R., … Visel, A. (2018). Ultraconserved Enhancers Are Required for Normal Development. Cell, 172(3), 491-499.e15. https://doi.org/10.1016/j.cell.2017.12.017
Gorkin, D. U., Barozzi, I., Zhao, Y., Zhang, Y., Huang, H., Lee, A. Y., Li, B., Chiou, J., Wildberg, A., Ding, B., Zhang, B., Wang, M., Strattan, J. S., Davidson, J. M., Qiu, Y., Afzal, V., Akiyama, J. A., Plajzer-Frick, I., Novak, C. S., … Ren, B. (2020). An atlas of dynamic chromatin landscapes in mouse fetal development. Nature, 583(7818), 744–751. https://doi.org/10.1038/s41586-020-2093-3
Snetkova, V., Ypsilanti, A. R., Akiyama, J. A., Mannion, B. J., Plajzer-Frick, I., Novak, C. S., Harrington, A. N., Pham, Q. T., Kato, M., Zhu, Y., Godoy, J., Meky, E., Hunter, R. D., Shi, M., Kvon, E. Z., Afzal, V., Tran, S., Rubenstein, J. L. R., Visel, A., … Dickel, D. E. (2021). Ultraconserved enhancer function does not require perfect sequence conservation. Nature Genetics, 53(4), 521–528. https://doi.org/10.1038/s41588-021-00812-3
 
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Variants of Core Histones: Modulators of Chromatin Structure and Function (Sandra Hake)

In this episode of the Epigenetics Podcast, we caught up with Sandra Hake from the Justus Liebig University in Giessen to talk about her work on variants of core histones and their role as modulators of chromatin structure and function.
The overarching goal of Sandra Hake's research group is to understand how changes in chromatin structure and composition can influence various DNA-based processes, such as gene expression, repair of DNA damage, cell cycle progression, and genome stability. Their work deals with the study of histone variants which, together with DNA, represent the building blocks of the smallest chromatin components, the nucleosomes. They also investigate whether mutations and/or post-translational histone modifications and the deregulation of histone variant networks influence the emergence of diseases, especially the emergence of tumors.
In this episode we discuss how Sandra Hake approaches the characterization and identification of novel histone variants like H3.3, H3.X and H3.Y, what it's like to work in such a small field like histone variants, and what is coming up next for the Hake lab.
 
References
Hake, S. B., Garcia, B. A., Duncan, E. M., Kauer, M., Dellaire, G., Shabanowitz, J., Bazett-Jones, D. P., Allis, C. D., & Hunt, D. F. (2006). Expression Patterns and Post-translational Modifications Associated with Mammalian Histone H3 Variants. Journal of Biological Chemistry, 281(1), 559–568. https://doi.org/10.1074/jbc.M509266200
Wiedemann, S. M., Mildner, S. N., Bönisch, C., Israel, L., Maiser, A., Matheisl, S., Straub, T., Merkl, R., Leonhardt, H., Kremmer, E., Schermelleh, L., & Hake, S. B. (2010). Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. Journal of Cell Biology, 190(5), 777–791. https://doi.org/10.1083/jcb.201002043
Bönisch, C., Schneider, K., Pünzeler, S., Wiedemann, S. M., Bielmeier, C., Bocola, M., Eberl, H. C., Kuegel, W., Neumann, J., Kremmer, E., Leonhardt, H., Mann, M., Michaelis, J., Schermelleh, L., & Hake, S. B. (2012). H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization. Nucleic Acids Research, 40(13), 5951–5964. https://doi.org/10.1093/nar/gks267
Link, S., Spitzer, R. M. M., Sana, M., Torrado, M., Völker-Albert, M. C., Keilhauer, E. C., Burgold, T., Pünzeler, S., Low, J. K. K., Lindström, I., Nist, A., Regnard, C., Stiewe, T., Hendrich, B., Imhof, A., Mann, M., Mackay, J. P., Bartkuhn, M., & Hake, S. B. (2018). PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex. Nature Communications, 9(1), 4300. https://doi.org/10.1038/s41467-018-06665-5
 
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DNA Methylation and Mammalian Development (Déborah Bourc'his)

In this episode of the Epigenetics Podcast, we caught up with Déborah Bourc'his from L'Institut Curie in Paris to talk about her work on the role of DNA methylation in mammalian development.
During her postdoc years Déborah Bourc'his was able to characterize DNMT3L, a protein with unknown function at that time. It turned out that this protein is the cofactor responsible for stimulating DNA methylation activity in both the male and the female germline. Later on she discovered a novel DNA methylation enzyme called DNMT3C, which was unknown because it was not properly annotated, there was no sign of expression, and it was only expressed in male fetal germ cells. Furthermore, this enzyme only evolved in rodents, as a defense against young transposons.
In this episode we discuss the story behind how Déborah Bourc'his was able to discover and characterize the DNA methylation enzymes DNMT3L and DNMT3C and their role in mammalian development.
 
References
R. Duffie, S. Ajjan, … D. Bourc’his (2014) The Gpr1/Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals (Genes & Development) DOI: 10.1101/gad.232058.113
Natasha Zamudio, Joan Barau, … Déborah Bourc’his (2015) DNA methylation restrains transposons from adopting a chromatin signature permissive for meiotic recombination (Genes & Development) DOI: 10.1101/gad.257840.114](https://doi.org/10.1101/gad.257840.114)
Marius Walter, Aurélie Teissandier, … Déborah Bourc’his (2016) An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells (eLife) DOI: 10.7554/eLife.11418](https://doi.org/10.7554/eLife.11418)
Joan Barau, Aurélie Teissandier, … Déborah Bourc’his (2016) The DNA methyltransferase DNMT3C protects male germ cells from transposon activity (Science (New York, N.Y.)) DOI: 10.1126/science.aah5143
Maxim V. C. Greenberg, Juliane Glaser, … Déborah Bourc’his (2017) Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth (Nature Genetics) DOI: 10.1038/ng.3718
Roberta Ragazzini, Raquel Pérez-Palacios, … Raphaël Margueron (2019) EZHIP constrains Polycomb Repressive Complex 2 activity in germ cells (Nature Communications) DOI: 10.1038/s41467-019-11800-x
Dura, M., Teissandier, A., Armand, M., Barau, J., Bonneville, L., Weber, M., Baudrin, L. G., Lameiras, S., & Bourc’his, D. (2021). DNMT3A-dependent DNA methylation is required for spermatogonial stem cells to commit to spermatogenesis [Preprint]. Developmental Biology. https://doi.org/10.1101/2021.04.19.440465
 
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Chromatin Analysis using Mass Spectrometry (Axel Imhof)

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

In this episode of the Epigenetics Podcast, we caught up with Steven Henikoff from the Fred Hutchinson Cancer Research Center in Seattle to talk about his work on Chromatin Profiling: From ChIP to CUT&RUN, CUT&Tag and CUTAC.
In the last few years Steven Henikoff has been developing methods which profile the chromatin landscape by using enzyme tethering. The quest first started with ChEC-Seq, which improved on Uli Laemmli's method of Chromatin endogenous cleavage (ChEC) but used sequencing as a read-out rather than southern blotting. Next, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) was developed by making a fusion protein of Protein A and micrococcal nuclease (MNase), making it possible to achieve antibody-targeted cleavage of chromatin fragments. And finally, Cleavage Under Targets & Tagmenation (CUT&Tag) was developed by using Transposase Tn5 instead of MNase, which adds sequencing adapters and fragments chromatin at the same time, streamlining the protocol even further.
In this episode we discuss how working on centromeres set the stage for Steven Henikoff’s subsequent work, how he developed CUT&RUN and CUT&Tag, what the advantages and disadvantages of those methods are and how he developed all those experiments at home in his garage.
 
References
Takehito Furuyama, Steven Henikoff (2009) Centromeric nucleosomes induce positive DNA supercoils (Cell) DOI: 10.1016/j.cell.2009.04.049
Kristina Krassovsky, Jorja G. Henikoff, Steven Henikoff (2012) Tripartite organization of centromeric chromatin in budding yeast (Proceedings of the National Academy of Sciences of the United States of America) DOI: 10.1073/pnas.1118898109
Jorja G. Henikoff, Jitendra Thakur, … Steven Henikoff (2015) A unique chromatin complex occupies young α-satellite arrays of human centromeres (Science Advances) DOI: 10.1126/sciadv.1400234
Epigenomics Methods: ChEC-Seq, CUT&RUN, AutoCUT&RUN, Improved CUT&RUN, CUT&Tag, Automated CUT&Tag, CUTAC, scCUT&Tag.
 
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