Epigenetics Podcast

Multiple challenges of ATAC-Seq, Points to Consider (Yuan Xue)

In this episode of the Epigenetics Podcast, we caught up with Active Motif’s own Yuan Xue to talk about some of the challenges of performing ATAC-Seq.
ATAC-Seq stands for Assay for Transposase-Accessible Chromatin with high-throughput sequencing and was initially described by Jason Buenrostro in 2013. The ATAC-Seq method relies on next-generation sequencing (NGS) library construction using the hyperactive transposase Tn5. NGS adapters are loaded onto the transposase, which allows simultaneous fragmentation of chromatin and integration of those adapters into open chromatin regions. ATAC-Seq is an attractive method to start your epigenetic journey. Whether you want to analyze the state of the chromatin in your sample or compare the chromatin state before and after a special treatment, ATAC-Seq allows you to investigate genome-wide chromatin changes and can offer guidelines about which epigenetic modification or transcription factor should be studied next in the follow-up experiments and which method should be used to study them.
In this Episode we go through the Protocol in detail and discuss potential challenges and points to pay attention to when starting your first ATAC-Seq experiment.
 
References
ATAC-Seq Resource Center
Complete Guide to Understanding and Using ATAC-Seq
Beginner’s Guide to Understanding Single-Cell ATAC-Seq
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
Cusanovich, D. A., Daza, R., Adey, A., Pliner, H. A., Christiansen, L., Gunderson, K. L., Steemers, F. J., Trapnell, C., & Shendure, J. (2015). Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science (New York, N.Y.), 348(6237), 910–914. https://doi.org/10.1126/science.aab1601
Podcast: ATAC-Seq, scATAC-Seq and Chromatin Dynamics in Single-Cells (Jason Buenrostro)
 
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The Effect of lncRNAs on Chromatin and Gene Regulation (John Rinn)

In this episode of the Epigenetics Podcast, we caught up with John Rinn from the University of Colorado in Boulder to talk about his work on the role of lncRNAs in gene expression and nuclear organization.
The Rinn Lab pioneered the approach of screening the human genome for long noncoding RNAs (lncRNAs). More recently, the lab has shifted focus from measuring the number of lncRNAs to finding lncRNAs that have a distinct biological function in human health and disease. One example of such a lncRNA is FIRRE, which is present in all animals, however the sequence is not conserved, except for in primates. FIRRE contains many interesting features, such as repeat sequences and CTCF binding sites. In absence of FIRRE, defects in the immune system can be observed and also some brain defects may also be observed.
 
References
Carter, T., Singh, M., Dumbovic, G., Chobirko, J. D., Rinn, J. L., & Feschotte, C. (2022). Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo. eLife, 11, e76257. Advance online publication. https://doi.org/10.7554/eLife.76257
Long, Y., Hwang, T., Gooding, A. R., Goodrich, K. J., Rinn, J. L., & Cech, T. R. (2020). RNA is essential for PRC2 chromatin occupancy and function in human pluripotent stem cells. Nature Genetics, 52(9), 931–938. https://doi.org/10.1038/s41588-020-0662-x
Kelley, D., & Rinn, J. (2012). Transposable elements reveal a stem cell-specific class of long noncoding RNAs. Genome biology, 13(11), R107. https://doi.org/10.1186/gb-2012-13-11-r107
Khalil, A. M., Guttman, M., Huarte, M., Garber, M., Raj, A., Rivea Morales, D., Thomas, K., Presser, A., Bernstein, B. E., van Oudenaarden, A., Regev, A., Lander, E. S., & Rinn, J. L. (2009). Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proceedings of the National Academy of Sciences, 106(28), 11667–11672. https://doi.org/10.1073/pnas.0904715106
Guttman, M., Amit, I., Garber, M., French, C., Lin, M. F., Feldser, D., Huarte, M., Zuk, O., Carey, B. W., Cassady, J. P., Cabili, M. N., Jaenisch, R., Mikkelsen, T. S., Jacks, T., Hacohen, N., Bernstein, B. E., Kellis, M., Regev, A., Rinn, J. L., & Lander, E. S. (2009). Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature, 458(7235), 223–227. https://doi.org/10.1038/nature07672
 
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Epigenetic Clocks and Biomarkers of Ageing (Morgan Levine)

 In this episode of the Epigenetics Podcast, we caught up with Morgan Levine from Altos lab to talk about her work on Epigenetic Clocks and Biomarkers of Ageing.
The Levine Lab focuses on deciphering mechanisms that lead to epigenetic ageing, which can be measured by epigenetic clocks. Epigenetic clocks were first described in 2011 by Bocklandt et al.. Later-on, the Horvath and the Hannum clock were described by using a combination of CpGs to calculate biological/epigenetic age in contrast to chronological age.
The Levine Lab themselves worked on generating an advanced version of an Epigenetic clock, called "DNAm PhenoAge" that will now be used, and not only in human samples. The team now moves to mouse models and to cells in a dish and using those models to investigate the mechanisms behind epigenetic aging.
 
References
Liu, Z., Leung, D., Thrush, K., Zhao, W., Ratliff, S., Tanaka, T., Schmitz, L. L., Smith, J. A., Ferrucci, L., & Levine, M. E. (2020). Underlying features of epigenetic aging clocks in vivo and in vitro. Aging cell, 19(10), e13229. https://doi.org/10.1111/acel.13229
Levine, M. E., Lu, A. T., Quach, A., Chen, B. H., Assimes, T. L., Bandinelli, S., Hou, L., Baccarelli, A. A., Stewart, J. D., Li, Y., Whitsel, E. A., Wilson, J. G., Reiner, A. P., Aviv, A., Lohman, K., Liu, Y., Ferrucci, L., & Horvath, S. (2018). An epigenetic biomarker of aging for lifespan and healthspan. Aging, 10(4), 573–591. https://doi.org/10.18632/aging.101414
Levine, M., McDevitt, R. A., Meer, M., Perdue, K., Di Francesco, A., Meade, T., Farrell, C., Thrush, K., Wang, M., Dunn, C., Pellegrini, M., de Cabo, R., & Ferrucci, L. (2020). A rat epigenetic clock recapitulates phenotypic aging and co-localizes with heterochromatin. eLife, 9, e59201. https://doi.org/10.7554/eLife.59201
Kuo, C. L., Pilling, L. C., Atkins, J. C., Masoli, J., Delgado, J., Tignanelli, C., Kuchel, G., Melzer, D., Beckman, K. B., & Levine, M. (2020). COVID-19 severity is predicted by earlier evidence of accelerated aging. medRxiv : the preprint server for health sciences, 2020.07.10.20147777. https://doi.org/10.1101/2020.07.10.20147777
 
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Epigenetic and Metabolic Regulation of Early Development (Jan Żylicz)

In this episode of the Epigenetics Podcast, we caught up with Jan Żylicz from the Novo Nordisk Foundation Center for Stem Cell Medicine to talk about his work on epigenetic and metabolic regulation of early development.
The focus of the Żylicz Lab is studying early development and how this process is influenced by epigenetic factors. In more detail, the Team focuses on the function of chromatin modifiers in this process. Primed pluripotent epiblasts in vivo show a distinct chromatin landscape that is characterized by high levels of histone H3 lysine 9 dimethylation (H3K9me2) and rearranged Polycomb-associated histone H3 lysine 27 trimethylation (H3K27me3) at thousands of genes along the genome. However, the function of only about 100 loci is impaired. The Żylicz Lab tries to understand this process behind and also the cause of this discrepancy.
 
References
Żylicz, J. J., Bousard, A., Žumer, K., Dossin, F., Mohammad, E., da Rocha, S. T., Schwalb, B., Syx, L., Dingli, F., Loew, D., Cramer, P., & Heard, E. (2019). The Implication of Early Chromatin Changes in X Chromosome Inactivation. Cell, 176(1–2), 182-197.e23. https://doi.org/10.1016/j.cell.2018.11.041
Dossin, F., Pinheiro, I., Żylicz, J. J., Roensch, J., Collombet, S., Le Saux, A., Chelmicki, T., Attia, M., Kapoor, V., Zhan, Y., Dingli, F., Loew, D., Mercher, T., Dekker, J., & Heard, E. (2020). SPEN integrates transcriptional and epigenetic control of X-inactivation. Nature, 578(7795), 455–460. https://doi.org/10.1038/s41586-020-1974-9
 
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Multiple challenges of CUT&Tag (Cassidee McDonough, Kyle Tanguay)

In this episode of the Epigenetics Podcast, we caught up with Active Motif scientists Casidee McDonough from Epigenetic Services and Kyle Tanguay from R&D to talk about technical details of the CUT&Tag protocol and current developments around this method in our R&D Team. 
CUT&Tag, which is short for Cleavage Under Targets and Tagmentation, is a molecular biology method that is used to investigate interactions between proteins and DNA and to identify DNA binding sites for their protein of interest. Although CUT&Tag is similar in some ways to ChIP assays, the starting material for CUT&Tag is live, permeabilized cells or isolated cell nuclei, rather than cells or tissue that have been crosslinked with formaldehyde as is the case when performing ChIP. The CUT&Tag method is very sensitive and has been reported to work with as few as 60 cells for some histone modifications. The ability to work with such small numbers of cells is an advantage for researchers working on specific cell types, such as rare neuronal populations, pancreatic islets, or stem cells that are difficult to obtain in large numbers. 
In this Episode we discuss the CUT&Tag workflow in detail, talk about the challenges and pitfalls, give guidelines on how to do a good CUT&Tag experiment and offer a glimpse into the future of CUT&Tag product development at Active Motif. 
 
References
Comprehensive Guide to Understanding and Using CUT&Tag Assays
Library QC for ATAC-Seq and CUT&Tag | AKA “Does My Library Look Okay?”
Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930 (2019). https://doi.org/10.1038/s41467-019-09982-5
Podcast: Chromatin Profiling: From ChIP to CUT&RUN, CUT&Tag and CUTAC (Steven Henikoff)
CUT&Tag-validated antibodies
 
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The Role of Histone Dopaminylation and Serotinylation in Neuronal Plasticity (Ian Maze)

In this episode of the Epigenetics Podcast, we caught up with Ian Maze from Ichan School of Medicine at Mount Sinai and a Howard Hughes Medical Institute (HHMI) Investigator to talk about his work on the role of histone dopaminylation and serotinylation in neuronal plasticity.
The Maze group focuses on understanding the complex interplay between chromatin regulatory mechanisms in brain and neuronal plasticity. The lab places an emphasis on psychiatric disorders associated with monoaminergic (e.g., serotonin, dopamine, etc.) dysfunction, such as major depressive disorder and drug addiction. In particular the Maze team has investigated cocaine addiction and its effect on chromatin by serotonylation and dopaminylation of Histone H3 Tails.
 
References
Maze, I., Covington, H. E., Dietz, D. M., LaPlant, Q., Renthal, W., Russo, S. J., Mechanic, M., Mouzon, E., Neve, R. L., Haggarty, S. J., Ren, Y., Sampath, S. C., Hurd, Y. L., Greengard, P., Tarakhovsky, A., Schaefer, A., & Nestler, E. J. (2010). Essential Role of the Histone Methyltransferase G9a in Cocaine-Induced Plasticity. Science, 327(5962), 213–216. https://doi.org/10.1126/science.1179438
Farrelly, L. A., Thompson, R. E., Zhao, S., Lepack, A. E., Lyu, Y., Bhanu, N. V., Zhang, B., Loh, Y.-H. E., Ramakrishnan, A., Vadodaria, K. C., Heard, K. J., Erikson, G., Nakadai, T., Bastle, R. M., Lukasak, B. J., Zebroski, H., Alenina, N., Bader, M., Berton, O., … Maze, I. (2019). Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3. Nature, 567(7749), 535–539. https://doi.org/10.1038/s41586-019-1024-7
Lepack, A. E., Werner, C. T., Stewart, A. F., Fulton, S. L., Zhong, P., Farrelly, L. A., Smith, A. C. W., Ramakrishnan, A., Lyu, Y., Bastle, R. M., Martin, J. A., Mitra, S., O’Connor, R. M., Wang, Z.-J., Molina, H., Turecki, G., Shen, L., Yan, Z., Calipari, E. S., … Maze, I. (2020). Dopaminylation of histone H3 in ventral tegmental area regulates cocaine seeking. Science, 368(6487), 197–201. https://doi.org/10.1126/science.aaw8806
 
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The Role of Blimp-1 in Immune-Cell Differentiation (Erna Magnúsdóttir)

In this episode of the Epigenetics Podcast, we caught up with Erna Magnúsdóttir from the University of Iceland to talk about her work on the role of Blimp-1 in immune-cell differentiation.
The Magnúsdóttir Lab is interested in how the mammalian genome is interpreted in a context dependent manner, leading to different cellular states, by using mouse primordial germ cells as well as mouse and human B-cells as model systems. More specifically, the team is interested in the Transcription Factor Blimp-1 and its effect on immune cell differentiation. Next to its function in immune cells, Blimp-1 also plays a role in Waldenström’s macroglobulinemia. The lab hopes to reveal the intricacies in disease progression and alteration in cellular states to increasingly aggressive tumor behavior.
 
References
Magnúsdóttir, E., Dietmann, S., Murakami, K. et al. A tripartite transcription factor network regulates primordial germ cell specification in mice. Nat Cell Biol 15, 905–915 (2013). https://doi.org/10.1038/ncb2798
Anderson, K.J., Ósvaldsdóttir, Á.B., Atzinger, B. et al. The BLIMP1—EZH2 nexus in a non-Hodgkin lymphoma. Oncogene 39, 5138–5151 (2020). https://doi.org/10.1038/s41388-020-1347-8
 
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Multimodal Characterization of Cellular Identity | CITE-Seq and Beyond (Peter Smibert, VP of 10X Genomics)

In this episode of the Epigenetics Podcast, we speak with Peter Smibert, Vice President of Biology at 10X Genomics to talk about an exciting new method in Multimodal Characterization of Cellular Identity using Barcoding.
During his time at the New York Genome Center, Peter Smibert was instrumental in the development of a new method called "Cellular Indexing of Transcriptomes and Epitopes by Sequencing" short CITE-Seq. This method enables the characterization of a cell's transcriptome, while at the same time, also allows the characterization of the cell's protein surface markers - at the single cell level. In CITE-Seq, sequencing adapters are coupled to antibodies that recognize surface proteins, which can then be detected by sequencing.
Further advancements of the CITE-Seq method led to the launch of BioLegend’s TOTAL-Seq and the integration of scATAC-Seq into the workflow. With the integration of scATAC-Seq in the CITE-Seq protocol, it is now possible to characterize single-cells along the path of the central dogma of biology, this is why the method called DOGMA-Seq.
 
References
https://cite-seq.com
Baron, M., Yanai, I. New skin for the old RNA-Seq ceremony: the age of single-cell multi-omics. Genome Biol 18, 159 (2017). https://doi.org/10.1186/s13059-017-1300-5
Stoeckius, M., Zheng, S., Houck-Loomis, B. et al. Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol 19, 224 (2018). https://doi.org/10.1186/s13059-018-1603-1
Stoeckius, M., Hafemeister, C., Stephenson, W. et al. Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14, 865–868 (2017). https://doi.org/10.1038/nmeth.4380
Mimitou, E.P., Cheng, A., Montalbano, A. et al. Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells. Nat Methods 16, 409–412 (2019). https://doi.org/10.1038/s41592-019-0392-0
 
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