Epigenetics Podcast

Unraveling Mechanisms of Chromosome Formation (Job Dekker)

In this episode of the Epigenetics Podcast, we caught up with Job Dekker from the University of Massachusetts Medical School to talk about his work on unraveling mechanisms of chromosome formation.
In 2002, during graduate school, Job Dekker was the first author on the paper describing the chromosome conformation capture (3C) method, which revolutionized the field of nuclear architecture. In the 3C protocol, chromatin is crosslinked using formaldehyde and then digested using a restriction enzyme. After ligating the digested blunt ends of crosslinked DNA fragments together they can be analyzed using qPCR. In the next couple of years 3C was further developed and methods like 4C, 5C, and Hi-C were published. This led to the generation of genome-wide contact maps which helped understand the 3-D organization of the nucleus.
Job Dekker’s research group is also part of the 4D Nucleome initiative, which is dedicated to understanding the structure of the human genome. More recent work of the lab includes analyzing interactions between and along sister chromatids with a method called SisterC and expanding their research to organisms like dinoflagellates to learn more about the basic organization principles of the genome.
In this episode, we discuss the story behind the idea of the chromosome conformation capture method, how close Job Dekker was to giving up on it, how the 3C methods evolved, the importance of data visualization, and we touch on parts of his current work on dinoflagellates.
 
References
Job Dekker, Karsten Rippe, … Nancy Kleckner (2002) Capturing Chromosome Conformation (Science) DOI: 10.1126/science.1067799
Josée Dostie, Todd A. Richmond, … Job Dekker (2006) Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements (Genome Research) DOI: 10.1101/gr.5571506
Erez Lieberman-Aiden, Nynke L. van Berkum, … Job Dekker (2009) Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome (Science) DOI: 10.1126/science.1181369
Amartya Sanyal, Bryan R. Lajoie, … Job Dekker (2012) The long-range interaction landscape of gene promoters (Nature) DOI: 10.1038/nature11279
Marlies E. Oomen, Adam K. Hedger, … Job Dekker (2020) Detecting chromatin interactions between and along sister chromatids with SisterC (Nature Methods) DOI: 10.1038/s41592-020-0930-9 • Job Dekker, Andrew S. Belmont, … 4D Nucleome Network (2017) The 4D nucleome project (Nature) DOI: 10.1038/nature23884 • The 4D Nucleome Project
Related Episodes
Hi-C and Three-Dimensional Genome Sequencing (Erez Lieberman Aiden)
Identification of Functional Elements in the Genome (Bing Ren)
Biophysical Modeling of 3-D Genome Organization (Leonid Mirny)
Epigenetics and X-Inactivation (Edith Heard)
Dosage Compensation in Drosophila (Asifa Akhtar)
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Transcription and Polycomb in Inheritance and Disease (Danny Reinberg)

In this episode of the Epigenetics Podcast, we caught up with Dr. Danny Reinberg from the New York University School of Medicine to talk about his work on transcription and polycomb in inheritance and disease.
Dr. Danny Reinberg is a pioneer in the characterization of transcription factors for human RNA polymerase II. In his groundbreaking work in the 1990s, he purified the essential transcription factors and reconstituted the polymerase in vitro on both naked DNA and chromatin.  Dr. Reinberg next started focusing on the polycomb repressive complex 2 (PRC2), which is the only known methyltransferase for lysine 27 on histone H3. He biochemically characterized the PRC2 subunits EZH1 and EZH2. More recently, Dr. Reinberg has been investigating the role of PRC2 in neurons. 
This interview discusses the story behind how Dr. Danny Reinberg started his research career by identifying the essential RNA polymerase transcription factors, how he discovered and characterized the polycomb repressive complex 2 (PRC2), and what his research holds for the future.
 
References
H. Lu, L. Zawel, … D. Reinberg (1992) Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II (Nature) DOI: 10.1038/358641a0
A. Merino, K. R. Madden, … D. Reinberg (1993) DNA topoisomerase I is involved in both repression and activation of transcription (Nature) DOI: 10.1038/365227a0
G. Orphanides, W. H. Wu, … D. Reinberg (1999) The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins (Nature) DOI: 10.1038/22350
Andrei Kuzmichev, Kenichi Nishioka, … Danny Reinberg (2002) Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein (Genes & Development) DOI: 10.1101/gad.1035902
Andrei Kuzmichev, Raphael Margueron, … Danny Reinberg (2005) Composition and histone substrates of polycomb repressive group complexes change during cellular differentiation (Proceedings of the National Academy of Sciences of the United States of America) DOI: 10.1073/pnas.0409875102
Ozgur Oksuz, Varun Narendra, … Danny Reinberg (2018) Capturing the Onset of PRC2-Mediated Repressive Domain Formation (Molecular Cell) DOI: 10.1016/j.molcel.2018.05.023
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The Epigenetics of COVID-19

In this episode of the Epigenetics Podcast, we caught up with Dr. Sandra Atlante and Dr. Carlo Gaetano from the Instituti Clinici Scientifici Maugeri in Pavia, Italy, to talk about the roles epigenetic mechanisms play in COVID-19.
In early 2020 a novel coronavirus, SARS-CoV-2, emerged in Wuhan, China. This coronavirus causes the coronavirus disease 2019 (COVID-19) and rapidly spread all over the globe. In a worldwide effort, scientists and doctors tried to find drugs and looked for vaccines to help contain the spreading of the virus. It seems that an overreaction of the immune system, the so called "cytokine storm," could be one of the major complications of this disease. This reaction is not directly linked to the viral infection but is an overreaction of the body's own immune system. Therefore, small molecules that regulate gene expression via chromatin modifying enzymes might help keep the immune system in check.
In this episode we discuss how Dr. Gaetano and Dr. Atlante set up studies to investigate the epigenetic response to a SARS-CoV-2 infection, which epigenetic factors play a role in disease progression, and what we can expect from mutations of the virus in the future.
 
References
Sandra Atlante, Alessia Mongelli, … Carlo Gaetano (2020) The epigenetic implication in coronavirus infection and therapy (Clinical Epigenetics) DOI: 10.1186/s13148-020-00946-x
 
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Epigenetic Reprogramming During Mammalian Development (Wolf Reik)

In this episode of the Epigenetics Podcast, we caught up with Dr. Wolf Reik, Director at the Babraham Institute in Cambridge, UK, to talk about his work on the role of epigenetic factors in cellular reprogramming.
In the beginning of his research career, Dr. Wolf Reik worked on cellular reprogramming during embryogenesis. Epigenetic marks like DNA methylation or post-translational modifications of histone tails are removed and reprogrammed during embryogenesis, which can limit the amount of epigenetic information that can be passed on to future generations. However, this process is sometimes defective, which can lead to transgenerational epigenetic inheritance.
More recently, the laboratory of Dr. Wolf Reik has done pioneering work in the emerging field of single-cell experimental methods. The Reik lab developed a single-cell reduced representation bisulfite sequencing (scRRBS) approach to investigate DNA methylation at single-cell resolution. They also developed an integrated multi-omics approach called single-cell nucleosome, methylation, and transcription sequencing (scNMT-Seq) to map chromatin accessibility, DNA methylation, and RNA expression at the same time during the onset of gastrulation in mouse embryos.
In this interview, we discuss the story behind how Dr. Wolf Reik almost discovered 5-hmC and how he later moved into developing single-cell methods like scRRBS and single-cell multi-omics approaches.
 
References
W. Reik, A. Collick, … M. A. Surani (1987) Genomic imprinting determines methylation of parental alleles in transgenic mice (Nature) DOI: 10.1038/328248a0
W. Dean, F. Santos, … W. Reik (2001) Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos (Proceedings of the National Academy of Sciences of the United States of America) DOI: 10.1073/pnas.241522698
Miguel Constância, Myriam Hemberger, … Wolf Reik (2002) Placental-specific IGF-II is a major modulator of placental and fetal growth (Nature) DOI: 10.1038/nature00819
Adele Murrell, Sarah Heeson, Wolf Reik (2004) Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops (Nature Genetics) DOI: 10.1038/ng1402
Irene Hernando-Herraez, Brendan Evano, … Wolf Reik (2019) Ageing affects DNA methylation drift and transcriptional cell-to-cell variability in mouse muscle stem cells (Nature Communications) DOI: 10.1038/s41467-019-12293-4
Tobias Messmer, Ferdinand von Meyenn, … Wolf Reik (2019) Transcriptional Heterogeneity in Naive and Primed Human Pluripotent Stem Cells at Single-Cell Resolution (Cell Reports) DOI: 10.1016/j.celrep.2018.12.099
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​In vivo Nucleosome Structure and Dynamics (Srinivas Ramachandran)

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
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Pioneer Transcription Factors and Their Influence on Chromatin Structure (Ken Zaret)

In this episode of the Epigenetics Podcast, we caught up with Dr. Ken Zaret, Professor in the Department of Cell and Developmental Biology at the Perelman School of Medicine, University of Pennsylvania, to talk about his work on pioneer transcription factors and their influence on chromatin structure.
Embryonic development is a complex process that needs to be tightly regulated. Multiple regulatory factors contribute to proper development, including a family of specialized regulatory proteins called "pioneer factors." Our guest Dr. Ken Zaret found that these pioneer factors are among the first proteins to bind to chromatin during development and that they can prime important regulatory genes for activation at a later developmental stage. Furthermore, he and his team showed that there might be a "pre-pattern" that exists in cells that determines their developmental fate.
Pioneer factors are not only important in embryonic development, they can also help restart transcription after mitosis. Dr. Zaret and his colleagues demonstrated that FoxA stays bound to chromosomes during mitosis, leading to a rapid reactivation of essential genes at the exit of mitosis.
In this interview, we discuss the story behind how Dr. Zaret discovered pioneer transcription factors like FoxA, how these factors are influenced by the chromatin environment, and how they function.
 
References
R. Gualdi, P. Bossard, … K. S. Zaret (1996) Hepatic specification of the gut endoderm in vitro: cell signaling and transcriptional control (Genes & Development) DOI: 10.1101/gad.10.13.1670
L. A. Cirillo, C. E. McPherson, … K. S. Zaret (1998) Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome (The EMBO journal) DOI: 10.1093/emboj/17.1.244
Lisa Ann Cirillo, Frank Robert Lin, … Kenneth S. Zaret (2002) Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4 (Molecular Cell) DOI: 10.1016/s1097-2765(02)00459-8
Kenneth S. Zaret (2020) Pioneer Transcription Factors Initiating Gene Network Changes (Annual Review of Genetics) DOI: 10.1146/annurev-genet-030220-015007
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The Role of Small RNAs in Transgenerational Inheritance in C. elegans (Oded Rechavi)

In this episode of the Epigenetics Podcast, we caught up with Dr. Oded Rechavi, Professor at the University of Tel Aviv, to talk about his work on the role of small RNAs in transgenerational inheritance in C. elegans.
The most prominent example of transgenerational inheritance is the Dutch famine of 1944 during World War II. Effects of this famine could be observed in the grandchildren of people that lived through this hunger winter, but the molecular mechanisms involved remain largely unknown. The guest of this podcast episode, Dr. Rechavi, has taken on the challenge to unravel parts of this puzzle by studying transgenerational epigenetics in C. elegans.
It was already known that small RNA molecules could play a role in passing on information from one generation to the next, but it was not clear what exactly was being inherited. Was it RNAs? Or chromatin modifications? Or something else?
Dr. Rechavi made several important discoveries in his journey to answer these questions. He started out by showing that RNAi provides an antiviral protection mechanism in C. elegans that can be passed on over multiple generations. He then went on to show that starvation in one generation leads to changes in the lifespan of future generations, and investigate how long this memory could last. Simple dilution of the parental RNA in future generations could not be the answer because the inherited phenotypes lasted much longer than would be possible if this were the case. This led Dr. Rechavi to the discovery that small RNAs were amplified in each generation, and the effect of a stimulus could affect multiple generations. More recently, Dr. Rechavi and his team studied the interplay of neurons and the germ line and how information can be passed on from the brain to the germ line.
In this interview, we cover how Dr. Rechavi chose C. elegans as a model organism, discuss his first major discoveries in the field of transgenerational effects of starvation, and what role epigenetic factors play in this process.
 
References
 
Oded Rechavi, Gregory Minevich, Oliver Hobert (2011) Transgenerational Inheritance of an Acquired Small RNA-Based Antiviral Response in C. elegans (Cell) DOI: 10.1016/j.cell.2011.10.042
Oded Rechavi, Leah Houri-Ze’evi, … Oliver Hobert (2014) Starvation-induced transgenerational inheritance of small RNAs in C. elegans (Cell) DOI: 10.1016/j.cell.2014.06.020
Leah Houri-Ze’evi, Yael Korem, … Oded Rechavi (2016) A Tunable Mechanism Determines the Duration of the Transgenerational Small RNA Inheritance in C. elegans (Cell) DOI: 10.1016/j.cell.2016.02.057
Itamar Lev, Uri Seroussi, … Oded Rechavi (2017) MET-2-Dependent H3K9 Methylation Suppresses Transgenerational Small RNA Inheritance (Current biology: CB) DOI: 10.1016/j.cub.2017.03.008
Leah Houri-Zeevi, Yael Korem Kohanim, … Oded Rechavi (2020) Three Rules Explain Transgenerational Small RNA Inheritance in C. elegans (Cell) DOI: 10.1016/j.cell.2020.07.022
 
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Development of Site-Specific ChIP Technologies (Hodaka Fujii)

In this episode of the Epigenetics Podcast, we caught up with Dr. Hodaka Fujii, Professor of Biochemistry and Genome Biology at Hirosaki University Graduate School of Medicine and School of Medicine, to talk about his work on the development of locus-specific ChIP technologies.
The goal of conventional chromatin immunoprecipitation (ChIP) assays is to find genomic locations of transcription factor binding or genome-wide profiles of histone tail modifications.  In contrast to that, the guest of this episode, Dr. Fujii, has developed methods such as insertional chromatin immunoprecipitation (iChIP) and engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) to identify the factors that are binding to specific sites on the genome.
In iChIP, LexA binding sites are inserted into the genomic region of interest. In parallel, the DNA-binding domain of LexA, fused with FLAG epitope tags and a nuclear localization signal, is expressed in the same cells. After crosslinking and chromatin preparation, the resulting chromatin is immunoprecipitated with an antibody against the tag. This allows proteins or RNA interacting with the region of interest to be analyzed with the appropriate downstream application. The enChIP takes a similar approach, but does not require insertion of the LexA binding sites. Instead, a FLAG-tagged dCas9 protein together with the respective guide RNA are used to target the region of the genome of interest. After the IP and the purification DNA, RNA, or proteins can be analyzed accordingly. The lack of the requirement of to insert the LexA binding sites into the genome makes enChIP much more straightforward than iChIP.
In this interview, we discuss the story behind how Dr. Fujii got into the field of epigenetics, how he developed iChIP, and how the method was improved over the years. Furthermore, we discuss the development of enChIP and how this can be used as an alternate method to Hi-C.
 
References
 
Akemi Hoshino, Satoko Matsumura, … Hodaka Fujii (2004) Inducible Translocation Trap (Molecular Cell) DOI: 10.1016/j.molcel.2004.06.017
Akemi Hoshino, Hodaka Fujii (2009) Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions (Journal of Bioscience and Bioengineering) DOI: 10.1016/j.jbiosc.2009.05.005
Toshitsugu Fujita, Hodaka Fujii (2013) Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR (Biochemical and Biophysical Research Communications) DOI: 10.1016/j.bbrc.2013.08.013
Toshitsugu Fujita, Miyuki Yuno, … Hodaka Fujii (2015) Identification of Non-Coding RNAs Associated with Telomeres Using a Combination of enChIP and RNA Sequencing (PLOS ONE) DOI: 10.1371/journal.pone.0123387
Toshitsugu Fujita, Miyuki Yuno, Hodaka Fujii (2016) Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins (Genes to Cells) DOI: 10.1111/gtc.12341
Toshitsugu Fujita, Miyuki Yuno, … Hodaka Fujii (2017) Identification of physical interactions between genomic regions by enChIP-Seq (Genes to Cells) DOI: 10.1111/gtc.12492
Toshitsugu Fujita, Fusako Kitaura, … Hodaka Fujii (2017) Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line (DNA Research) DOI: 10.1093/dnares/dsx023
 
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