Epigenetics Podcast

​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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Regulation of Chromatin Organization by Histone Chaperones (Geneviève Almouzni)

In this episode of the Epigenetics Podcast, we caught up with Geneviève Almouzni, Ph.D., Research Director at the CNRS at Institut Curie in Paris, to talk about her work on the regulation of chromatin organization by histone chaperones.
Geneviève Almouzni got her Ph.D. from Université Pierre-et-Marie-Curie in 1988 under the supervision of Marcel Méchali. She then moved to the United States to work as a postdoc in the National Institutes of Health in the laboratory of Professor Alan Wolffe. In 1994, she returned to Paris and became a Junior Group Leader at Institut Curie and became a Group Leader there in 2000. In 2013, she took over the direction of research at the Institut Curie and became the third woman to hold this position, after Marie Curie and Irène Joliot-Curie.
Geneviève Almouzni’s research focuses on the assembly of chromatin and the identification of histone chaperones. Histone chaperones are necessary for the establishment and maintenance of chromatin, as they help to assemble the nucleosomes out of the core histones and DNA. This occurs both when the polymerase transcribes through a nucleosome and after DNA replication and repair.
The Almouzni group has identified and characterized multiple histone chaperones, including CAF-1, HirA, and HJURP. Furthermore, they investigated how post-translational modifications on soluble histones influence the final epigenetic state of the nucleosome and the reassembly of chromatin after DNA replication. In the last couple of years, the group has focused on the unraveling the link between the structure of chromatin at centromeres and cancer.
In this interview, we discuss the focus of the Almouzni lab on histone chaperones, how the lab was able to identify its first one with CAF-1, how histone PTMs on soluble histones influence the deposition on the DNA, and how the chromatin on centromeres is involved in cancer.
 
References
 
Dominique Ray-Gallet, Jean-Pierre Quivy, … Geneviève Almouzni (2002) HIRA Is Critical for a Nucleosome Assembly Pathway Independent of DNA Synthesis (Molecular Cell) DOI: 10.1016/S1097-2765(02)00526-9
Pierre-Henri L. Gaillard, Emmanuelle M.-D. Martini, … Geneviève Almouzni (1996) Chromatin Assembly Coupled to DNA Repair: A New Role for Chromatin Assembly Factor I (Cell) DOI: 10.1016/S0092-8674(00)80164-6
Jean-Pierre Quivy, Danièle Roche, … Geneviève Almouzni (2004) A CAF-1 dependent pool of HP1 during heterochromatin duplication (The EMBO Journal) DOI: 10.1038/sj.emboj.7600362
 
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How the "Fragile Nucleosome" Science Community Came to Life (Christine Cucinotta, Melvin Noe Gonzalez)

In this episode of the Epigenetics Podcast, we caught up with Dr. Christine Cucinotta and Dr. Melvin Noe Gonzalez to talk about how they brought the #fragilenucleosome seminar series and Discord channel to life.
 
Christine Cucinotta and Melvin Noe Gonzales are part of the organizing committee of the independent scientific community "Fragile Nucleosome." This community consists of a Discord channel with more than 1,000 members, a biweekly seminar series, a mentoring program, and a journal club series. The Fragile Nucleosome is organized exclusively by early-career scientists, without external sponsors or under the roof of a single graduate program or university.
 
In this interview, Christine and Melvin share the story on how the Fragile Nucleosome community got started, what has happened so far, and what the future plans are for the #fragilenucleosome.
 
 
References
#fragilenucleosome on Twitter
Fragile Nucleosome Discord Channel
Fragile Nucleosome on generegulation.org
Christine Cucinotta on Twitter
Melvin Noe Gonzalez on Twitter
 
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Epigenetic Influence on Memory Formation and Inheritance (Isabelle Mansuy)

In this episode of the Epigenetics Podcast, we caught up with Professor Isabelle Mansuy, Ph.D., from the University of Zürich and the ETH Zürich, to talk about her work on epigenetic influences on memory formation and inheritance.
 
Dr. Mansuy received her Ph.D. from the Friedrich Miescher Institute, Basel, Switzerland in 1994. After doing a postdoc at the Center for Neurobiology and Behavior at the Howard Hughes Medical Institute at the Columbia University in New York, she moved to Zürich and became Assistant Professor in Neurobiology at the Department of Biology at the Swiss Federal Institute of Technology in 1998. In 2004 Dr. Mansuy became Professor at the Brain Research Institute of the University Zurich, where, in 2007, she became Managing Director. Since 2013 she has been a full Professor in Neuroepigenetics at the University of Zürich and at the ETH in Zürich.
 
Dr. Isabelle Mansuy's work centers around the formation of memories and how those memories are inherited. She started to work on memory formation in the beginning of her research career, where she investigated the influence of calcineurin and Zif268 in this process. In the early 2010s she pivoted and transitioned to work on transgenerational epigenetic inheritance. To investigate this field of research she created an unbiased experiment that allowed her to study the transgenerational influence of early life stress, which she was able to observe for across up to 4 generations through the germline.
 
If you want to learn more about the challenges and obstacles that needed to be overcome to create this novel experimental approach to tackle the questions of and which epigenetic factors might influence transgenerational epigenetic inheritance, don't miss out on this episode.
 
 
References
Karsten Baumgärtel, David Genoux, … Isabelle M. Mansuy (2008) Control of the establishment of aversive memory by calcineurin and Zif268 (Nature Neuroscience) DOI: 10.1038/nn.2113
Tamara B. Franklin, Holger Russig, … Isabelle M. Mansuy (2010) Epigenetic Transmission of the Impact of Early Stress Across Generations (Biological Psychiatry) DOI: 10.1016/j.biopsych.2010.05.036
Johannes Gräff, Bisrat T. Woldemichael, … Isabelle M. Mansuy (2012) Dynamic histone marks in the hippocampus and cortex facilitate memory consolidation (Nature Communications) DOI: 10.1038/ncomms1997
Eloïse A. Kremer, Niharika Gaur, … Isabelle M. Mansuy (2018) Interplay between TETs and microRNAs in the adult brain for memory formation (Scientific Reports) DOI: 10.1038/s41598-018-19806-z
Katharina Gapp, Ali Jawaid, … Isabelle M. Mansuy (2014) Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice (Nature Neuroscience) DOI: 10.1038/nn.3695
Katharina Gapp, Saray Soldado-Magraner, … Isabelle M. Mansuy (2014) Early life stress in fathers improves behavioural flexibility in their offspring (Nature Communications) DOI: 10.1038/ncomms6466
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Influence of Dynamic RNA Methylation on Gene Expression (Chuan He)

In this episode of the Epigenetics Podcast, we caught up with Dr. Chuan He, John T. Wilson Distinguished Service Professor at University of Chicago, to talk about his work on the influence of dynamic RNA methylation on gene expression. RNA methylation is an important biological process, and cellular RNA methylation levels can have profound impacts on normal cellular differentiation and cancer cell proliferation.
Dr. He received his Ph.D. from MIT in 2000 and went on to do his postdoctoral work at Harvard University. He then became Assistant Professor at the University of Chicago in 2002, was promoted to Associate Professor in 2008, and in 2014 he became the John T. Wilson Distinguished Service Professor at the University of Chicago.  From 2012 to 2017 he was Director of the Institute for Biophysical Dynamics at the University of Chicago.
Chuan He's current research focuses on understanding the reversible RNA modification m6A. This modification was discovered in the 1980s, but work from Dr. He's laboratory showing that m6A was indeed a transient epigenetic modification by the discovery of the first m6A demethylase FTO in 2011 rekindled the interest in this modification. In the following years Dr. He and his team identified and characterized additional m6A enzymes, including the m6A eraser ALKBH5, the m6A readers YTH and HNRNP, and the m6A writer complex METTL3/14. 
METTL3/14 is a core complex in this regulatory network, and it requires an accessory factor WTAP, which mediates cellular m6A RNA methylation. The current work in the He lab focuses on how the methylation selectivity of this complex is achieved.
In this interview, we discuss the story of how the He lab discovered the members of the family of proteins that read, write, and erase RNA modifications and how those RNA modifications act in the field of epigenetics.
 
References
 
Guifang Jia, Cai-Guang Yang, … Chuan He (2008) Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO (FEBS letters) DOI: 10.1016/j.febslet.2008.08.019
Guifang Jia, Ye Fu, … Chuan He (2011) N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO (Nature Chemical Biology) DOI: 10.1038/nchembio.687
Guanqun Zheng, John Arne Dahl, … Chuan He (2013) ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility (Molecular Cell) DOI: 10.1016/j.molcel.2012.10.015
Jianzhao Liu, Yanan Yue, … Chuan He (2014) A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation (Nature Chemical Biology) DOI: 10.1038/nchembio.1432
 
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