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

Related Episodes

• Hi-C and Three-Dimensional Genome Sequencing (Erez Lieberman Aiden)

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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

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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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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

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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

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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

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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

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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

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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

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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

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