4DN Investigator Spotlight: Christine Disteche
Tracking DNA Organization During Embryo Development and Disease Progression
“I’ve always been fascinated by chromosomes,” says 4D Nucleome researcher Dr. Christine Disteche. And with good reason: chromosomes contain all the genetic information necessary to control cell function and development. As an embryo grows, complex processes of molecular signaling direct what kind of cells and organs will develop by controlling which genes are turned on and off. The way that chromosomes, and the DNA they contain, are organized inside the cellular nucleus may have a role in determining cell development as part of these processes. Dr. Disteche, as part of the University of Washington 4-Dimensional Genomic Organization of Mammalian Embryogenesis Center, is studying the mechanisms behind embryo development and what role DNA organization plays in determining cell fate. The ambitious project combines many different types of analyses that follow cells in mouse embryos as they grow and mature. The researchers are studying how different cell types develop by measuring the activity of genetic pathways within individual cells and combining those results with where those cells are in the embryo and how their DNA is organized. This project is one of the first to study these processes in living embryos, with previous work focusing only on cultures of isolated cells.
Dr. Disteche’s research has benefited from the new tools and resources that are being created by the 4D Nucleome program. She reflects that, when the program started, “it was like all of a sudden I had all these things that could be done to really look at the nucleus and look at the chromosomes and see their structure at the molecular level.” These new methods to track cell type-specific development and monitor DNA organization will allow her team to understand the underlying processes of how genetic mutations lead to different DNA arrangement in the nucleus that can cause diseases. She is interested in sex-linked genes and their roles in sex-based differences in diseases, particularly those found on the X chromosome, as well as imprinting disorders caused by incorrect activation or silencing of parental genes. Finding new methods to modify how DNA is organized in the nucleus could help find potential treatment strategies for these diseases.
Dr. Disteche has always been driven by her curiosity. Her journey into research was inspired both by an innate desire to understand the world around her as well as both of her parents, who were also in academia. Her mother in particular was a role model, as she was a chemist at a time when the field was heavily male-dominated. Dr. Disteche reflects that “she gave me a lot of confidence that I could do everything just like the boys would do it.” Now a mentor to her own trainees, Dr. Disteche stresses the importance of being excited by the questions that they are pursuing, and always encourages her students to take on projects of interest even if the path is challenging.
Learn more about the research of Dr. Disteche and her collaborators here.
4DN Investigator Spotlight: Katherine Pollard
Modeling Nuclear DNA to Understand our Hearts, Brains, and Beyond
The microscopic DNA inside our cells, packaged into a structure called chromatin, is impossible to see with the naked eye. However, changes in chromatin arrangement in different cells might affect entire organs, though the processes underlying these effects remain largely unknown. For example, the most common type of birth defects are congenital heart defects, which are often linked to genetic mutations and can require extensive treatment and surgeries for affected children. 4D Nucleome (4DN) researcher Dr. Katherine Pollard, along with a team of 4DN researchers at the Gladstone Institutes, aims to uncover how chromatin organization changes as heart cells develop. Her research involves studying stem cells, which could mature into heart cells, and measuring how different regions of chromatin interact with each other during this maturation process. These data are used to build deep learning models that can predict how chromatin will fold within the cellular nucleus and what the effect of genetic variations may be, shedding light on the cellular mechanisms that link chromatin organization to heart cell function. By predicting changes in how a mutation will affect chromatin organization, this model could help provide diagnoses of the underlying issues behind a heart defect, and open new possibilities for targeted treatments.
These models will not only be useful to predict effects of mutations on heart cells, says Dr. Pollard; her lab has already begun studying models of childhood brain cancer and autism spectrum disorders to understand how chromatin organization affects those diseases. As part of the 4DN predictive modeling working group, Dr. Pollard’s work has been enhanced by the ability to get feedback from other researchers doing similar work across many institutions. 4DN has not only aided her work by building connections to other scientists but has also generated new methods to sequence the genome and map how chromatin is organized within the cellular nucleus. As building predictive models of the genome requires large amounts of data, progress in measuring and mapping chromatin are critical to advancing her work.
Dr. Pollard advises that, to find success in research as well as a scientific career, it is important to follow your passions, and “sometimes that means doing things that aren’t the well-trodden path.” Throughout her career, she has followed her scientific interests though the fields of anthropology, math, computer science, and biology, building strong networks of other women researchers in fields that used to have much less representation. When she was beginning her research career, the field of genome-wide bioinformatics didn’t exist: the human genome was sequenced for the first time during her graduate studies. “Science moves really quickly, technology moves really quickly,” says Dr. Pollard, “and what you’ll be doing ten to twenty years from now is really hard to predict.”
Learn more about Dr. Pollard’s research here.
Photo: Michael Short/Gladstone Institutes
4DN Investigator Spotlight: Huaiying Zhang
"Just go for it!": Understanding Cancers Without Common Genetic Mutations
Cancer cells are characterized by their unlimited growth, while healthy cells only divide a limited number of times. This limit blocks excess cell replication and prevents DNA from building up harmful mutations. Cells control how many times they can divide using telomeres, long sequences of DNA that cap each end of chromosomes in the nucleus, which shorten each time a cell divides until the telomere is gone. When a cell senses the lack of telomeres, natural genetic processes prevent further DNA replication and cell division. Most cancer cells get around this mechanism by activating the protein telomerase, which extends telomeres and allows cells to continue dividing and build up more cancerous mutations. However, around 10% of cancer cells do not activate telomerase, instead rebuilding telomeres through other pathways that involve abnormal organization of DNA in the cell nucleus. There is no common genetic mutation among this type of cancer cells, making it challenging to develop targeted treatments.
Dr. Huaiying Zhang, Assistant Professor in Department of Biological Sciences at Carnegie Mellon University, studies these alternative pathways and their underlying mechanisms. Her work has already shown that in these cancer cells, telomeres cluster together into droplets that include template DNA for telomeres and the cellular components needed to repair DNA. As part of the 4D Nucleome program, she aims to discover how the biophysical process that drives this clustering, called phase separation, can be targeted as a potential cancer treatment. This ambitious project wouldn’t be possible without the collaborative efforts of researchers across many scientific disciplines, says Dr. Zhang, who values the interdisciplinary nature of the research in her lab as well as with researchers across the 4D Nucleome program. A chemical engineer by training and now studying cancer biology, she is excited to work with other biologists and genomic researchers to bring together many kinds of expertise to tackle these challenging questions.
How does a chemical engineer end up researching the mechanisms of genetic abnormalities in cancer? “I’m just a curious person,” says Dr. Zhang. By following that curiosity to see how one research question leads into the next, studying the chemical properties of cell membranes transitioned to her work into other aspects of cell biology. Whether it was changing fields from engineering to biology, starting an academic career while balancing a family, or leading a project that will tackle challenging questions about cancer biology, Dr. Zhang spoke to the importance of not overthinking these scientific journeys, believing that it is important to “just go for it!”
Learn more about Dr. Zhang’s research here.
4D Nucleome: Successes in Advancing Understanding of Nuclear Organization and Health
Inside the nucleus of every cell in the human body, over six feet of DNA – the genome – folds together into a dynamic structure called chromatin that packs into a space thirty times smaller than a grain of salt. Understanding how chromatin organization is controlled and how it affects gene activity is critical to understanding healthy cell function and may reveal new ways to treat disease. The Common Fund’s 4D Nucleome (4DN) program was launched in 2015 to investigate how chromatin organization occurs in different types of cells, how it changes over time, and how it relates to health and disease.
In the first phase of the 4DN program, researchers successfully developed many new technologies and refined existing approaches to measure and model nuclear chromatin organization. The field of genomic research is being propelled forward by tools such as cleavage under targets and tagmentation (CUT&Tag), a method to measure how and where proteins bind to DNA. CUT&Tag is faster, cheaper, and higher resolution than existing methods, and has been cited in over 400 studies. 4DN also created the 4DN Data Portal as a resource to make data on nuclear chromatin organization available to the entire research community, which hosts data from over 4,600 experiments generated by researchers from both inside and outside the 4DN consortium. These data are in high demand, with over 250 dataset downloads each month, and the 4DN Data Portal provides dozens of software packages and pipelines to expand the types of analyses possible with genomic data.
As 4DN research builds off the discoveries of Phase 1 and digs deeper into nuclear chromatin organization in mammalian tissues, 4DN researchers will continue to find new insights into the relationship between chromatin and human health. Studies using these tools are already demonstrating links between chromatin structures and disease, such as new findings from 4DN researchers on how mutated genes that can cause cancer are normally tucked away in the nucleus, making them inactive, but are exposed and activated by disruptions in chromatin structures in cancer cells. Through its contributions to new technologies, data resources, and genomic studies, the 4DN program is leaving a lasting impact on the field of nuclear chromatin organization and will expand that impact in Phase 2.
1. Elucidating the Structure and Function of the Nucleus - the NIH Common Fund 4D Nucleome Program. Roy AL, Conroy RS, Taylor VG, Mietz J, Fingerman IM, Pazin MJ, Smith P, Hutter CM, Singer DS, Wilder EL. Molecular Cell, 2023. doi: 10.1016/j.molcel.2022.12.025 Epub 2023 January 13.
Open Access Link (Active through March 4th, 2023)
2. CUT&Tag for Efficient Epigenomic Profiling of Small Samples and Single Cells. Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG, Ahmad K, Henikoff S. Nat Commun, 2019 Apr 29;10(1):1930. doi: 10.1038/s41467-019-09982-5.
3. Activation of Proto-Oncogenes by Disruption of Chromosome Neighborhoods. Hnisz D, Weintraub AS, Day DS, Valton AL, Bak RO, Li CH, Goldmann J, Lajoie BR, Fan ZP, Sigova AA, Reddy J, Borges-Rivera D, Lee TI, Jaenisch R, Porteus MH, Dekker J, Young RA. Science, 2016 Mar 25;351(6280):1454-1458. doi: 10.1126/science.aad9024. Epub 2016 Mar 3.
Differences in DNA Organization Among Cell Types Give Insight into Neurological Disease
The goal of the NIH Common Fund’s 4D Nucleome (4DN) program is to study the three-dimensional organization of the cell’s nucleus over time. Inside the nucleus, several feet of DNA are packed with protein into structures called chromatin. Chromatin can change shape at specific locations to affect how the genes in those locations are regulated. 4DN researchers aim to advance the scientific community’s understanding of how different types of chromatin organization influence gene regulation and cell function. It is currently difficult to study how chromatin organization differs among cell types, so cell-type specific changes to chromatin structure in disease often go unnoticed.
To address this issue, a 4DN research team led by Dr. Ana Pombo developed immunoGAM (immuno Genome Architecture Mapping). This is an extension of their previously established GAM technique, which uses extremely thin, frozen sections of the nucleus precisely cut at various angles to determine which chromatin locations are near each other in space. ImmunoGAM expands the GAM technique to analyze specific cell types within a tissue, without the need to isolate these cells from their native tissue environment and using samples containing a small overall number of cells. Using immunoGAM, the research team found that specialized chromatin structures exist within mouse brain cells, and that these structures reflect specialized functions of each cell type.
One interesting discovery was related to DNA segments that encode very long genes. These genes are often involved in specialized cell functions. Although typically DNA is tightly condensed inside the nucleus, very long genes were observed to undergo ‘domain melting,’ an event where the DNA became less condensed, only in specific cell types. These ‘melted’ genes were also found to be highly active in a cell-type specific fashion. For example, Nrxn3 is a very long gene, and in dopaminergic neurons, a special type of brain cell, it was found to ‘melt’ and be active. However, in other brain cell types, the Nrxn3 gene was condensed and not active. Knowing how the gene is regulated in different cell types is particularly important, because the gene is dysregulated in neurological conditions like autism spectrum disorder and schizophrenia. These findings show the importance of examining cell-type specific chromatin organization to better understand gene regulation, cell function, and neurological disease.
Cell-type specialization is encoded by specific chromatin topologies. Winick-Ng W, Kukalev A, Harabula I, Zea-Redondo L, Szabó D, Meijer M, Serebreni L, Zhang Y, Bianco S, Chiariello AM, Irastorza-Azcarate I, Thieme CJ, Sparks TM, Carvalho S, Fiorillo L, Musella F, Irani E, Triglia ET, Kolodziejczyk AA, Abentung A, Apostolova G, Paul EJ, Franke V, Kempfer R, Akalin A, Teichmann SA, Dechant G, Ungless MA, Nicodemi M, Welch L, Castelo-Branco G, Pombo A. Nature, 2021 Nov;599(7886):684-691. doi: 10.1038/s41586-021-04081-2. Epub 2021 Nov 17.
Watch a 4DN Scientific Webinar Series presentation by author Dr. Warren Winick-Ng about this work on YouTube.
4D Nucleome researchers ‘sniff out’ how SARS-CoV-2 causes loss of smell
When the COVID-19 pandemic began, one of its telltale symptoms was a loss of sense of smell. This loss was not caused by the usual stuffy nose, and for some sufferers it lingered even after the virus was gone. Furthermore, olfactory sensory neurons, the specialized cells responsible for our sense of smell, are rarely infected by the SARS-CoV-2 virus that causes COVID-19. Researchers from the Common Fund’s 4D Nucleome (4DN) program have shed light on this mystery by looking at our DNA. The 4DN program strives to understand the three-dimensional organization of the cell’s nucleus in space and time. In the nucleus, the cell’s DNA, which encodes its genetic instructions, is packaged along with proteins into compact structures called chromatin. Changes in how chromatin is packed into the nucleus can alter gene regulation and cell function.
A team of researchers led by 4D Nucleome grantee Dr. Stavros Lomvardas discovered that nuclear architecture was a missing link between SARS-CoV-2 infection and loss of smell. They observed that genes located in olfactory sensory neurons were downregulated after the SARS-CoV-2 virus infected other nearby cells, meaning the proteins encoded by those genes were produced less often. While some of these genes returned to their usual levels of activity by the tenth day of infection, genes responsible for encoding key proteins in the process of smelling were still downregulated. To explain the persistent gene downregulation, the researchers separated out the nuclei from human olfactory sensory neurons to examine the chromatin architecture. The architecture of key genes related to our sense of smell was disrupted in people infected by SARS-CoV-2, compared to that of uninfected people.
These findings help explain why COVID-19 sufferers may report a decreased sense of smell, even though olfactory sensory neurons are not largely targeted by the virus. It also explains why a loss of smell can continue after viral infection has passed. SARS-CoV-2 can alter the nuclear organization and function of cells it does not infect, resulting in decreased protein production and possibly acting as a “nuclear memory” in cases where loss of smell persists. More studies are needed to identify the molecule (or molecules) that signal for the nuclear reorganization in olfactory sensory neurons, and to explore how the changeable structure of odor perception genes may result in its use as an early indicator of other human conditions.
Non-cell autonomous disruption of nuclear architecture as a potential cause of COVID-19 induced anosmia. Zazhytska M, Kodra A, Hoagland DA, Frere J, Fullard JF, Shayya H, McArthur NG, Moeller R, Uhl S, Omer AD, Gottesman ME, Firestein S, Gong Q, Canoll PD, Goldman JE, Roussos P, tenOever BR, Overdevest JB, Lomvardas S. Cell, 2022, doi:10.1016/j.cell.2022.01.024. Epub 2022 Feb 1.
New and improved SPRITE: Now with RNA!
For the Common Fund’s 4D Nucleome (4DN) program, understanding the three-dimensional organization of the cell’s nucleus in space and time isn’t limited to studies of our genetic material, DNA. Other biomolecules, such as RNA and protein, play important roles in nuclear structure and function, including gene regulation. While previous studies have found distinct structural compartments within the nucleus, the full spectrum of these structures and their effect on nuclear function are still unknown. For researchers studying RNA, questions remain regarding whether RNA molecules contribute to these structural compartments, where in the nucleus they contribute, and by what mechanisms. Previous studies suggest that a particular type of RNA that does not encode directions for making proteins, called non-coding RNA (ncRNA), plays an important role in gene regulation and nuclear structure. However, a lack of methods to measure interactions between RNA and DNA in three-dimensional space has made this difficult to demonstrate.
A 4DN research team, led by Dr. Mitchell Guttman, helped scientists study RNA’s role in nuclear organization through a new method, RD-SPRITE (RNA & DNA Split-Pool Recognition of Interactions by Tag Extension). RD-SPRITE generates three-dimensional maps of DNA like its predecessor, SPRITE, but improves the sensitivity for mapping the location of thousands of nuclear RNAs. For the first time, the researchers showed that ncRNAs in the nuclei of mouse cells can act as “seeds” that drive localization of other ncRNAs and protein molecules to specific locations within the nucleus. Through this “seeding,” ncRNAs can contribute to the formation of nuclear compartments. This discovery uncovers a new role for RNA in nuclear organization and functions such as gene regulation. While this study focused on using RD-SPRITE to understand the role of ncRNAs, the method has future applications in understanding the roles of other types of RNA in nuclear organization and function.
RNA promotes the formation of spatial compartments in the nucleus. Quinodoz SA, Jachowicz JW, Bhat P, Ollikainen N, Banerjee AK, Goronzy IN, Blanco MR, Chovanec P, Chow A, Markaki Y, Thai J, Plath K, Guttman M. Cell, 2021 Nov 11;184(23):5775-5790.e30. doi 10.1016/j.cell.2021.10.014. Epub 2021 Nov 4.
Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus. Quinodoz SA, Ollikainen N, Tabak B, Palla A, Schmidt JM, Detmar E, Lai MM, Shishkin AA, Bhat P, Takei Y, Trinh V, Aznauryan E, Russell P, Cheng C, Jovanovic M, Chow A, Cai L, McDonel P, Garber M, Guttman M. Cell, 2018 July 26;174(3):744-757.e24. doi: 10.1016/j.cell.2018.05.024. Epub 2018 Jun 7.
4D Nucleome Program Makes Awards for Stage 2
The NIH Common Fund's 4D Nucleome (4DN) program has made awards for its second stage. A total of thirty awards have been made for six initiatives. The 4DN program will continue to support research on the three-dimensional organization of the nucleus and how it shifts over time (the fourth dimension). Stage 1 of 4DN successfully developed important technologies and datasets that will be leveraged in Stage 2. The 4DN Organization and Function in Human Health and Disease initiative supports ten projects to monitor and/or manipulate the 4D nucleome in the context of human health and disease. Additionally, six projects from early stage investigators are supported by the New Investigator Projects on 4DN Organization and Function in Human Health and Disease. The 4DN program will focus on further developing single cell analysis technology to address the fourth dimension of time. This will be carried out through the eight projects of the Real-Time Chromatin Dynamics and Function initiative. As part of the 4DN Centers for Data Integration, Modeling and Visualization, four research centers will generate comprehensive maps of genome structure, dynamics and function. The 4DN Organizational Hub (https://www.4dnucleome.org/) will continue to administer and coordinate the consortium, and the 4DN Data Coordination & Integration Center will continue to be responsible for data collection, storage, curation, and dissemination (https://data.4dnucleome.org/).
Read more about the awards here!
A “Micro” Technology Yields Big Details
The nucleus is the control center of the cell, packed with 6 linear feet of DNA in a space narrower than the width of a human hair. Since it began, research supported by the 4D Nucleome (4DN) program has improved our understanding of how DNA is packaged and organized in the crowded space of the nucleus. Advancements by the program have prompted scientists to develop new methods to get an even clearer picture of how the DNA is organized. These methods are beginning to give us a good grasp on how large areas of DNA are packaged into different compartments, where portions of DNA that would be far apart in a piece of DNA stretched out long, are actually located close to each other in the nucleus, similar to if you scrunched up a long piece of string and placed it in a ball. Understanding how DNA is organized in three dimensions has informed scientists how that organization can alter which genes are turned on or off, an important factor in many diseases such as cancer. We can now see how large portions of DNA are situated, but can more information be gathered by looking at how smaller regions of DNA in the nucleus are organized?
A team of researchers led by Drs. Xavier Darzacq and Job Dekker, whose research is supported by the 4DN program, developed a new method, called Micro-C, to provide a better image of how smaller regions of DNA are organized in three dimensional space. Looking at the DNA in a mouse model system, the team demonstrated that smaller-than-previously-thought regions of DNA can control gene activity and that the closeness of these small regions is important in recruiting proteins to control how genes are turned on or off. A companion study mirrored these studies demonstrating that Micro-C is suitable for human cells as well. The development of this new method will help strengthen our understanding of how genes are spatially controlled in the crowded nucleus and may provide opportunities for developing future treatments as we learn more about gene activity regulation in healthy and diseased cells.
Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding. Tsung-Han S Hsieh, Claudia Cattoglio, Elena Slobodyanyuk, Anders S Hansen, Oliver J Rando, Robert Tjian, Xavier Darzacq. Molecular Cell, 2020 May 7. 78, 539-553
Ultrastructural Details of Mammalian Chromosome Architecture. Nils Krietenstein, Sameer Abraham, Sergey V Venev, Nezar Abdennur, Johan Gibcus, Tsung-Han S Hsieh, Krishna Mohan Parsi, Liyan Yang, René Maehr, Leonid A Mirny, Job Dekker, Oliver J Rando. Molecular Cell, 2020 May 7. 78, 554-565
Are There Cellular Checkpoints on the Road to Cancer?
One of the key events during cancer metastasis is a process called the epithelial-to-mesenchymal transition (EMT). EMT occurs when genetic changes allow tumor cells to break away and move into different body regions, potentially leading to new tumors in the body. Previous experiments identified specific “stages” of EMT based on the activity of a small number of genes. However, several new studies indicate these stages may not be as distinct as previously thought. Understanding how EMT occurs is a fundamental goal of cancer biology, as it may lead to new cancer treatment options. The Common Fund 4D Nucleome (4DN) program is primed to help by providing scientists with the tools and resources to monitor the changes in gene activity from groups of cells in tumors as well as small changes in individual cells.
4DN program researchers used advanced techniques to look at gene activity over time in single cells. Instead of finding distinct stages linked to EMT, they showed that EMT is actually a continuum of changes within the cell, with multiple genes getting turned on or off at different times. Many of these genes control how our cells make specific proteins that help hold cells in place. By turning these genes off, it may allow tumor cells to break free and move to new regions within the body. The findings also showed why previous experiments indicated EMT occurred in discrete stages. The researchers discovered “checkpoints” within the continuum where different factors regulate how a cell proceeds through the EMT. If any regulatory factors were disrupted, the progress through EMT could stall, making it look as though there were discrete stages to EMT. It was only by looking at single cells over time that the 4DN researchers could explore the continuum of changes and the factors regulating them without being confused by combined results from large groups of cells. These findings begin to shed light on the genetic changes that govern not only EMT in cancer, but diverse biological processes in development and disease. By learning more about the driving forces behind EMT, researchers can start to explore new cancer treatments that prevent or slow metastasis.
A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition. McFaline-Figueroa JL, Hikk AJ, Qui X, Jackson D, Shendure J, Trapnell C. Nature Genetics, 51, 2019, 1389-1398.
4DN Investigator Spotlight: Ana Pombo
Building tools to map our DNA
Dr. Ana Pombo is a Professor at the Berlin Institute for Medical Systems Biology at the Max Delbruck Center for Molecular Medicine. Her lab developed a pioneering technique called Genome Architecture Mapping (GAM). This technology combines an imaging-based approach with a high-throughput method called genomics to map which regions of the genome are physically close to one another in the cell nucleus. With GAM, users can simultaneously view the nuclei of cells and capture position information in three-dimensions for different regions of DNA inside the nuclei that come together for proper gene regulation. Dr. Pombo joined 4DN to improve GAM and has so far succeeded in streamlining the protocol to make it cheaper, faster, and easier to use. For example, GAM requires very few cells and can be used to study many different and difficult-to-study cell types, such as neurons, an improvement over existing technology. Her lab is now collecting large datasets that will hopefully reveal the full power of GAM. One dataset includes 3,000 nuclear images that will be analyzed and compared to other technologies to gain new biological insights. In a parallel project, Dr. Pombo’s lab will also apply GAM to look at rare cell types in complex tissues such as the brain.
Dr. Pombo has worked on 3D genome organization for her entire research career. Although she originally considered a career in conventional architecture, her curiosity about how things work with respect to how they are structured led to a career in genomic architecture. Her passion is studying structures like the nucleus, where many variables must work together for the complex genome to function across different cell types. While much progress has been made in the field since Dr. Pombo first started, she explains that observations made during the time of her graduate and postdoctoral training are now back in the spotlight thanks to technological advances that are enabling deeper investigation of basic questions. Dr. Pombo has focused on developing these cutting-edge techniques. For example, she developed cryoFISH, a precursor to GAM, to see where specific genes were located in the nucleus. This technology could only analyze a few genes at a time, so she developed GAM to study all genomic locations simultaneously and gain a holistic understanding of how genomic systems work. Dr. Pombo credits the 4DN program as an outstanding opportunity to engage with peers, including internationally, over new technologies and research findings in the field of nuclear architecture and function.
4DN Investigator Spotlight: Bing Ren
Expanding our research toolbox
Dr. Bing Ren is a professor of cellular and molecular medicine at the University of California in San Diego (UCSD). As a member of the NIH Common Fund’s 4D Nucleome (4DN) program, Dr. Ren’s research team is developing new experimental techniques and computational methods to help us understand how all of our DNA – our genome – is organized in 3-dimensional space inside the nucleus of each of our cells. His team is also exploring how that organization changes over time, the fourth dimension. Dr. Ren hopes to uncover what changes in genome architecture mean for our health. This is no easy task. The research team is learning how to make purposeful changes to genome architecture in the nuclei of a variety of cell types and laboratory model systems. Observing how these changes affect cell function will help the 4DN program relate specific features of genome architecture to normal development and disease.
Dr. Ren’s interest in the field of genomic architecture and gene regulation started when he was a graduate student, and that interest never left him. He began working on how cells control which genes are turned on or off in different insects, such as fruit flies, and other cellular models and has applied concepts from that research to new research models for the 4DN program. In our interview, Dr. Ren spoke of how excited he is to be part of the 4DN program. “It is a great opportunity to interact with other researchers working in the same field, but now with a more organized network setting.” The program was designed so that its participating research groups could apply different tools to the same set of biological materials to provide a broader and more cohesive understanding of the genome features identified by those tools. This benchmarking process will allow scientists across the world to obtain a more holistic picture of the importance of genomic architecture and its role in human health and disease.
4DN Investigator Spotlight: Bradley Cairns
Dr. Bradley Cairns is the Chair of the Department of Oncological Sciences at the Huntsman Cancer Institute at the University of Utah. Dr. Cairns brings a unique view to the Common Fund 4D Nucleome (4DN) program as his research aims to understand the relationship between chromatin architecture and biological development and how changes that occur early in development may impact diseases that start much later in life. In order to study the impact of nuclear organization on development, Dr. Cairns uses the zebrafish as a model system, as gene transcription is highly regulated during embryo development in zebrafish compared to other model organisms. For example, Dr. Cairns can use the biological fact that in zebrafish all the cells in the developing organism are identical up until gene transcription starts when 1000 cells are developed. Dr. Cairns can then move any of these identical cells to different parts within the developing zebrafish and they will grow into cells specific for their new location, not where they came from. He is studying how these cells have the developmental potential to become any cell type and how can the nuclear organization prime cells for differences based on the environment they are in. These types of studies may serve as a road map for the potential mapping of genes in specific chromatin structures and support the idea that if we can monitor a change in genomic transcription or architecture during development, it may lead to an adverse biological outcome, such as a disease, later in life.
Dr. Cairns is a strong believer that “science is more productive and more fun when you can do it with more people”, which is why he wanted to become part of the 4DN program. He was excited to become part of a scientific community with similar interests, but is glad he can bring his unique perspective by focusing primarily on organisms as they develop instead of focusing on single cells. He feels that one of the biggest values of the 4DN program is providing access to new protocols and technologies through the joint analysis project to help with the benchmarking of scientific studies.
Learn more about Dr. Cairns 4DN project.
Pumping the brakes on heart disease: How DNA dynamics may influence early and late stage heart health
Nearly 1% of all live births are impacted by congenital heart disease (CHD) and over 500 regions of our DNA are associated with cardiovascular disease (CVD). These facts indicate the important role DNA plays in heart health, from developing embryos to adults. By learning how different DNA regions are “turned on/off” in heart cells throughout development and over time, researchers may identify factors that influence heart disease and how heart disease may be treated. However, these studies are challenging as there is a question of how developing heart cells used in research experiments compare to those found in a developing embryo.
Research supported by the Common Fund 4D Nucleome (4DN) program from the lab of Dr. Charles Murry provided an important step in overcoming these challenges. Dr. Murry and his colleagues integrated several research techniques, some of which were developed by the 4DN program, to show that derived human heart cells used in research experiments were very similar to naturally occurring cells with respect to the 3-dimensional architecture of all their DNA – or genomes – and whether certain genes were turned on or off. This information gives researchers confidence when using derived cells in experiments that model diseases of the heart. Overall, the methods developed in this research represent a valuable resource for the study of both heart development and heart disease by revealing genome-wide changes in developing heart cells as well as the dynamics of different DNA regions and which genes are active at different times.
Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory. Bertero A, Fields P, Ramani V, Bonora G, Yardimci G, Reinecke H, Pabon L, Noble W, Shendure J, Murry C. Nature Communications, 2019. doi: 10.1038/s41467-019-09483-5
DNA is packed differently in each tiny cell nucleus
DNA is organized inside the nucleus of a cell like a ball of cooked spaghetti. The organization of DNA can determine whether a gene encoded by the DNA gets turned on or off. Scientists can use high-resolution techniques such as chromosome conformation capture (Hi-C) to examine how pieces of DNA are organized to fit inside the space of the nucleus.
In a recent article, 4D Nucleome program-supported research combined Hi-C with an imaging method – high-throughput fluorescence in situ hybridization – to physically map and visualize regions of DNA. These combined techniques yielded information on the interactions of more than hundred pairs of genes in individual cells. This unique approach revealed that the folding and looping of DNA in 3-dimensional space varies cell-to-cell, and that there is diversity in the frequency of interactions between different regions of DNA. Furthermore, the two copies of a gene – one inherited from each parent – are independent of each other and not packaged in a specific part of the nucleus together. These results suggest DNA packaging and organization is highly variable across a population and within single cells.
This study provides novel insights into the organization of the nucleus, which is a hallmark of the 4D Nucleome program. In addition, these findings lay the groundwork for functional analyses of how genes are organized in a cell. This is important because the organization of genes affects the role they play in health and disease.
Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization. Finn EH, Pegoraro G, Brandão HB, Valton AL, Oomen ME, Dekker J, Mirny L, Misteli T. 2019. Cell. Mar 7;176(6):1502-1515.e10. doi: 10.1016/j.cell.2019.01.020.
In the News:
Genome Organization Variation Uncovered With Molecular Mapping Methods, Genomeweb
4DN Investigator Spotlight: Andrew Belmont
Dr. Andrew Belmont is a Professor of Cell and Developmental Biology as well as a Professor of Biophysics and Quantitative Biology at the University of Illinois at Urbana-Champaign. He researchers how movement and organization of DNA within the cell nucleus play a role in normal biological functions and how changes in these properties may influence diseases, such as cancer. Dr. Belmont explains that while people have been able to view structures within a cell nucleus for over 120 years, we are only beginning to understand what these structures actually mean and how they change with time. Dr. Belmont is adding to our understanding with a genomic read-out tool his team developed, called TSA-Seq, to measure the position of different compartments in the nucleus, a task that could previously be done though time consuming microscopy experiments. With this tool and other experiments, Dr. Belmont's laboratory showed that after a gene is "turned on or off" in the nucleus, it can move to different nuclear locations. As part of the NIH Common Fund's 4D Nucleome (4DN) program, Dr. Belmont is developing new methods that combine microscopy with genome-wide mapping techniques to study the movement of DNA in the nucleus after a gene is activated. Exploring this mechanism will help researchers better understand how our bodies work and may help them develop targets for diseases that impact DNA movement and organization in the nucleus. "It's like a puzzle" Dr. Belmont explains, "and you're just trying to understand the puzzle, and it's a difficult puzzle so it takes longer."
Dr. Belmont has long been assembling puzzle pieces. His very first summer job as a college student was drug testing races horses from a Philadelphia local track. Coming from a family of medical professionals, Dr. Belmont obtained a medical degree. At first, he did not know what scientists did, but he joined a lab as a medical student and has been involved in biomedical research ever since.
Throughout his career, Dr. Belmont has seen the difficulties of getting a group of people together to accomplish a goal. He explains that his work on the 4DN program has been "wonderful because it is a privilege to work with so many really interesting people who have established all these methods and systems and to be part of a team where everyone enjoys trying to do better science."
Learn more about Dr. Belmont's 4DN research project.
4DN Investigator Spotlight: Jennifer Phillips-Cremins
Dr. Jennifer Phillips-Cremins is an Assistant Professor at the University of Pennsylvania. She researches the brain and how neurons (nerve cells) in the brain connect and dictate brain function. Dr. Phillips-Cremins is interested in how 3D genome folding governs neuron properties in healthy states and how mis-folding contributes to neurological disorders such as Alzheimer’s, Huntington’s Disease, and Friederich’s Ataxia. Dr. Phillips-Cremins explains that chromatin organization is tightly controlled by cells, and when the chromatin becomes “tangled up,” it can no longer perform its function in the neurons, leading to disease. A lot is known about genome folding patterns over very long time scales during cell development, but very little is known about how those structures change over shorter time scales. Dr. Phillips-Cremins is funded by the 4D Nucleome program (4DN) to engineer tools that can visualize and alter chromatin structures on demand over very short time scales to provide insight into the relationship of chromatin folding and gene regulation. She is developing her tools ultimately to be used in neurons in the brain. She says that the 4DN community has helped advance her research by providing a network of people dedicated to understanding genome folding and who are developing a vast array of tools and model systems to understand changes in genome folding and its relevance to human health. Since the founding of the 4D Nucleome program, our ability to study and understand the 3D structure of the genome has expanded. This is important because neurological disorders are notoriously difficult to treat. “This is an area where we can use what we’re good at and combine it with something that’s needed in this world right now . . . there are very few treatments for these severe disorders,” Dr. Phillips-Cremins explained. She is driven by a commitment to make discoveries that help people with neurological disorders and uses this commitment as a criterion when developing new research projects.
In addition to helping others, Dr. Phillips-Cremins enjoys the challenges of her research and the opportunity to keep learning. She loves the idea of pushing herself to see what she’s capable of and persevering through difficulties. This comes through in her love of sports, serving as a point guard on her college basketball team and running a marathon during her PhD studies. Regarding her challenging research projects, she said, “Science is very humbling. The more I learn through the process, the more I realize there is so much further to go.”
Learn more about Dr. Phillips-Cremins’ 4DN research project.
Scientists Develop Molecular Tweezers with CRISPR-GO
One of the goals of the 4D Nucleome (4DN) program is to understand how organization of all the DNA in the nucleus – called the genome – affects cell function. To truly understand this relationship, scientists need to be able to manipulate genome organization to observe the effect on which genes are turned on or off. In a recent article in Cell, 4DN investigator Dr. Stanley Qi and his research team describe a new technique called CRISPR-genome organization (CRISPR-GO) to manipulate the organization of DNA in the cell nucleus. Described as “molecular tweezers,” CRISPR-GO allows scientists to grab a specific section of DNA and move it to a new targeted location in the nucleus. This tool works rapidly and is inducible and reversible, making it very useful for exploring the effects of genome reorganization on many genes. CRISPR-GO allowed the team to explore whether genes behave differently in different parts of the nucleus. They found that genes relocated to the periphery of the nucleus or to blobs of proteins and RNA in the nucleus called Cajal bodies were turned off, indicating a role for these regions in gene regulation. CRISPR-GO is a valuable tool for studying the relationship between genome organization and cell function that could improve our understanding of diseases associated with abnormal genome organization to help develop new treatments for these diseases.
Reference: CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization. Wang H, Xu X, Nguyen CM, Liu Y, Gao Y, Lin X, Daley T, Kipniss NH, La Russa M, and Qi LS. November 2018. Cell 175: 1405-1417.
In the News: Researchers modify CRISPR to reorganize genome, Stanford Medicine News Center
3D Mapping Strategy Reveals Framework for Gene Expression
Chromatin is a complex of genomic DNA and proteins that make up the chromosomes within the nucleus of a cell. The organization of genomic material into chromatin is presumed to play an important role in regulating expression of genes. However, the precise relationship between spatial genome organization and expression of resident genes in health and disease remains unclear. Toward understanding 3D genome architecture and its relationship to gene regulation, 4D Nucleome researcher Dr. Yijun Ruan, Ph.D., a Jackson Laboratory Professor, Florine Deschenes Roux Chair and Director of Genome Sciences, and his team worked with international collaborators to identify a framework in which genes are organized and transcribed at the chromosomal level.
For these studies, the authors used advanced 3D genome mapping technologies and simulation, as well as super-resolution microscopy. These models revealed higher order chromosome folding and specific chromatin interactions, mediated by the chromatin proteins CTCF and cohesin. These chromatin structures suggest a “barrier” between genes being actively transcribed and those that are not. Importantly, these studies further uncovered potential mechanistic links between genetic mutations associated with specific disease and chromatin topology.
“The significance of this paper lies in our advanced 3D genome mapping strategy,” Dr. Ruan said, “which allowed us to reveal, for the first time, the higher-order and detailed topological structures of the human genome mediated by CTCF and cohesin, and the relation to gene transcription regulation carried out by RNA polymerase II. This publication is also timely adding new excitement to the recently initiated 4D Nucleome program by NIH." For additional information, read The Jackson Laboratory news release.
Reference: CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription. Tang Z, Luo OJ, Li X, Zheng M, Zhu JJ, Szalaj P, Trzaskoma P, Magalska A, Wlodarczyk J, Ruszczycki B, Michalski P, Piecuch E, Wang P, Wang D, Tian SZ, Penrad-Mobayed M, Sachs LM, Ruan X, Wei CL, Liu ET, Wilczynski GM, Plewczynski D, Li G, Ruan Y. Cell. (7) 11611 – 27.
New Tools to Study Cellular Phase Separation
4D Nucleome program-funded investigator Dr. Clifford Brangwynne and his team have developed two new tools to study and control cellular phase separation and allow phase mapping in living cells. Watch the video to learn about "corelets" and "Casdrop".
Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds. Bracha D, Walls MT, Wei MT, Zhu L, Kurian M, Avalos JL, Toettcher JE, and Brangwynne CP. Nov 2018. Cell 175:6 1467-1480.
Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome. Shin Y, Chang YC, Lee DSW, Berry J, Sanders DW, Ronceray P, Wingreen NS, Haataja M, and Brangwynne C. Nove 2018. Cell 175:6 1481-1491.
In the News:
New tools illuminate the liquid forces at play in living cells, Princeton News
Misfolded DNA Contributes to Neurodegenerative Disease
Over 1% of our DNA is made up of repeated sequences, which equals tens of thousands of short repeat tracts. Most repeat sequences are stable in length, but a small subset tend to expand or contract in length during processes such as DNA replication and repair. Over 30 human neurodegenerative disorders are associated with DNA repeat instability, including fragile X syndrome, Huntington’s disease, and Friedreich’s ataxia. For the disease-associated repeats, healthy individuals have a “normal” number of repeats, while those with the disease have repeats expanded beyond a threshold length. Researchers have long sought to understand why some DNA repeats are prone to expansion, while others are not.
The 3D arrangement of DNA impacts how information encoded by the DNA is “read” and used by the cell. To explore whether DNA folding plays a role in repeat instability, 4DN Program-funded researcher Dr. Jennifer Phillips-Cremins and her team analyzed the folding pattern of unstable regions of DNA repeats. They found that nearly all the repeat sequences associated with human disease are located at the boundaries between discreet 3D regions of the genome. To explore this in the context of human disease, they created genome folding maps around the FMR1 gene, the gene associated with fragile X syndrome, in samples from patients and healthy individuals. In samples from patients (which contained expanded repeats) they found misfolding at the expanded repeat tracts disrupted the boundary between domains, leading to the FMR1 gene being turned off. This study shows that regions of DNA repeats associated with human diseases can localize to genome domain boundaries and can disrupt 3D genome structure and gene function. Improved understanding of the link between DNA repeat instability and genome folding can aid in development of treatments for repeat expansion disorders.
Disease-Associated Short Tandem Repeats Co-Localize with Chromatin Domain Boundaries. Sun, JH, Zhou, L, Emerson, DJ, Phyo, SA, Titus, KR, Gong, W, Gilgenast, TG, Beagan, JA, Davidson, BL, Tassone, F, and Phillips-Cremins, JE. Cell 175, 38-40. September 2018.
Chromatin Domains Go on Repeat in Disease. Bruneau, BG and Nora, EP. Cell 175:1, 224-238. September 2018.
In the News:
Penn Researchers: Class of Neurological Disorders Share 3D Genome Folding Pattern, Science Magazine
Making the Nucleus Measure Up
The dynamic 3D organization of DNA in the tiny nucleus of a cell plays a critical role in determining which genes are turned on in a cell (gene expression), which affects cell function. Determining the 3D organization of DNA in the nucleus of live cells has been extremely difficult. To overcome this obstacle, the NIH Common Fund 4D Nucleome (4DN) program is developing new tools to explore nuclear organization in relation to cell function and human health. 4DN-funded investigator Dr. Andrew Belmont and a team of researchers have developed a new technique called “TSA-seq” that can measure the positions of genes in the nucleus relative to nuclear landmarks such as the nuclear lamina (surrounds the nucleus) or nuclear speckles (found in the center of the nucleus). The technique works by targeting an enzyme called “horseradish peroxidase” to a specific nuclear structure. The enzyme generates reactive molecules that label the surrounding DNA, with DNA closer to the enzyme being more heavily labeled. The DNA is isolated and sequenced, and the amount of labeling at each gene can then be used to calculate how close each gene is to the tagged nuclear structure. This information can be used to build a genome-wide 3D picture of nuclear organization. Combining TSA-seq with measurements of gene expression showed that nuclear speckles tend to be “hot zones” of gene activity, with more of the genes close to the nuclear speckles being active. This finding suggests that even small changes in the position of a gene, that move the gene closer to or farther from a nuclear speckle, could have significant consequences on gene expression and cell function.
Reference: Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler. Chen, Y, Zhang, Y, Wang, Y, Zhang, L, Brinkman, EK, Adam, SA, Goldman, R, van Steesel, B, Ma, J, and Belmont, A. Journal of Cell Biology. August 2018.
In the News: Researchers develop ‘cytological ruler’ to build 3D map of human genome, Science Daily
Turning on Genes is No Lonely Job
At any given time, only some of the genes in our DNA are turned on, or “active,” while others are turned off. Making sure only the correct genes are turned on in the correct cells is critical for our health. Scientists aim to understand how proteins in cells coordinate to turn a gene on, which could help identify strategies for treating improper regulation of gene activity. Two 4D Nucleome program-funded studies recently published in the journal Science explore the protein interactions that help turn a gene on. Both studies found that proteins cluster together at regions in the DNA called enhancers and interact to trigger gene activation.
In one study, Dr. Ibrahim Cisse and a team of researchers used live-cell super-resolution microscopy to view single molecules of proteins in mouse cells. They were able to view the interactions of a protein complex called Mediator, which helps kick-start transcription, and RNA polymerase II, the protein that carries out transcription by copying DNA into RNA. They found that both Mediator and RNA polymerase II group into stable clusters forming liquid-like droplets, a process known as phase-separation. Protein interactions were found to be brief, with proteins able to move in and out of the droplets, and droplets able to fuse together. They propose that Mediator droplets cluster at enhancers and fuse with RNA polymerase II droplets, allowing interactions between Mediator and RNA polymerase II that spur transcription to turn on genes.
In another study, led by Dr. Robert Tijan and Dr. Xavier Darzacq, the research team used live-cell single-molecule imaging to explore how proteins called transcription factors bind to the DNA enhancer and interact to initiate gene activation. They found that transcription factors also form high-concentration clusters that localize at the enhancer to stabilize DNA binding, recruit RNA polymerase II, and activate transcription. The interactions between transcription factors and RNA Polymerase II were rapid, reversible, and selective, making them a potential class of drug targets for regulating the process of gene activation.
Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Cho, WK, Spille, JH, Hecht, M, Lee, C, Li, C, Grube, V, and Cisse, II. Science. 361, 412-415. 2018 July 27.
Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Chong, S, Dugast-Darzacq, C, Liu, Z, Dong, P, Dailey, GM, Cattoglio, C, Heckert, A, Banala, S, Lavis, L, Darzacq, X, and Tijan, R. Science. 361 (6400). 2018 July 27.
In the News:
a href="https://www.sciencenews.org/article/it-may-take-village-proteins-turn-genes" target="_blank" title="Links to article in Science News">It may take a village (of proteins) to turn on genes, Science News
Viewing the Moment a Gene Turns On
Genes are the segments of our DNA that code for proteins that determine our traits. Over 90% of our DNA does not encode genes and was long considered “junk” DNA that had no known purpose. We now know that much of this DNA does have a purpose, for example, some of this non-coding DNA contains enhancers- regions of DNA that help “turn on” genes to ultimately produce proteins. The timing of turning a gene on is very important for normal development and issues with timing can lead to development of disease. Enhancers are typically located far away from the gene they turn on, and how the enhancers find their target genes within the nucleus of the cell and how they interact with gene- coding regions to result in protein production is not well understood. In a recent study by 4D Nucleome program-funded investigator Dr. Thomas Gregor and his research team, a live imaging approach was used to track the position of an enhancer and its target gene in developing fly embryos, while also monitoring gene activity. Using this technique, they were able to observe the moment when a gene was turned on. The results showed that close proximity between the enhancer and target gene was required not only to turn the gene on, but also to keep the gene active. When the enhancer disconnected from the target gene, the gene turned off. They also found that when the gene was turned on, the structure formed by the enhancer and target gene became more compact, and the results suggest changes in the 3D DNA arrangement improve the stability of this structure, allowing the gene to remain active. The results of this study improve our understanding of how gene activity is regulated and may help provide insight into how improper regulation of gene activity leads to developmental defects and disease.
Reference: Dynamic interplay between enhancer-promoter topology and gene activity. Chen, H, Levo, M, Barinov, L, Fujioka, M, Jaynes, JB, and Gregor, T. Nature Genetics. 2018.
In the news:
Imaging in living cells reveals how ‘junk DNA’ switches on a gene, Princeton University
DNA Enhancer Video Rules Out Hit-and-Run Activity, Genetic Engineering & Biotechnology News
New Technique for 3D Genome Mapping
The 3D organization of DNA in the nucleus of a cell plays an important role in determining which genes are turned on in that cell. This has important implications for human health, as problems with DNA organization are linked to human diseases such as cancer and early aging. Understanding how the DNA is organized in healthy cells is a critical step in identifying targets and developing treatments for abnormal nuclear organization. A current method for mapping 3D genome organization uses a technique called “proximity ligation” in which regions of DNA that are very close together, or “touching,” are linked together and then sequenced to determine where these DNA “touches” occur. This technique mostly identifies interactions of DNA regions within the same chromosome. However, imaging of the genome using microscopy techniques has shown that there are interactions between chromosomes and that these interactions tend to occur at discrete regions of the nucleus known as nuclear bodies. This indicates limitations of proximity ligation techniques in identifying interactions between chromosomes that occur over longer-range distances. In addition, both proximity ligation and microscopy techniques are limited to measuring simultaneous contacts between a small number of DNA regions, making it difficult to develop a comprehensive model of global genome organization.
A recent study led by NIH Common Fund 4D Nucleome Program-funded investigator Dr. Mitchell Guttman, developed a new technique for detecting simultaneous genome-wide interactions within the nucleus, called Split-Pool Recognition of Interactions by Tag Extension (SPRITE). SPRITE works by linking interacting DNA, RNA, and proteins in cells, isolating the nuclei, fragmenting the chromatin, “barcoding” interacting molecules within a complex, and sequencing and matching the areas with identical “barcodes” to identify interacting regions. Unlike proximity ligation and microscopy techniques, SPRITE is not limited in the number of simultaneous DNA interactions that it can identify. Using SPRITE, they were able to detect interactions that occur across larger distances than those found by proximity ligation techniques. They found two “hubs” of interactions between chromosomes, both associated with nuclear bodies: an inactive gene-poor hub that organizes around the nucleolus and an active gene-rich hub that organizes around regions called “nuclear speckles.” Using the SPRITE results, they created a global model of 3D genome organization, in which nuclear bodies act as inter-chromosomal hubs that shape the 3D packaging of DNA in the nucleus.
Reference: Higher-order inter-chromosomal hubs shape three-dimensional genome organization in the nucleus. Quinodoz, SA, Ollikainen, N, Tabak, B, Palla, A, Schmidt, JM, Detmar, E, Lai, MM, Shishkin, AA, Bhat, P, Takei, Y, Trinh, V, Aznauryan, E, Russell, P, Cheng, C, Jovanovic, M, Chow, A, Cai, L, McDonel, P, Garber, M, and Guttman, M. Cell. 2018.
In the News: Biologists Create 3D Maps of DNA Within the Innermost Parts of a Cell, SciTech Daily
Packaging Chromosomes for Cell Division
As a cell moves through the cell cycle, the shape of the DNA in the nucleus changes dramatically, from a ball of DNA during normal cell activities to distinct X-shaped chromosomes as the cell prepares to divide. This change in shape is important for the orderly passage of one copy of the DNA to each new cell. Before the cells begin to divide, the DNA compacts into the dense X-shaped chromosomes that are made up of consecutive DNA loops. How the compaction of loops occurs is not well understood. One of the goals of the 4D Nucleome program is to determine how the structure of DNA in the nucleus of a cell changes over time.
In a recent study in Science, a team led by 4DN-funded researchers Dr. Leonid Mirny and Dr. Job Dekker combined techniques that are used to determine where regions of the genome “touch” with imaging and modeling techniques at one-minute time intervals to investigate how DNA rearrangement occurs before cell division. Based on their results, they propose a model in which cells use a protein called “condensin” to drive the compaction of DNA. Condensin proteins create DNA loops by pushing DNA through their ring-like structures. In this model, condensin I creates wide loops in the DNA that are then split into smaller loops by condensin II. The loops twist around a condesin scaffold in a structure resembling a spiral staircase, creating a condensed helix of consecutive DNA loops that makes up the X-structure of the chromosomes and results in formation of compact units of DNA that can be easily divided between new cells. Understanding how the organization of DNA changes throughout the cell cycle is critical for determining how problems with cell cycle-associated DNA rearrangement (such as chromosome breakage) lead to human diseases such as cancer.
Reference: A pathway for mitotic chromosome formation. Gibcus, JH, Samejima, K, Goloborodko, A, Samejima, I, Naumova, N, Nuebler, J, Kanemaki, MT, Xie, L, Paulson, JR, Earnshaw, WC, Mirny, LA, Dekker, J. Science. 2018 Jan 18. doi: 10.1126/science.aao6135.
In the news:
Packing a Genome, Step-by-Step, Howard Hughes Medical Institute
How Cells Pack Tangled DNA Into Neat Chromosomes, Quanta Magazine
Loop Loss in the Human Genome
DNA is organized in the small nucleus of a cell in the form of a DNA-protein complex called chromatin. The protein “cohesin" helps maintain DNA organization by tethering two regions of DNA on the same chromosome to form loops. The loops have long been thought to regulate which genes are turned on by controlling the distance between DNA promoters and enhancers. Promoters are regions of DNA that generally occur before a gene and serve as a landing space for the molecular machinery needed to activate the gene. Enhancers are regions of DNA that can increase gene activity when in close contact with the target gene’s promoter.
In one study, a group of researchers led by 4D Nucleome program-funded investigator Erez Lieberman Aiden used a technique called chromosome conformation capture (Hi-C) to map the formation of DNA loops throughout the genome at 20-minute intervals during the loss and recovery of cohesin. They found that cohesin removal led to the loss of loops. However, this surprisingly had modest effects on gene activity, with only a few genes experiencing significant changes in activity. They also observed formation of a separate group of cohesin-independent loops and links between different chromosomes. The results suggest that cohesin-dependent loops play only a modest role in regulating interactions between promoters and enhancers. Based on these results, the team proposes a revised model in which a combination of cohesin-dependent and -independent loops regulate gene activity.
In another study led by 4DN-funded researcher Leonid Mirny and Transformative Collaborative Project Awardee Francois Spitz, the protein responsible for loading cohesin onto chromatin, Nipbl, was deleted in a mouse model. The resulting changes in chromatin organization were then identified using a Hi-C technique. Nipbl deletion led to significant changes in chromatin organization, including loss of cohesin-dependent loops and enhancement of compartments made up of chromatin regions with similar activity. The loss of cohesin-dependent loops allowed formation of smaller chromatin compartments with fewer contacts between active and inactive chromatin regions. The results contradict a model of chromatin organization in which DNA loops combine to form larger compartments. Instead, the authors propose a revised model in which genomic compartment formation is interrupted by cohesin-dependent loops that can bring regions of different chromatin activities together to drive gene activity. Although these studies, used different approaches, they led to similar conclusions. Understanding how the 3-dimensional structure of chromatin is controlled over time and affects gene activity can lead to better treatment of human diseases linked to abnormal chromatin organization.
Cohesin Loss Eliminates All Loop Domains. Rao, SSP, Huang, S, St Hilaire, BG, Engreitz, JM, Perez, EM, Kieffer-Kwon, K, Sanborn, AL, Johnstone, SE, Bascom, GD, Bochkov, ID, Huang, X, Shamim, MS, Shin, J, Turner, D, Ye, Z, Omer, AD, Robinson, JT, Schlick, T, Bernstein, BE, Casellas, R, Lander, ES, and Lieberman Aiden, E. Cell (171), 305-320. 2017 October 5.
Two independent modes of chromatin organization revealed by cohesin removal. Schwarzer, W, Abdennum, n, Goloborodko, A, Pekowska, A, Fudenberg, G, Moe-Mie, Y, Fonesca, NA, Huber, W, Haering, CH, Mirny, L, and Spitz, F. Nature (551), 51-56. 2017 November 2.
In the News: Watch the human genome fold itself in four dimensions, Science News
Dynamic DNA Loops Affect How Cells Become Specialized
Genomic DNA is packaged and organized in the tiny nucleus of the cell as chromatin (a complex of DNA and histone proteins). The 3-dimensional organization of chromatin in the nucleus affects which genes are expressed and at what times. A complex network of chromatin loops is involved in coordinating changes in transcription during cell development. Chromatin loops can bring enhancers (sections of DNA that promote transcription when bound by proteins called transcription factors) closer to their target genes in the genome. How chromatin architecture changes as cells differentiate into specialized cell types and how these changes affect cell-type-specific gene expression and cellular function are not well understood.
A team of researchers, including 4DN program-funded researcher Erez Lieberman Aiden, used a technique called in situ chromosome conformation capture (Hi-C) to create high-resolution genome-wide looping maps to compare the chromatin structure of cells before and after differentiation to become specialized immune cells (macrophages). Hi-C is a method of detecting frequencies of contact between all mappable regions of the human genome. Following differentiation, they found genes at loops that were newly formed (“gained loops”) or newly activated by changes in chromatin architecture (“activated loops”) have increased expression. The gained and activated loops form multi-loop activation “hubs” that create long-range interactions between active enhancers and promoters and have increased binding of transcriptional regulators, thus facilitating transcription. The multi-loop hubs occur at genes known to play a role in macrophage development and function, indicating a role in regulating gene transcription during cell differentiation. This study could have broader implications for how chromosome organization instructs transcription in other cellular contexts and throughout human development.
Reference: Static and Dynamic DNA Loops form AP-1-Bound Activation Hubs during Macrophage Development. Phanstiel, DH, Van Bortle, K, Spacek, D, Hess, GT, Shamim, MS, Machol, I, Love, MI, Lieberman Aiden, E, Bassik, MC, Snyder, MP. Molecular Cell. 2017 September 21. 67(6): 1037-1048.
The 4D Nucleome Project
The 4D Nucleome Program aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of understanding how the nucleus is organized and functions. The program will develop and benchmark robust experimental and computational approaches for measuring genome conformation and nuclear organization, and illuminate how they contribute to gene regulation and other genome functions. These efforts will lead to new insights into how the genome is organized, maintained, expressed and replicated, in both normal and disease states.
Reference: The 4D Nucleome Project. Dekker J, Belmont A, Guttman M, Leshyk V, Lis J, Lomvardas S, Mirny L, O’Shea C, Park P, Ren B, Ritland Politz C, Shendure J, Zhong S, & the 4D Nucleome
Network. Nature. 2017 September 14.
Visualizing the Nucleus in 3D
How DNA is packaged in the nucleus determines how it is used and how genes are expressed. DNA is condensed and packaged as chromatin (a complex of DNA and proteins called histones), which constantly changes as genes are expressed. Understanding chromatin packaging may reveal the structural code for how genes are turned on or off in human health and disease. For example, understanding chromatin packaging could be used to make cancer cells with abnormally structured chromatin “remember” how to be healthier through repackaging chromatin. Toward understanding chromatin packaging in the nucleus, 4D Nucleome (4DN) program grantee Dr. Clodagh O’Shea collaborated with fellow 4DN grantee Dr. Mark Ellisman to develop a new approach to visualize chromatin in 3D space. This method, called ChromET, combines electron microscopy tomography (EMT) and a labeling method that enhances the visualization of DNA in human cell lines. An electron microscope uses a beam of electrons to create an image of a sample and is capable of seeing much smaller objects than a traditional light microscope. The 4DN researchers used ChromET to show that chromatin is flexibly disordered and packed together at different concentrations in the nucleus. This is different from the textbook model of rigid higher-order chromatin folding. This new model of diverse chromatin structures – able to bend at various lengths and achieve different packing concentrations – is important because it provides an explanation for how different parts of the genome could be fine-tuned to make different structures, at different times, with different functions. This research brings us one step closer to discovering how the structural code of our genomes could be used to advance medical care.
Reference: ChromEMT: Visualizing 3D chromatin structure and compaction of the human genome in interphase and mitotic cells. Ou, HD, Phan S, Deerinck TJ, Thor A, Ellisman, MH, O’Shea CC. Science. 2017 July 28.
In the News: Salk Institute Press Release, NIH Press Release, Study: DNA Folding Patterns Revealed The Scientist
Genes on the Move
By studying cell-to-cell variability in dividing cells, a research team led by 4D Nucleome (4DN) grantees Drs. Peter Fraser and Amos Tanay revealed that genes don't have a fixed location in the cell, but are constantly moving. The cell cycle is a series of events during which a cell grows, replicates its chromosomes, and then divides. Chromosome conformation capture is a technique used to detect physical interactions between different segments of chromosomes. It can be used to investigate the 3D folding of chromosomes, and to identify where genes on those chromosomes are positioned in the nucleus. Most chromosome conformation capture experiments are done using millions of cells in different phases of the cell cycle, all mixed together. In this study, researchers used chromosome conformation capture and statistical analysis to look at the 3D folding of chromosomes in thousands of individual mouse embryonic stem cells, all separated from one another. Surprisingly, they found that genes don’t have a fixed location in the cell nucleus – rather, genes are continuously moving and change their positions as they progress through different stages of the cell cycle. This is important because changes in gene location can result in changes in gene activity, which can influence human health and disease. Studying chromosomal organization could illuminate how changes in the location of a gene can affect normal development as well as various diseases.
Reference: Cell-cycle dynamics of chromosomal organization at single-cell resolution. Nagano T, Lubling Y, Várnai C, Dudley C, Leung W, Baran Y, Mendelson Cohen N, Wingett S, Fraser P, Tanay A. Nature. 2017 July 6. 547(7661):61-67.
In the News: Cell cycle: Continuous chromatin changes, Nature. Music of the Genes Rises from the Cell Cycle, Genetic Engineering & Biotechnology News.
Genome Organization: Unpacked
4D Nucleome grantees have combined imaging and genomic methods to discover new insights about certain proteins that impact gene expression and nuclear organization. Chromatin is a substance found within our chromosomes that consists of DNA and protein. The function of chromatin is to efficiently package over 6 feet of DNA into a remarkably small volume – the nucleus of a cell – and protect the DNA structure and sequence. Formation of chromatin structures and nuclear organization are not random, it turns out that chromatin is organized in chromosomal “neighborhoods” within the nucleus. Packaging of DNA into chromatin in these neighborhoods can control gene expression and DNA replication. Yet, the principles behind genome organization in the nucleus are not well understood. Understanding nuclear architecture is important because abnormalities in genome organization are associated with diseases, including cancer and premature aging. One way to study genome organization is through chromosome conformation capture, a technique that is used to detect physical interactions between different segments of the genome. Another method is fluorescent in situ hybridization (FISH), which researchers use to visualize and map the cell’s genetic material through imaging. Although both provide a wealth of information, bringing these approaches together (correlating imaging technologies with genomics and sequencing) will allow researchers to sequence information that they see.
Toward this aim, 4D Nucleome grantees Drs. Arjun Raj, Jennifer E. Phillips-Cremins, and Gerd A. Blobel have used FISH imaging and chromosome capture genomic methods to discover new insights about bromodomain and extraterminal motif (BET) proteins, which help regulate gene expression by controlling chromatin and nuclear organization. Although there are several different types of BET proteins, the role of each type and how they interact with chromatin is not well understood. The study of BET proteins is important because BET inhibitors are a class of drugs with anti-cancer, immunosuppressive, and other effects in clinical trials. In this study, they found that a certain type of BET protein contributes to the formation of boundaries, which help chromatin form chromosomal neighborhoods. This raises the possibility that BET inhibitors can influence gene expression by disrupting boundary function. Using imaging and genomic sequencing methods to gain a clearer understanding of BET proteins will help researchers understand how BET inhibitors work, bringing us one step closer to treating disease.
Reference: The BET Protein BRD2 Cooperates with CTCF to Enforce Transcriptional and Architectural Boundaries. Hsu SC, Gilgenast TG, Bartman CR, Edwards CR, Stonestrom AJ, Huang P, Emerson DJ, Evans P, Werner MT, Keller CA, Giardine B, Hardison RC, Raj A, Phillips-Cremins JE, Blobel GA. Molecular Cell. 2017 April 6. 66(1):102-116.
Studying the Mystery of Genome Organization in Development
Researchers from the Common Fund’s 4DN Program have developed a new technology to peer inside the cell at the earliest stages of development, revealing important differences in how the DNA from the mother and the father are organized and potentially providing clues about how the multitude of cell types in an organism can arise from a single cell. During fertilization, maternal and paternal DNA coexist in the zygote, which later develops into an embryo following the instructions encoded in the DNA. Understanding how DNA from both parents is organized in the zygote is important because 3D organization of DNA is one way that a cell can control gene expression. Tight regulation of gene expression in these early developmental stages is key to tissue, organ, and organism development. One way to study genome organization is through chromosome conformation capture (Hi-C), a technique that is used to detect physical interactions between sequences of the genome. However, using this method to study the nuclear organization of a single zygote or embryo is challenging because it usually requires hundreds, thousands, or millions of cells.
To overcome this challenge, 4D Nucleome grantee Dr. Leonid Mirny, and collaborators, have developed a new form of Hi-C technology to analyze cells at a single cell level - a significant technological advancement in this field. Using this method, they found that nuclear architecture is uniquely reorganized during the ococyte to zygote transition in mice and that maternal and paternal DNA are packaged differently in mouse zygotes. The oocyte to zygote transition refers to a critical period of development, during which fundamental changes in nuclear function take place as the egg and sperm give rise to an embryonic genome. This exciting knowledge of the zygotic “ground state” will contribute to our understanding of how cells give rise to an astounding number of different cell types that make up an organism.
In the News: Mom’s and Dad’s Genes Packed Differently in Fertilized Egg, Genetic Engineering & Biotechnology News. In the beginning there was order, Nature.
Reference: Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition. Flyamer IM, Gassler J, Imakaev M, Brandão HB, Ulianov SV, Abdennur N, Razin SV, Mirny LA, Tachibana-Konwalski K. Nature. 2017 April 6. 544(7648):110-114.
Understanding Chromosome Structure and Cancer
DNA is not randomly arranged in the nucleus. Instead, nuclear organization is tightly controlled. For example, insulated neighborhoods are loops of DNA that function to maintain normal expression of genes within and outside of the loop. Defects in nuclear organization and the folding of the human genome have been linked to cancer. In a new study, 4D Nucleome investigator Dr. Job Dekker, Ph.D., Howard Hughes Medical Institute Investigator, University of Massachusetts Medical School, and collaborators investigated the causal relationship between chromosome structure and oncogene activation. Oncogenes are genes that have the potential to cause cancer, when activated through dysregulation of gene expression. Using DNA sequences from tumors and targeted mutations in cancer cell lines, they show that disruption of insulated neighborhoods can activate oncogenes. This suggests that disruption of chromatin architecture is causally linked to the formation of tumors. This work represents a step toward understanding 3D chromosome structure and the authors conclude that “understanding these regulatory processes may provide new approaches to therapeutics that have on impact an aberrant chromosome structure”.
Reference: Activation of proto-oncogenes by disruption of chromosome neighborhoods. Hnisz D, Weintraub AS, Day DS, Valton AL, Bak RO, Li CH, Goldmann J, Lajoie BR, Fan ZP, Sigova AA, Reddy J, Borges-Rivera D, Lee TI, Jaenisch R, Porteus MH, Dekker J, Young RA. Science. (6280) 1454 – 8.
A Guiding Light to Study Protein Assembly in Living Cells
4D Nucleome grantee Dr. Clifford Brangwynne and collaborators have developed a new tool that uses light to manipulate matter inside living cells. Called optoDroplet, this tool helps explain the physics and chemistry behind how cells assemble a mysterious structure called a membraneless organelle. An organelle is a specialized part of a cell having some specific function. For example, the nucleus is an organelle that holds most of the cell’s genetic information. Organelles like the nucleus are walled off from the rest of the cell by a membrane. The cell also uses membraneless organelles that resemble liquid droplets and exhibit dynamic behavior, such as rapid assembly and disassembly of protein building blocks that make up the organelle. When these mechanisms go awry, aggregates of the protein building blocks can form. Protein aggregation is associated with a number of diseases, including amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease) and Alzheimer's disease. Understanding the process by which proteins condense into these droplet-like, membraneless organelles may be used to develop of interventions and treatments for diseases connected with protein aggregation. To better understand this process, Dr. Brangwynne’s group developed optoDroplet. This new tool relies on optogenetics, which involves proteins whose behavior can be altered by exposure to light. Using mouse and human cells, researchers showed that they could create membraneless organelles by switching on the light-activated proteins. They were also able to use this tool to generate protein aggregates, similar to those found in some diseases. The optoDroplet system will help researchers understand the basic mechanisms that underlie self-assembly of membraneless organelles in healthy living cells and may reveal how cells become diseased when this process goes awry.
In the news: New tool shines a light on protein condensation in living cells, Light-Operated Tool Will Offer New Insights Into Protein Assembly in ALS
Reference: Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets. Shin Y, Berry J, Pannucci N, Haataja MP, Toettcher JE, Brangwynne CP. Cell. 2017, January 12.
Study Reveals Elaborate X-inactivated Chromosome Structure
X-inactivation is the process by which one of the two X chromosomes present in female mammals is inactivated. This prevents them from having twice as many X chromosomes gene products as males, who only have a single X chromosome. A Barr body is the structure inside of the nucleus that consists of the inactive X chromosome. Although the Barr body appears to be a condensed blob under a microscope, a new study from 4D Nucleome grantee Job Dekker, Ph.D. and collaborators reveals a highly elaborate structure. Using a variety of methods, including chromosome conformation capture technologies and mouse models, they found that the inactive X chromosome is actually composed of two distinct lobes of inactive DNA. They also found that these lobes were separated by highly repetitive segments of DNA called “microsatellite repeats”. When these microsatellite repeats were removed, the bi-lobed chromosome structure vanished. In another finding of this study, a limited number of active genes in these lobes were separated by topologically associated domains (TADs), which are regions of the genome where DNA interactions frequently occur. This finding is significant because it suggests that TADs may organize gene expression in the inactive X chromosome, at least in neural progenitor cells.
“This is the most detailed molecular view we’ve been able to obtain of the DNA inside the Barr body,” said Dekker. “Under a microscope, the inactive X chromosome is very different than other chromosomes; it looks like a condensed, undefined, inactive ‘blob.’ Our study, using a range of experimental approaches including imaging and genomic methods, describes something else entirely: a highly organized and elaborate structure, rich in features that may silence or activate genes all along the chromosome.”
In the news: Read the press release from the University of Massachusetts Medical School here.
Reference: Structural organization of the inactive X chromosome in the mouse. Giorgetti L, Lajoie BR, Carter AC, Attia M, Zhan Y, Xu J, Chen CJ, Kaplan N, Chang HY, Heard E, Dekker J. Nature. 2016 July 28.
Bursting onto the Transcription Scene
Transcription occurs when a particular segment of DNA is expressed into RNA. This process appears to take place in intermittent bursts and has been observed in organisms ranging from bacteria to humans. Transcriptional bursting is a term that describes this highly variable occurrence. Enhancers are short regions of DNA that can significantly increase the likelihood that a gene will be transcribed. There are hundreds of thousands of enhancers in the human genome, many of which precisely regulate patterns of gene expression that are required for the differentiation and growth of cells and tissues. To better understand the relationship between transcriptional bursts and enhancers, Dr. Michael Levine, a 4D Nucleome grantee, used quantitative analysis and live-imaging methods in Drosophila embryos in real time. Using this model organism, they report that enhancers regulate the frequency of transcriptional bursts and that strong enhancers produce more bursts than weak ones. They also report that shared enhancers can drive coordinated bursting of two different reporter genes, suggesting the importance of chromosome architecture in control of gene expression.
In the news: Dynamic enhancer–promoter interactions for transcriptional bursting.
Reference: Enhancer Controls of Transcriptional Bursting. Fukaya T, Lim B, Levine M. Cell. 2016 June 9.
Keeping Chromosomes in the Loop
Chromosomes are compacted over 100-fold to form highly condensed structures during cell division, yet the active process of chromosome compaction into loops is not well understood. 4D Nucleome grantee Dr. Leonid Mirny, a professor of physics in MIT’s Institute for Medical Engineering and Sciences, and collaborators have developed model that explains how chromosomes condense into these compact structures. Using computer simulations of chromosomes, the team investigated whether loops could produce the compact chromosomes seen in dividing cells. Their work, reported in three papers published in Cell Reports, eLife, and Biophysical Journal, suggests that chromosome organization relies on proteins that act as molecular motors that pull strands of DNA into increasingly larger loops. The authors report that proteins thought to hold DNA together, cohesion and condensin, can also transform loosely tangled chromosomes into a series of small loops that condense each chromosome. This allows compacted chromosomes to remain segregated from one another. A similar model explains how chromosomes are organized when cells are not dividing, suggesting that this loop extrusion model helps to control which genes are expressed. “Nobody has ever directly observed this mechanism of loop extrusion. If it exists, it will solve lots of problems,” says Dr. Leonid Mirny. “We will know how chromosomes condense, how they segregate, how genes talk to enhancers. Lots of things can be solved by this mechanism.”
Compaction and segregation of sister chromatids via active loop extrusion. Goloborodko A, Imakaev MV, Marko JF, Mirny L. Elife. 2016 May 18.
Chromosome Compaction by Active Loop Extrusion. Goloborodko A, Marko JF, Mirny LA. Biophysical Journal. 2016 May 24.
Formation of Chromosomal Domains by Loop Extrusion. Fudenberg G, Imakaev M, Lu C, Goloborodko A, Abdennur N, Mirny LA. Cell Reports. 2016 May 31.
CRISPRainbow: Observing the Genome in Living Color
4D Nucleome investigators Drs. Thoru Pederson and David Grunwald, and colleagues, have developed a new technology called CRISPRainbow. While most researchers use CRISPR for editing genomes, Pederson, Grunwald and colleagues used CRISPR/Cas9 technology to tag specific locations of the genome, label these locations with fluorescent proteins, and track them in real time under a microscope. Existing technologies are only capable of following three genomic locations at a time in living cells, but CRISPRainbow allows researchers to tag and track up to seven different genomic locations in live cells. "Computers cooperating with spectral filters in the microscope read out combinations of colors and display them as a color that you request," explains Thoru Pederson, Ph.D. "For example, red and green can be yellow. Using the three primary colors and this approach, which is called computational coloring, we can generate an additional three colors.” A seventh label, white, is accomplished by combining all three primary colors. Fluorescent labels such as these are important for studying chromosome dynamics and movements of the genome, which may have important biological consequences.
Learn more about this study from the University of Massachusetts Medical School.
In the news: Read about this study in Genetic Engineering & Biotechnology News.
Reference: Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T. Nature Biotechnology. 2016 April 18.
This page last reviewed on March 21, 2023