Friday, April 20 8:30AM – 4:30PM Prentice Women’s Hospital, Conference Room L Keynote “Illuminating Biology at the Nanoscale and Systems Scale by Imaging” Xiaowei Zhuang, PhD Howard Hughes Medical Institute
The seven-year, $6.4 million grant supports leaders who have made significant contributions in cancer research and are pursuing areas with unusual potential to move the field forward. Chandel, whose work focuses on cellular organelles called mitochondria, will be exploring the mechanisms of mitochondrial metabolism that contribute to tumor formation and investigating related enzymes that may be targeted for future therapies.
“In the last five to ten years, the idea that the metabolism of cancer cells might be different than the metabolism of normal cells has emerged,” he said. “We believe that mitochondrial metabolism is central to tumorigenesis. If that’s true, we have to figure out how that works.”
Mitochondria are known for their ability to produce energy by making a molecule called adenosine triphosphate (ATP). Chandel’s lab has demonstrated that the organelles also have other important responsibilities, such as generating molecules called reactive oxygen species (ROS) that support cell proliferation and adaptation to hypoxia. Thus ROS can activate signaling inside cancer cells that leads to tumor growth, an interaction Chandel plans to study with the support of the new grant.
“We’re going to use CRISPR technology to conduct forward genetic screens in mammalian cells to explain mitochondrial biology in the context of cancer,” he explained.
In previous research, published in the journal eLife, Chandel’s group also showed that metformin, a widely used drug used to treat type II diabetes, can inhibit mitochondrial complex I and reduce human cancer cell growth in mouse models.
“We developed tools that uncovered that this drug can be repurposed as an anticancer agent,” Chandel said. “With the NCI’s award, we’ll continue these studies.”
Image shown above DNA loops help to keep local regions of the genome together. (M. Imakaev/G. Fudenberg/N. Naumova/J. Dekker/L. Mirny)
Leonid Mirny swivels in his office chair and grabs the power cord for his laptop. He practically bounces in his seat as he threads the cable through his fingers, creating a doughnut-sized loop. “It’s a dynamic process of motors constantly extruding loops!” says Mirny, a biophysicist here at the Massachusetts Institute of Technology in Cambridge.
He argues that DNA is constantly being slipped through ring-like motor proteins to make loops. This process, called loop extrusion, helps to keep local regions of DNA together, disentangling them from other parts of the genome and even giving shape and structure to the chromosomes.
Scientists have bandied about similar hypotheses for decades, but Mirny’s model, and a similar one championed by Erez Lieberman Aiden, a geneticist at Baylor College of Medicine in Houston, Texas, add a new level of molecular detail at a time of explosive growth for research into the 3D structure of the genome. The models neatly explain the data flowing from high-profile projects on how different parts of the genome interact physically — which is why they’ve garnered so much attention.
But these simple explanations are not without controversy. Although it has become increasingly clear that genome looping regulates gene expression, possibly contributing to cell development and diseases such as cancer, the predictions of the models go beyond what anyone has ever seen experimentally.
For one thing, the identity of the molecular machine that forms the loops remains a mystery. If the leading protein candidate acted like a motor, as Mirny proposes, it would guzzle energy faster than it has ever been seen to do. “As a physicist friend of mine tells me, ‘This is kind of the Higgs boson of your field’,” says Mirny; it explains one of the deepest mysteries of genome biology, but could take years to prove.
And although Mirny’s model is extremely similar to Lieberman Aiden’s — and the differences esoteric — sorting out which is right is more than a matter of tying up loose ends. If Mirny is correct, “it’s a complete revolution in DNA enzymology”, says Kim Nasmyth, a leading chromosome researcher at the University of Oxford, UK. What’s actually powering the loop formation, he adds, “has got to be the biggest problem in genome biology right now”.
Geneticists have known for more than three decades that the genome forms loops, bringing regulatory elements into close proximity with genes that they control. But it was unclear how these loops formed.
Several researchers have independently put forward versions of loop extrusion over the years. The first was Arthur Riggs, a geneticist at the Beckman Research Institute of City of Hope in Duarte, California, who first proposed what he called “DNA reeling” in an overlooked 1990 report1. Yet it’s Nasmyth who is most commonly credited with originating the concept.
As he tells it, the idea came to him in 2000, after a day spent mountain climbing in the Italian Alps. He and his colleagues had recently discovered the ring-like shape of cohesin2, a protein complex best known for helping to separate copies of chromosomes during cell division. As Nasmyth fiddled with his climbing gear, it dawned on him that chromosomes might be actively threaded through cohesin, or the related complex condensin, in much the same way as the ropes looped through his carabiners. “It appeared to explain everything,” he says.
Nasmyth described the idea in a few paragraphs in a massive, 73-page review article3. “Nobody took notice whatsoever,” he says — not even John Marko, a biophysicist at Northwestern University in Evanston, Illinois, who more than a decade later developed a mathematical model that complemented Nasmyth’s verbal argument4.
Mirny joined this loop-modelling club around five years ago. He wanted to explain data sets compiled by biologist Job Dekker, a frequent collaborator at the University of Massachusetts Medical School in Worcester. Dekker had been looking at physical interactions between different spots on chromosomes using a technique called Hi-C, in which scientists sequence bits of DNA that are close to one another and produce a map of each chromosome, usually depicted as a fractal-like chessboard. The darkest squares along the main diagonal represent spots of closest interaction.
The Hi-C snapshots that Dekker and his collaborators had taken revealed distinct compartmentalized loops, with interactions happening in discrete blocks of DNA between 200,000 and 1 million letters long5.
These ‘topologically associating domains’, or TADs, are a bit like the carriages on a crowded train. People can move about and bump into each other in the same carriage, but they can’t interact with passengers in adjacent carriages unless they slip between the end doors. The human genome may be 3 billion nucleotides long, but most interactions happen locally, within TADs.
Mirny and his team had been labouring for more than a year to explain TAD formation using computer simulations. Then, as luck would have it, Mirny happened to attend a conference at which Marko spoke about his then-unpublished model of loop extrusion. (Marko coined the term, which remains in use today.) It was the missing piece of Mirny’s puzzle. The researchers gave loop extrusion a try, and it worked. The physical act of forming the loops kept the local domains well organized. The model reproduced many of the finer-scale features of the Hi-C maps.
When Mirny and his colleagues posted their finished manuscript on the bioRxiv preprint server in August 2015, they were careful to describe the model in terms of a generic “loop-extruding factor”. But the paper didn’t shy away from speculating as to its identity: cohesin was the driving force behind the looping process for cells not in the middle of dividing, when chromosomes are loosely packed6. Condensin, they argued in a later paper, served this role during cell division, when the chromosomes are tightly wound7.
A key clue was the protein CTCF, which was known to interact with cohesin at the base of each loop of uncondensed chromosomes. For a long time, researchers had assumed that loops form on DNA when these CTCF proteins bump into one another at random and lock together. But if any two CTCF proteins could pair, why did loops form only locally, and not between distant sites?
Mirny’s model assumes that CTCFs act as stop signs for cohesin. If cohesin stops extruding DNA only when it hits CTCFs on each side of a growing loop, it will naturally bring the proteins together.
But singling out cohesin was “a big leap of faith”, says biophysicist Geoff Fudenberg, who did his PhD in Mirny’s lab and is now at the University of California, San Francisco. “No one has seen these motors doing these things in living cells or even in vitro,” he says. “But we see all of these different features of the data that line up and can be unified under this principle.”
Experiments had shown, for example, that reducing the amount of cohesin in a cell results in the formation of fewer loops8. Overactive cohesin creates so many loops that chromosomes smush up into structures that resemble tiny worms9.
The authors of these studies had trouble making sense of their results. Then came Mirny’s paper on bioRxiv. It was “the first time that a preprint has really changed the way people were thinking about stuff in this field”, says Matthias Merkenschlager, a cell biologist at the MRC London Institute of Medical Sciences. (Mirny’s team eventually published the work in May 2016, in Cell Reports6.)
Lieberman Aiden says that the idea of loop extrusion first dawned on him during a conference call in March 2015. He and his former mentor, geneticist Eric Lander of the Broad Institute in Cambridge, Massachusetts, had published some of the most detailed, high-resolution Hi-C maps of the human genome available at the time10.
During his conference call, Lieberman Aiden was trying to explain a curious phenomenon in his data. Almost all the CTCF landing sites that anchored loops had the same orientation. What he realized was that CTCF, as a stop sign for extrusion, had inherent directionality. And just as motorists race through intersections with stop signs facing away from them, so a loop-extruding factor goes through CTCF sites unless the stop sign is facing the right way.
His lab tested the model by systematically deleting and flipping CTCF-binding sites, and remapping the chromosomes with Hi-C. Time and again, the data fitted the model. The team sent its paper for review in July 2015 and published the findings three months later11.
Mirny’s August 2015 bioRxiv paper didn’t have the same level of experimental validation, but it did include computer simulations to explain the directional bias of CTCF. In fact, both models make essentially the same predictions, leading some onlookers to speculate on whether Mirny seeded the idea. Lieberman Aiden insists that he came up with his model independently. “We submitted our paper before I ever saw their manuscript,” he says.
There are some tiny differences. The cartoons Mirny uses to describe his model seem to suggest that one cohesin ring does the extruding, whereas Lieberman Aiden’s contains two rings, connected like a pair of handcuffs (see ‘The taming of the tangles’). Suzana Hadjur, a cell biologist at University College London, calls this mechanistic nuance “absolutely fundamental” to determining cohesin’s role in the extrusion process.
Neither Lieberman Aiden nor Mirny say they have a strong opinion on whether the system uses one ring or two, but they do differ on cohesin’s central contribution to loop formation. Mirny maintains that the protein is the power source for looping, whereas Lieberman Aiden summarily dismisses this idea. Cohesin “is a big doughnut”, he says. It doesn’t do that much. “It can open and close, but we are very, very confident that cohesin itself is not a motor.”
Instead, he suspects that some other factor is pushing cohesin around, and many in the field agree. Claire Wyman, a molecular biophysicist at Erasmus University Medical Centre in Rotterdam, the Netherlands, points out that cohesin is only known to consume small amounts of energy for clasping and releasing DNA, so it’s a stretch to think of it motoring along the chromosome at the speeds required for Mirny’s model to work. “I’m willing to concede that it’s possible,” she says. “But the Magic 8-Ball would say that, ‘All signs point to no’.”
One group of proteins that might be doing the pushing is the RNA polymerases, the enzymes that create RNA from a DNA template. In a study online in Nature this week12, Jan-Michael Peters, a chromosome biologist at the Research Institute of Molecular Pathology in Vienna, and his colleagues show that RNA polymerases can move cohesin over long distances on the genome as they transcribe genes into RNA. “RNA polymerases are one type of motor that could contribute to loop extrusion,” Peters says. But, he adds, the data indicate that it cannot be the only force at play.
Frank Uhlmann, a biochemist at the Francis Crick Institute in London, offers an alternative that doesn’t require a motor protein at all. In his view, a cohesin complex might slide along DNA randomly until it hits a CTCF site and creates a loop. This model requires only nearby strands of DNA to interact randomly — which is much more probable, Uhlmann says. “We do not need to make any assumptions about activities that we don’t have experimental evidence for.”
Researchers are trying to gather experimental evidence for one model or another. At the Lawrence Livermore National Laboratory in California, for example, biophysicist Aleksandr Noy is attempting to watch loop extrusion in action in a test tube. He throws in just three ingredients: DNA, some ATP to provide energy, and the bacterial equivalent of cohesin and condensin, a protein complex known as SMC.
“We see evidence of DNA being compacted into these kinds of flowers with loops,” says Noy, who is collaborating with Mirny on the project. That suggests that SMC — and by extension cohesin — might have a motor function. But then again, it might not. “The truth is that we just don’t know at this point,” Noy says.
The experiment that perhaps comes the closest to showing cohesin acting as a motor was published in February13. David Rudner, a bacterial cell biologist at Harvard Medical School in Boston, Massachusetts, and his colleagues made time-lapse Hi-C maps of the bacterium Bacillus subtilis that reveal SMC zipping along the chromosome and creating a loop at a rate of more than 50,000 DNA letters per minute. This tempo is on par with what researchers estimate would be necessary for Mirny’s model to work in human cells as well.
Rudner hasn’t yet proved that SMC uses ATP to make that happen. But, he says, he’s close — and he would be “shocked” if cohesin worked differently in human cells.
For now, the debate rages about what cohesin is, or is not, doing inside the cell — and many researchers, including Doug Koshland, a cell biologist at the University of California, Berkeley, insist that a healthy dose of scepticism is still warranted when it comes to Mirny’s idea. “I am worried that the simplicity and elegance of the loop-extrusion model is already filling textbooks, coronated long before its time,” he says.
And although it may seem an academic dispute among specialists, Mirny notes that if it his model is correct, it will have real-world implications. In cancer, for instance, cohesin is frequently mutated and CTCF sites altered. Defective versions of cohesin have also been implicated in several rare human developmental disorders. If the loop-extruding process is to blame, says Mirny, then perhaps a better understanding of the motor could help fix the problem.
But his main interest remains more fundamental. He just wants to understand why DNA is configured in the way it is. And although his model assumes a lot of things about cohesin, Mirny says, “The problem is that I don’t know any other way to explain the formation of these loops.”
Chuan He, a CR-PSOC investigator, was one of three recipients of the 2017 Paul Marks Prize for Cancer Research. The award, given by Memorial Sloan Kettering (MSK), recognizes promising investigators aged 45 or younger at the time of nomination for their efforts in advancing cancer research. He will receive an award of $50,000 and give a scientific presentation at a symposium held at MSK.
Dr. He is the John T. Wilson Distinguished Service Professor in Chemistry, Biochemistry, and Molecular Biology at the University of Chicago; director of U of C’s Institute for Biophysical Dynamics; and a Howard Hughes Medical Institute (HHMI) investigator. He is also director of the Synthetic and Functional Biomolecules Center at Peking University in China.
He is an expert in the field of cancer epigenetics and RNA modification biology. Epigenetics involves variations in the way that genes are expressed that don’t affect the actual DNA sequence. “The human genome contains 3 billion base pairs but only roughly 20,000 genes,” he says. “We have tens of trillions of cells, and about 200 different tissue or cell types. Epigenetics facilitates cell differentiation into different identities, despite having the same genetic sequence in an individual human being.”
His major contribution to the field is that he was the first to put forward the idea that modifications to RNA are reversible and can control gene expression. Control of RNA, the molecule that carries DNA’s “message” to the protein-making machinery of the cell, is one of the major ways that affects the outcomes of gene expression.
“When I started this work back in 2008 and 2009, we knew that proteins called writers could install modifications to RNA molecules that altered their function, but no one knew that there were also proteins called erasers that could undo these changes,” Dr. He explains. His team went on to identify for the first time the eraser proteins, and in later work characterized a series of reader proteins that explain how RNA methylation functioned.
“This research laid down the mechanistic pathways for our current understanding of how these modifications impact biological outcomes, including those related to cancer,” he says. “Cancer and other diseases can hijack aberrant RNA methylation to gain a survival advantage, allowing cells to proliferate and grow out of control.”
These types of RNA changes are known to play a role in many types of cancer, including endometrial cancer, acute myelogenous leukemia, and glioblastoma. Dr. He’s work forms some of the foundations for developing potential future therapies that target RNA methylation effectors against human cancer.
For the first time, researchers control cells’ chromatin to prevent cancer from adapting to treatment
Chicago Region Physical Sciences Oncology Center investigator Vadim Backman has developed an effective new strategy for treating cancer, which has wiped out the disease to near completion in cellular cultures in the laboratory.
The treatment works by controlling chromatin, a group of macromolecules — including DNA, RNA, and proteins — that houses genetic information within cells and determines which genes get suppressed or expressed. In the case of cancer, chromatin has the ability to regulate the capacity of cancer cells to find ways to adapt to treatment by expressing genes that allow the cancer cells to become resistant to treatment.
Backman’s solution alters chromatin’s structure in a way that prevents cancer from evolving to withstand treatment, making the disease an easier target for existing drugs. If the cells cannot evolve to resist chemotherapy, for example, they die. After having shown great potential to fight cancer in cellular cultures, the treatment is now undergoing studies in an animal model.
“If you think of genetics as hardware, then chromatin is the software,” said Backman, the Walter Dill Scott Professor of Biomedical Engineering at Northwestern’s McCormick School of Engineering. “Complex diseases such as cancer do not depend on the behavior of individual genes, but on the complex interplay among tens of thousands of genes. By targeting chromatin, we can modulate global patterns in gene expression.”
Cancer has many distinctive features, but one trait underlies them all: its relentless ability to survive. Even as it is bombarded by the immune system and treatments such as chemotherapy, immunotherapy, and radiation, cancer might shrink or slow its proliferation, but it rarely disappears.
“How does cancer jump through so many hoops to survive? It’s a highly improbable event, if you think about it,” Backman said. “But there is one thing that all cancers do. They have a phenomenal ability to change, to adapt, to evolve in order to evade the treacherous conditions they frequently have to face during the process of their growth or in the face of treatment.”
Backman’s goal does not lie in discovering new drugs or treatment options. Instead his team aims to stop cancer’s adaptive behavior to boost the effectiveness of current treatments. The key is in the chromatin.
Found in cells, chromatin is a disordered chain polymer that is packed together at different densities throughout a cell’s nucleus. Through a combination of imaging, simulations, systems modeling, and in vivo experiments, Backman’s team discovered that this packing-density of chromatin in cancer cells produced predictable changes in gene expression. The more heterogeneous and disordered the packing density, the more likely cancer cells were to survive, even in the face of chemotherapy. The more ordered and conservative the packing density, however, the more likely the cells would die from cancer treatment.
“Just by looking at the cell’s chromatin structure, we could predict whether or not it would survive,” Backman said. “Cells with normal chromatin structures die because they can’t respond; they can’t explore their genome in search of resistance. They can’t develop resistance.”
The study leverages an imaging technique developed in Backman’s laboratory last year. Called Partial Wave Spectroscopic (PWS) microscopy, the technique allows researchers to examine chromatin in living cells in real time. More importantly, researchers can use PWS to peer inside of chromatin at the never-before-seen 20-200 nanometer length scale, which is exactly where chromatin undergoes a transformation when cancer is formed. Backman also worked with Szleifer, an expert on molecular dynamics simulations, to model how chromatin reacted to different stimuli.
A “macro” approach
When scientists finished decoding the human genome in 2003, many thought the findings would help them design smarter medicines by either knocking out or turning on genes.
“As significant as changing a gene is, it’s only one part of the story,” Backman said. “The second part is that the gene has to be expressed, amplified, or suppressed. We have about 20,000 genes, and the number of genomic states that a cell can have is absolutely astronomical.”
The large number of possible combinations of genes and genomic states is so high that knocking out one gene most likely cannot prevent or fully treat a complex disease such as cancer. His laboratory, instead, takes a “macrogenomics” approach. By altering chromatin packing densities, Backman’s team can control the overall behavior of biological systems.
“Think of the number of mutations that have been documented in cancer. We’re talking about thousands,” Backman said. “Just changing one gene is not going to give you cancer, and it’s not going to cure cancer either. But we can rewrite the software by using chromatin engineering to manipulate the genetic code.”
“This work helps establish how the physical environment within the cell nucleus shapes gene expression patterns for many genes simultaneously,” said Luay Almassalha, graduate student in Backman’s laboratory and a first author on the paper. “Analogous to how the organization of a city shapes the collective behavior of thousands of people, the physiochemical rules of chromatin that we uncovered govern the collective behavior of the genome.”
Finding a way to alter chromatin
Throughout the study, Backman and his team realized that they could control chromatin by changing the electrolytes present in the cell’s nucleus. His team screened multiple existing drug compounds to find promising candidates that could alter the physical environment inside cell nuclei in order to modulate the spatial arrangement of chromatin packing density.
Two of the drugs the team pinpointed, Celecoxib and Digoxin, are FDA-approved immunological agents already on the market. Physicians prescribe them for arthritis and heart conditions, respectively, but both have a side effect of altering chromatin packing density.
Backman combined these compounds, which he calls “chromatin protection therapeutics,” or CPT compounds, with chemotherapy to treat seven different types of cancer in cell cultures. Monitoring the cancer cells with PWS, Backman saw “something remarkable.”
“Within two or three days, nearly every single cancer cell died because they could not respond,” he said. “The CPT compounds don’t kill the cells; they restructure the chromatin. If you block the cells’ ability to evolve and to adapt, that’s their Achilles’ heel.”
A new tool to fight disease
Though the treatment is promising, much more testing is needed before it can be part of cancer treatment in humans.
“There is a big difference between cell cultures and humans,” Backman added. “You never know how the environment inside the human body will affect cancer’s behavior or if there will be unforeseen side effects. But, having said that, in seven different types of cancer, we see the same story unfold each and every time. That’s very promising.”
If Backman’s team can alter chromatin in cancer, they believe that the same technique could potentially work in other complex diseases, such as Alzheimer’s disease and atherosclerosis. Chromatin could also be controlled to have the opposite effect — to make normal cells more plastic and adaptable to behave like stem cells. By prolonging the lives of stem cells or turning normal cells into stem cells, researchers might have a new tool to repair brain and spinal cord damage. Although he does not yet have the data, Backman cannot help but wonder about how this discovery could change the future of medicine.
“Genetic changes are permanent, but this type of modulation is more like software,” Backman said. “By definition, it’s reversible. You could reprogram a neuron, and then remove the stimulus and allow it to go back to its normal state. If chromatin is software, then we are saying there is room to write new codes.”
Thursday, September 28, 2017 at 5:30 PM
Pancoe-NSUHS Life Sciences Pavilion, Abbott Auditorium
2200 Campus Drive, Evanston, IL
Join us to discuss the next wave of cancer therapies.
Moderated panel discussion and an audience Q&A.
Jonathan D. Licht, MD
Director, University of Florida Health Cancer Center
Marshall E. Rinker, Sr. Foundation and David B. and Leighan R. Rinker Chair
Professor of Medicine, Biochemistry and Molecular Biology
University of Florida
Lucy A. Godley, MD, PhD
Professor of Medicine – Hematology/Oncology
Cancer Research Center, Committee on Cancer Biology
University of Chicago
Thomas Meade, PhD
Eileen M. Foell Professor of Cancer Research
Professor of Chemistry, Molecular Biosciences, Neurobiology, Biomedical Engineering, Radiology
Carole Baas, PhD
National Advocate, Physical Sciences-Oncology Network, Division of Cancer Biology, National Cancer Institute
Founding Editor, Convergent Science Physical Oncology
In the paper “Chromatin and lamin A determine two different mechanical response regimes of the cell nucleus”, recently published in the journal Molecular Biology of the Cell (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E16-09-0653), Dr. Andrew Stephens and collaborators from the Northwestern CR-PS-OC report experiments establishing differential roles played by the nuclear lamin proteins of the nuclear envelope and chromatin in the mechanics of the cell nucleus. By directly stretching nuclei of human cells, the team of researchers from the labs of Robert Goldman and John Marko at Northwestern University showed that chromatin – the chromosomes themselves – control the initial small-strain (< 30% lengthening) mechanics of the cell nucleus, with the lamin network coming into play for deformations of more than 30%. Dr. Ed Banigan showed that the experimental results are well-explained by a model in which the nuclear envelope is treated as a polymer “shell” and where the interior chromatin is modeled as a crosslinked polymer network.
The team also reports experiments showing that the state of the chromatin – whether it is more heterochromatic or euchromatic – affects nuclear stiffness. Given that the balance of heterochromatin and euchromatin, along with nuclear morphology and stability, are altered in many diseases, this new paper suggests that chromatin mechanics may play a much larger role in controlling nuclear stiffness and shape than has been previously appreciated.
Center for Theoretical Biological Physics and Departments of Physics and Astronomy, Chemistry, and Biosciences
Rice University, Houston, Texas
The Genetics and Biophysics of Epithelial-Mesenchymal Transition (EMT): Can Theory Help Cancer Biology?
Understanding epithelial-mesenchymal transitions during cancer metastasis remains a major challenge in modern biology. Thanks to the rapidly growing body of cell behavior observations and progress in mapping the key regulatory genetic networks associated with these decisions, it is now confirmed that the genetic network that regulates the epithelial-mesenchymal transitions is also able to create an epithelial-mesenchymal hybrid phenotype (E/M). These hybrid cells possess mixed epithelial and mesenchymal characteristics, enabling specialized capabilities such as collective cell migration. On the gene network level, it is now understood that the coexistence of and transitions between the different phenotypes are regulated by a decision unit composed of two highly interconnected chimeric modules: the miR-34/SNAI and the miR-200/ZEB mutual inhibition feedback circuits. A new tractable theoretical framework to model and decode the operating principles governing these decision units will be presented. This approach connects between the knowledge about intracellular pathways and observations of cellular behavior, and advances towards understanding the logic of cancer decision making. Finally we devise a mechanism-based theoretical model that links cell–cell communication via Notch-Delta-Jagged signaling with the regulation of EMT. We demonstrate that while both Notch-Delta and Notch-Jagged signaling can induce EMT in a population of cells, only Jagged-dominated Notch signaling, but not Delta-dominated signaling, can lead to the formation of clusters containing hybrid E/M cells.
Read the review from Nature Reviews Molecular Cell Biology below:
The positioning of nucleosomes along the DNA with regard to various genetic elements is thought to affect transcription by competing with transcription factors for binding DNA. Using a high-resolution chemical approach to map nucleosomes, Voong et al. provide new insights into the interplay between nucleosomes, transcription and splicing.
Looking to improve on the accuracy of the common nucleosome-mapping method MNase-seq, the authors developed a genome-wide nucleosome-mapping approach that determines nucleosome centre (dyad) positions at nucleotide resolution based on chemical cleavage of the DNA. The method requires substituting Ser47 of histone H4, which flanks the nucleosome centre with a Cys residue (H4S47C), which can be covalently bound by a copper-chelating compound. The copper ions direct cleavage of nucleosome DNA near the dyad by hydroxyl radicals, and the resulting DNA fragments are subjected to deep sequencing.
The authors substituted most of the endogenous H4 proteins in mouse embryonic stem (ES) cells with H4S47C. MNase-seq nucleosome maps generated in H4S47C and wild-type ES cells, as well as previously generated maps from different organisms, were generally in agreement. They showed the presence of nucleosome-depleted regions (NDRs) upstream of transcription start sites (TSSs) and at transcription termination sites (TTSs) in actively transcribed genes. By contrast, the chemical map revealed generally high nucleosome occupancy spanning the TSS, coding sequence and TTS of actively transcribed genes.
To investigate how nucleosome positioning correlates with RNA polymerase II (Pol II) elongation kinetics, the authors used an available global run-on and sequencing (GRO-seq) data set, from which sites of Pol II accumulation can be inferred. Alignment of Pol II accumulation at promoter-proximal sites with the chemically determined nucleosome positions revealed that occupancy of the +1 position relative to the TSS was positively associated with Pol II pausing.
The rate of transcription can influence co-transcriptional splicing. In contrast to recent studies, which found that nucleosomes have higher occupancy around exon centres, the chemical map revealed that nucleosomes are enriched at exon boundaries. Importantly, at all of the expressed genes (regardless of expression levels), Pol II accumulation correlated with nucleosome occupancy at exon boundaries, which is indicative of Pol II stalling at exon–intron junctions close to the nucleosome centre, where the strongest DNA–histone interactions occur.
It is still unclear whether pluripotency transcription factors can bind to their target sites when the DNA is bound by nucleosomes. The chemical map showed that the pluripotency factors OCT4, SOX2, Nanog and Krüppel-like factor 4 bind to their target sites within nucleosomes and modulate nucleosomes in the flanking regions. This suggests that they function as pioneer factors, which can induce chromatin opening and the formation of NDRs.
This study supports a dynamic function of nucleosome in gene regulation. At both promoter-proximal regions and exon–intron junctions, nucleosomes could function as transient barriers for Pol II progression, thereby regulating the kinetics of transcription elongation and splicing.
written by Eytan Zlotorynski | Published online 19 Dec 2016; doi:10.1038/nrm.2016.167
New nanoscale imaging technique allows researchers to study chromatin in live cells
When scientists finished decoding the human genome in 2003, they thought the findings would help us better understand diseases, discover genetic mutations linked to cancer, and lead to the design of smarter medicine. Now it’s 13 years later, and not all of these ideas have not yet come to fruition.
“We thought that understanding genes would answer all of our questions,” said Chicago-Region Physical Sciences Oncology Center investigator Vadim Backman. “But it’s not that simple.”It turns out that genes are just one piece of a much larger puzzle. To help put this puzzle together, Backman has developed a new way to image chromatin, a complex of macromolecules — including DNA, RNA, and proteins — within living cells that house genetic information and determines which genes get expressed.
Chromatin’s organization plays a major role in many molecular processes, including DNA transcription, replication, and repair. The structures within chromatin that regulate these processes span from nucleosomal (10 nanometers) to chromosomal (longer than 200 nanometers) length scales.
Backman’s imaging technique can capture nanoscopic information from dozens of nuclei within seconds, as seen here in cancer cells.
Little is known about chromatin’s dynamics between these length scales due to lack of imaging techniques. Because they require toxic fluorescent dyes to enhance contrast, previous techniques could not image chromatin in living cells without killing or perturbing the cells. Understanding this missing length scale is crucial, however, because it is the exact area where chromatin undergoes a transformation when cancer is formed.
“Changes in chromatin’s structure have been linked to the regulation in genes often implicated in cancer,” Backman said. “The organization of chromatin correlates both with the formation of tumors and their invasiveness. We want to understand how chromatin regulates these genes.”
Backman’s new imaging technique allows researchers to peer inside of chromatin at the missing, mysterious length scale (20-200 nanometers). Not only is the technique label-free, allowing researchers to study chromatin within unharmed, living cells, but it does so with high-throughput and at very low cost.
Supported by the National Science Foundation, National Institutes of Health, and Chicago Biomedical Consortium, the research is described online on October 4 in the Proceedings of the National Academy of Sciences. Luay Almassalha, Greta Bauer, John Chandler, and Scott Gladstein, all graduate students in Backman’s laboratory, served as co-first authors on the paper. Northwestern Engineering’s Igal Szleifer, the Christine Enroth-Cugell Professor of Biomedical Engineering, and Hariharan Subramanian, research assistant professor in biomedical engineering, also contributed to the work.
“Now we can look at live, healthy cells unperturbed and see their dynamic processes,” Almassalha said. “We can see how the chromatin is organized and how it responds to stimuli, such as drug treatments.”
Called live cell Partial Wave Spectroscopic (PWS) microscopy, the technique detects chromatin by using scattered light. Particles smaller than the diffraction limit of light cannot be visualized but their presence and organization can be sensed by analyzing the light they scatter. The approach can measure the nano-architecture in live cells within seconds, opening the door for large-scale screens. Researchers can run high-throughput screens on thousands of compounds and drugs, for example, and watch how they affect cells in real time.
“We know that chromatin is a major player in complex diseases,” Backman said. “We just haven’t had the techniques to study it. Now we can watch and record these dynamic processes as they unfold.”