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.
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.
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.
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.
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.
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.
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.
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.