Menu

Close

Live-cell imaging of marked chromosome regions reveals dynamics of mitotic chromosome resolution and compaction

John K Eykelenboom, Marek Gierlinski, Zuojun Yue, Nadia Hegarat, Hillary Pollard, Tatsuo Fukagawa, Helfrid Hochegger, Tomoyuki U Tanaka

Preprint posted on April 20, 2018 https://doi.org/10.1101/305391

and

Quantitative imaging of chromatin decompaction in living cells

Elisa Dultz, Roberta Mancini, Guido Polles, Pascal Vallotton, Frank Alber, Karsten Weis

Preprint posted on April 22, 2018 https://doi.org/10.1101/219253

It takes two to (un)tangle: Dual Lac/Tet operator arrays report on chromatin dynamics in live cells

Selected by Carmen Adriaens, Gautam Dey

By Gautam Dey and Carmen Adriaens

 

Interphase cell nuclei are compartmentalized into loose, transcribed euchromatin and compact, largely untranscribed heterochromatin. The consensus is that for a given gene to be transcribed, the active chromatin needs to decompact further through changes in the chromatin landscape. Another form of chromatin dynamics occurs during cell division. Indeed, to divide correctly, the DNA is neatly folded into an extreme state of compaction (the mitotic chromosome) after replication. In two recent independent studies, researchers have used a two-color live cell reporter system to study chromatid separation before and chromatin compaction during mitosis, and the process of decompaction upon transcriptional activation. Since the experimental setup is very similar, we will discuss these papers jointly and highlight the different insights obtained from this live cell imaging system.

 

The experimental setup

At a specific site in the genome, a lactose operon array (LacO) is integrated in the DNA. The repeats can be recognized by the Lac inhibitor protein (LacI), which is here overexpressed and tagged with a green fluorescent protein (GFP) to be visualized as a single dot per lacO in the cells. At another site in the DNA, a Tet operon, TetO, is integrated and its cognate protein (the Tet repressor, TetR) is tagged and visualized with the red fluorescent reporter mCherry. The two dots (green and red) can be followed using microscopy in live cells, to enable the monitoring and modeling of chromatin dynamics at high resolution in real time in different biological settings.

 

Figure 1: (A) Experimental set-up in human HT-1080 cells used by Eykelenboom et al. with sample images in (B), scale bar = 10 µm. (C) Experimental set-up in budding yeast used by Dultz et al. with sample images on the right. Taken from Figure 1 of Eykelenboom et al. 2018 and Figure 1 of Dultz et al. 2018 under Creative Commons CC-BY-4.0 licenses.

 

What are the papers about?

Eykelenboom et al. used CRISPR to integrate the Tet and Lac operator arrays separated by a 250 kb interval on one arm of chromosome 5 in HT-1080 human cells. Adding in a 4x-mCherry-tagged Tet repressor and an EGFP-tagged Lac repressor enabled them to follow dynamics of chromatid segregation and compaction throughout the unperturbed cell cycle in single cells. Imaging the 2 green and 2 red spots, representing the chromatids after replication, revealed 4 discernible states: non-resolved, partially resolved, resolved and compacted.

Surprisingly, cells begin to cycle between the partially resolved and unresolved states rather early in G2. This cycle is driven by an apparent antagonism between cohesin holding chromatids together on one hand, and WAPL trying to remove cohesin from the chromosome arms on the other. Exploiting a fortuitous side effect of the integration sites of their operator arrays, the authors showed that the timing of separation depends on the local cohesin concentration. Inhibiting topoisomerase II activity, and thereby preventing the resolution of DNA catenanes, caused partially resolved chromatids to revert to the unresolved state early in mitosis. Finally, the authors showed that condensin II depletion caused defects in sister chromatid separation, while condensin I depletion affected only the final compaction stage.

In a second paper, Dultz and colleagues used very similar experimental principles to study chromatin decompaction upon transcriptional activation. The authors integrated the LacO and TetO repeats on either side of the budding yeast GAL locus and studied chromatin dynamics by measuring distances between red and green dots in different conditions. In the absence of glucose and galactose, the locus is inactive, but derepressed. In the presence of glucose, the locus is actively repressed. In both scenarios, the distance between the dots was small, reflecting an inactive chromatin state. When, on the other hand, the cells were grown in or induced with galactose, the locus became highly active and the 3 linked genes (GAL7, GAL10 and GAL1) in the locus became transcribed. In this active state, the green and red dots lining the GAL locus on either side reversibly increased in distance, reminiscent of decompaction and transcriptional activation.

Next, the authors asked whether these observations are dependent specifically on either spatial or linear decompaction. Spatial decompaction is primarily mediated by post-translational modifications of the histones and hence manipulation of nucleosome-nucleosome interactions, whereas in linear decompaction the wrapping density changes through eviction and remodeling of nucleosomes at the site of transcription. They found that a change in histone modifications, and, by extension, inter-nucleosomal interactions, does not dramatically alter the decompaction dynamics. Conversely, both manipulation of transcription itself and nucleosomal density do.

These observations led to the hypothesis that decompaction of chromatin is principally mediated by the number of nucleosomes evicted. To test this, the authors set up an exogenous reporter system 10 kb upstream of the original locus and measured changes in distances between dots when no, one, or two open reading frames were present. In this way, they found that the extent of decompaction indeed correlates with the length of the transcribed region. Finally, the authors concluded that although decompaction is transcription dependent, the inverse isn’t necessarily true: cells can continue to transcribe the GAL locus even in the absence of significant changes in distances between the two reporter dots.

 

Takeaways and questions for the future

It’s a very exciting time for the field of chromatin reorganization and dynamics! To take just one example, new experimental approaches have validated an old molecular model for condensin function1 and mapped a pathway to mitotic chromosome organization mediated by condensin I and II2. In parallel, a proliferation of live cell reporters have uncovered layers of cell cycle regulation that were hitherto invisible to bulk assays3,4. Taken together, these advances represent the perfect storm to preface an analysis of chromosome dynamics in single cells as they progress through the cell cycle.

The most surprising finding from the first paper (Eykelenboom et al.) is that chromosomes cycle between unresolved and partially resolved states through most of their G2 phase, driven by an apparent concentration-dependent antagonism between cohesin and WAPL.

What then is the purpose of this (futile?) cycle, and how is it broken during prophase? Saturated feedback loops of this type can be used to generate switch-like responses (also termed zero-order ultrasensitivity5). In turn, then, one could ask: is it necessary to have a switch-like transition between unresolved and resolved chromatids during prophase?

Moreover, the authors’ observations complement other recent work2 helping to resolve long-standing questions in the field about the individual and collective roles of the two condensin complexes.

It has long been recognized that actively transcribed chromatin is less compact than inactive chromatin, and much is known about the packaging and histone modifications responsible for these states. However, being able to visualize actual decompaction directly in live cells is impressive: Dultz et al. leverage the ultra-simplicity of the experimental setup to reveal the solution of the chicken-and-egg problem of loose chromatin during transcription.

Yet, there may be some caveats to these experiments as well: because the GAL locus is highly active when growing in galactose and the regulation of the system is rather unique compared to the rest of the yeast genome (ON/OFF), these observations may confer a local rather than a global rule. The physical decompaction (i.e. the increase in physical distance between the two arrays) in other loci may not be as striking as here, and will potentially be more difficult to study.

Furthermore, we note that both studies employ population averaging to draw certain conclusions, faced with high levels of cell-to-cell variability. Other recent work6 shows that chromosomal contacts observed by high-throughput sequencing approaches such as Hi-C occur as infrequently as in ~10% of cells only (as observed by microscopy), highlighting the extraordinary differences even within relatively homogenous or synchronized populations of cells. Additional work will be required to understand the sources of this variability and its functional consequences, if any.

 

References

  1. Ganji, M. et al. Real-time imaging of DNA loop extrusion by condensin. Science 360, 102–105 (2018).
  2. Gibcus, J. H. et al. A pathway for mitotic chromosome formation. Science 359, eaao6135 (2018).
  3. Chao, H. X. et al. Evidence that the cell cycle is a series of uncoupled, memoryless phases. bioRxiv 283614 (2018). doi:10.1101/283614
  4. Spencer, S. L. et al. The Proliferation-Quiescence Decision Is Controlled by a Bifurcation in CDK2 Activity at Mitotic Exit. Cell 155, 369–383 (2013).
  5. Ferrell, J. E. & Ha, S. H. Ultrasensitivity part I: Michaelian responses and zero-order ultrasensitivity. Trends Biochem. Sci. 39, 496–503 (2014).
  6. Finn, E. et al. Heterogeneity and Intrinsic Variation in Spatial Genome Organization. biorXiv (2017). doi: https://doi.org/10.1101/171801

 

Tags: cell cycle, chromatin dynamics, live cell imaging, teamwork makes the dream work, transcription

Posted on: 15th May 2018

(No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the bioengineering category:

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    Optical determination of absolute membrane potential

    Julia R. Lazzari-Dean, Anneliese M.M. Gest, Evan Miller



    Selected by James Marchant

    Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

    Mikhail H. Hanewich-Hollatz, Zhewei Chen, Jining Huang, et al.



    Selected by Pavithran Ravindran

    1

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Defining the design requirements for an assistive powered hand exoskeleton

    Quinn A Boser, Michael R Dawson, Jonathon S Schofield, et al.



    Selected by Joanna Cross

    Imaging mechanical properties of sub-micron ECM in live zebrafish using Brillouin microscopy

    Carlo Bevilacqua, Héctor Sánchez Iranzo, Dmitry Richter, et al.



    Selected by Stephan Daetwyler

    1

    DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

    Joshua A. Weinstein, Aviv Regev, Feng Zhang



    Selected by Theo Sanderson

    2

    Polyacrylamide Bead Sensors for in vivo Quantification of Cell-Scale Stress in Zebrafish Development

    Nicole Traeber, Klemens Uhlmann, Salvatore Girardo, et al.



    Selected by Jacky G. Goetz

    SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

    Jocelyn Y. Kishi, Brian J. Beliveau, Sylvain W. Lapan, et al.



    Selected by Yen-Chung Chen

    HIF1-alpha expressing cells induce a hypoxic-like response in neighbouring cancer cells

    Hannah Harrison, Henry J Pegg, Jamie Thompson, et al.



    Selected by Anh Hoang Le

    Zebrafish as a model to investigate the effects of exercise in cancer

    Alexandra Yin, Nathaniel R. Campbell, Lee W. Jones, et al.



    Selected by Jacky G. Goetz

    A 3D model of human skeletal muscle innervated with stem cell-derived motor neurons enables epsilon-subunit targeted myasthenic syndrome studies

    Mohsen Afshar Bakooshli, Ethan S Lippmann, Ben Mulcahy, et al.



    Selected by Chris Demers

    Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

    Tsung-li Liu, Srigokul Upadhyayula, Daniel E Milkie, et al.



    Selected by Arnaud Monnard, Gautam Dey

    Also in the biophysics category:

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

    C. Favard, J. Chojnacki, P. Merida, et al.



    Selected by Amberley Stephens

    Microfluidic protein isolation and sample preparation for high resolution cryo-EM

    Claudio Schmidli, Stefan Albiez, Luca Rima, et al.



    Selected by David Wright

    ENDOSOMAL MEMBRANE TENSION CONTROLS ESCRT-III-DEPENDENT INTRA-LUMENAL VESICLE FORMATION

    Vincent Mercier, Jorge Larios, Guillaume Molinard, et al.



    Selected by Nicola Stevenson

    1

    Planar differential growth rates determine the position of folds in complex epithelia

    Melda Tozluoğlu, Maria Duda, Natalie J Kirkland, et al.

    AND

    Buckling of epithelium growing under spherical confinement

    Anastasiya Trushko, Ilaria Di Meglio, Aziza Merzouki, et al.



    Selected by Sundar Naganathan

    2

    Microtubules stabilize intercellular contractile force transmission during tissue folding



    Selected by Ivana Viktorinová

    Dynamic Aha1 Co-Chaperone Binding to Human Hsp90

    Javier Oroz, Laura J Blair, Markus Zweckstetter



    Selected by Reid Alderson

    1

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Mechanical Stretch Kills Transformed Cancer Cells

    Ajay Tijore, Mingxi Yao, Yu-Hsiu Wang, et al.



    Selected by Joseph Jose Thottacherry

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Force inference predicts local and tissue-scale stress patterns in epithelia

    Weiyuan Kong, Olivier Loison, Pruthvi Chavadimane Shivakumar, et al.



    Selected by Sundar Naganathan

    Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision

    Simao Coelho, Jongho Baek, Matthew S Graus, et al.



    Selected by Lars Hubatsch

    Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

    Edoardo Salladini, Claire Debarnot, Vincent Delauzun, et al.



    Selected by Tessa Sinnige

    Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision

    Simao Coelho, Jongho baek, Matthew S Graus, et al.



    Selected by Sam Barnett

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    Also in the cell biology category:

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

    Shih-Jui Hsu, Emily C. Stow, James R. Simmons, et al.



    Selected by Maiko Kitaoka

    1

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

    Aviel Even, Giovanni Morelli, Chiara Scaramuzzino, et al.



    Selected by Stephen Royle

    1

    HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

    C. Favard, J. Chojnacki, P. Merida, et al.



    Selected by Amberley Stephens

    Hepatocyte-specific deletion of Pparα promotes NASH in the context of obesity

    Marion Regnier, Arnaud Polizzi, Sarra Smati, et al.



    Selected by Pablo Ranea Robles

    Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases

    King Faisal Yambire, Lorena Fernandez-Mosquera, Robert Steinfeld, et al.



    Selected by Sandra Franco Iborra

    1

    Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

    Lauren Saunders, Abhishek Mishra, Andrew J Aman, et al.



    Selected by Hannah Brunsdon

    1

    Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

    Masashi Yukawa, Masaki Okazaki, Yasuhiro Teratani, et al.



    Selected by I. Bouhlel

    A pair of E3 ubiquitin ligases compete to regulate filopodial dynamics and axon guidance

    Nicholas P Boyer, Laura E McCormick, Fabio L Urbina, et al.



    Selected by Angika Basant

    1

    SorCS1-mediated Sorting of Neurexin in Dendrites Maintains Presynaptic Function

    Luis Filipe Ribeiro, Ben Verpoort, Julie Nys, et al.



    Selected by Carmen Adriaens

    1

    ENDOSOMAL MEMBRANE TENSION CONTROLS ESCRT-III-DEPENDENT INTRA-LUMENAL VESICLE FORMATION

    Vincent Mercier, Jorge Larios, Guillaume Molinard, et al.



    Selected by Nicola Stevenson

    1

    Microtubules stabilize intercellular contractile force transmission during tissue folding



    Selected by Ivana Viktorinová

    Synthetic pluripotent bacterial stem cells

    Sara Molinari, David L. Shis, James Chappell, et al.



    Selected by Lorenzo Lafranchi

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Also in the molecular biology category:

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Slide-seq: A Scalable Technology for Measuring Genome-Wide Expression at High Spatial Resolution

    Samuel G Rodriques, Robert R Stickels, Aleksandrina Goeva, et al.

    AND

    High-density spatial transcriptomics arrays for in situ tissue profiling

    Sanja Vickovic, Goekcen Eraslan, Johanna Klughammer, et al.



    Selected by Carmen Adriaens

    Optical determination of absolute membrane potential

    Julia R. Lazzari-Dean, Anneliese M.M. Gest, Evan Miller



    Selected by James Marchant

    MicroRNA-mediated control of developmental lymphangiogenesis

    Hyun Min Jung, Ciara Hu, Alexandra M Fister, et al.



    Selected by Rudra Nayan Das

    Microfluidic protein isolation and sample preparation for high resolution cryo-EM

    Claudio Schmidli, Stefan Albiez, Luca Rima, et al.



    Selected by David Wright

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Structures of the Otopetrin Proton Channels Otop1 and Otop3

    Kei Saotome, Bochuan Teng, Che Chun (Alex) Tsui, et al.



    Selected by David Wright

    Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

    Evgeny Zatulovskiy, Daniel F. Berenson, Benjamin R. Topacio, et al.



    Selected by Zaki Ahmad

    1

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

    Anushila Chatterjee, Cydney N Johnson, Phat Luong, et al.



    Selected by Yasmin Lau

    On-site ribosome remodeling by locally synthesized ribosomal proteins in axons

    Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, et al.



    Selected by Srivats Venkataramanan

    MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

    Emily Yun-Chia Chang, James P Wells, Shu-Huei Tsai, et al.



    Selected by Katie Weiner

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Unlimited genetic switches for cell-type specific manipulation

    Jorge Garcia-Marques, Ching-Po Yang, Isabel Espinosa-Medina, et al.



    Selected by Rafael Almeida

    1

    Close