DNA loop extrusion by human cohesin

Science ◽  
2019 ◽  
Vol 366 (6471) ◽  
pp. 1338-1345 ◽  
Author(s):  
Iain F. Davidson ◽  
Benedikt Bauer ◽  
Daniela Goetz ◽  
Wen Tang ◽  
Gordana Wutz ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Atpase Activity ◽  
Chromatin Structure ◽  
Adenosine Triphosphatase ◽  
Gene Recombination ◽  
Loop Formation ◽  
Base Pairs ◽  
Topologically Associating Domains ◽  
Dna Loop ◽  
Eukaryotic Genomes

Eukaryotic genomes are folded into loops and topologically associating domains, which contribute to chromatin structure, gene regulation, and gene recombination. These structures depend on cohesin, a ring-shaped DNA-entrapping adenosine triphosphatase (ATPase) complex that has been proposed to form loops by extrusion. Such an activity has been observed for condensin, which forms loops in mitosis, but not for cohesin. Using biochemical reconstitution, we found that single human cohesin complexes form DNA loops symmetrically at rates up to 2.1 kilo–base pairs per second. Loop formation and maintenance depend on cohesin’s ATPase activity and on NIPBL-MAU2, but not on topological entrapment of DNA by cohesin. During loop formation, cohesin and NIPBL-MAU2 reside at the base of loops, which indicates that they generate loops by extrusion. Our results show that cohesin and NIPBL-MAU2 form an active holoenzyme that interacts with DNA either pseudo-topologically or non-topologically to extrude genomic interphase DNA into loops.

scholarly journals A major role for Eco1 in regulating cohesin-mediated mitotic chromosome folding

2019 ◽  
Author(s):  
Lise Dauban ◽  
Rémi Montagne ◽  
Agnès Thierry ◽  
Luciana Lazar-Stefanita ◽  
Olivier Gadal ◽  
...  
Keyword(s):  
Mitotic Chromosome ◽  
Dna Looping ◽  
Loop Formation ◽  
Dna Loops ◽  
Sister Chromatids ◽  
Topologically Associating Domains ◽  
Dna Loop ◽  
Main Barrier ◽  
Structural Maintenance Of Chromosomes ◽  
Mitotic Chromatin

AbstractUnderstanding how chromatin organizes spatially into chromatid and how sister chromatids are maintained together during mitosis is of fundamental importance in chromosome biology. Cohesin, a member of the Structural Maintenance of Chromosomes (SMC) complex family, holds sister chromatids together 1–3 and promotes long-range intra-chromatid DNA looping 4,5. These cohesin-mediated DNA loops are important for both higher-order mitotic chromatin compaction6,7 and, in some organisms, compartmentalization of chromosomes during interphase into topologically associating domains (TADs) 8,9. Our understanding of the mechanism(s) by which cohesin generates large DNA loops remains incomplete. It involves a combination of molecular partners and active expansion/extrusion of DNA loops. Here we dissect the roles on loop formation of three partners of the cohesin complex: Pds5 10, Wpl1 11 and Eco1 acetylase 12, during yeast mitosis. We identify a new function for Eco1 in negatively regulating cohesin translocase activity, which powers loop extrusion. In the absence of negative regulation, the main barrier to DNA loop expansion appears to be the centromere. Those results provide new insights on the mechanisms regulating cohesin dependent DNA looping.

scholarly journals CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression

2020 ◽  
Author(s):  
Martin Franke ◽  
Elisa de la Calle-Mustienes ◽  
Ana Neto ◽  
Rafael Acemel ◽  
Juan Tena ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Long Range ◽  
Chromatin Structure ◽  
Ctcf Binding ◽  
Developmental Gene ◽  
Developmental Genes ◽  
Chromatin Interactions ◽  
Topologically Associating Domains ◽  
Regulatory Landscapes

Abstract CTCF is an 11-zinc-finger DNA-binding protein that acts as a transcriptional repressor and insulator as well as an architectural protein required for 3D genome folding. CTCF mediates long-range chromatin looping and is enriched at the boundaries of topologically associating domains, which are sub-megabase chromatin structures that are believed to facilitate enhancer-promoter interactions within regulatory landscapes. Although CTCF is essential for cycling cells and developing embryos, its in vitro removal has only modest effects over gene expression, challenging the concept that CTCF-mediated chromatin interactions and topologically associated domains are a fundamental requirement for gene regulation. Here we link the loss of chromatin structure and gene regulation in an in vivo model and during animal development. We generated a ctcf knockout mutant in zebrafish that allows us to monitor the effect of CTCF loss of function during embryo patterning and organogenesis. CTCF absence leads to loss of chromatin structure in zebrafish embryos and affects the expression of thousands of genes, including many developmental genes. In addition, chromatin accessibility, both at CTCF binding sites and cis-regulatory elements, is severely compromised in ctcf mutants. Probing chromatin interactions from developmental genes at high resolution, we further demonstrate that promoters fail to fully establish long-range contacts with their associated regulatory landscapes, leading to altered gene expression patterns and disruption of developmental programs. Our results demonstrate that CTCF and topologically associating domains are essential to regulate gene expression during embryonic development, providing the structural basis for the establishment of developmental gene regulatory landscapes.

scholarly journals CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression

2020 ◽  
Author(s):  
Martin Franke ◽  
Elisa De la Calle-Mustienes ◽  
Ana Neto ◽  
Rafael D. Acemel ◽  
Juan J. Tena ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Long Range ◽  
Chromatin Structure ◽  
Ctcf Binding ◽  
Developmental Gene ◽  
Developmental Genes ◽  
Chromatin Interactions ◽  
Topologically Associating Domains ◽  
Regulatory Landscapes

CTCF is an 11-zinc-finger DNA-binding protein that acts as a transcriptional repressor and insulator as well as an architectural protein required for 3D genome folding1–5. CTCF mediates long-range chromatin looping and is enriched at the boundaries of topologically associating domains, which are sub-megabase chromatin structures that are believed to facilitate enhancer-promoter interactions within regulatory landscapes 6–12. Although CTCF is essential for cycling cells and developing embryos13,14, its in vitro removal has only modest effects over gene expression5,15, challenging the concept that CTCF-mediated chromatin interactions and topologically associated domains are a fundamental requirement for gene regulation16–18. Here we link the loss of chromatin structure and gene regulation in an in vivo model and during animal development. We generated a ctcf knockout mutant in zebrafish that allows us to monitor the effect of CTCF loss of function during embryo patterning and organogenesis. CTCF absence leads to loss of chromatin structure in zebrafish embryos and affects the expression of thousands of genes, including many developmental genes. In addition, chromatin accessibility, both at CTCF binding sites and cis-regulatory elements, is severely compromised in ctcf mutants. Probing chromatin interactions from developmental genes at high resolution, we further demonstrate that promoters fail to fully establish long-range contacts with their associated regulatory landscapes, leading to altered gene expression patterns and disruption of developmental programs. Our results demonstrate that CTCF and topologically associating domains are essential to regulate gene expression during embryonic development, providing the structural basis for the establishment of developmental gene regulatory landscapes.

The thermodynamics of DNA loop formation, from J to Z

2013 ◽  
Vol 41 (2) ◽  
pp. 513-518 ◽  
Author(s):  
Stephen D. Levene ◽  
Stefan M. Giovan ◽  
Andreas Hanke ◽  
Massa J. Shoura
Keyword(s):  
Dna Looping ◽  
Biological Processes ◽  
Loop Formation ◽  
Base Pairs ◽  
Dna Molecule ◽  
Dna Loop ◽  
Sensitive Function ◽  
Statistical Mechanical ◽  
Single Dna Molecule ◽  
Dna Cyclization

The formation of DNA loops is a ubiquitous theme in biological processes, including DNA replication, recombination and repair, and gene regulation. These loops are mediated by proteins bound at specific sites along the contour of a single DNA molecule, in some cases many thousands of base pairs apart. Loop formation incurs a thermodynamic cost that is a sensitive function of the length of looped DNA as well as the geometry and elastic properties of the DNA-bound protein. The free energy of DNA looping is logarithmically related to a generalization of the Jacobson–Stockmayer factor for DNA cyclization, termed the J factor. In the present article, we review the thermodynamic origins of this quantity, discuss how it is measured experimentally and connect the macroscopic interpretation of the J factor with a statistical-mechanical description of DNA looping and cyclization.

Localization of Adenosine Triphosphatase Activity in the Phleom of Nicotiana Tabacum

1972 ◽  
Vol 30 ◽  
pp. 230-231
Author(s):  
James Cronshaw ◽  
Jamison E. Gilder
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Higher Plants ◽  
High Surface ◽  
Root Tip ◽  
Animal Cells ◽  
Physiological Processes ◽  
Tip Cells ◽  
Atpase Localization

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.

scholarly journals No major thermogenic role for (Na+ + K+)-dependent adenosine triphosphatase apparent in hepatocytes from hyperthyroid rats

1982 ◽  
Vol 202 (3) ◽  
pp. 661-665 ◽  
Author(s):  
D G Clark ◽  
M Brinkman ◽  
O H Filsell ◽  
S J Lewis ◽  
M N Berry
Keyword(s):  
Oxygen Consumption ◽  
Oxygen Uptake ◽  
Atpase Activity ◽  
Heat Production ◽  
Adenosine Triphosphatase ◽  
Heat Output ◽  
Liver Parenchymal ◽  
Dependent Atpase ◽  
Parenchymal Cells ◽  
Isolated Liver

(Na+ + K+)-dependent ATPase activity, heat production and oxygen consumption were increased by 59%, 62% and 75% respectively in hepatocytes from tri-iodothyronine-treated rats. Ouabain at concentrations of 1 and 10 mM decreased oxygen uptake by 2-8% in hepatocytes from euthyroid rats and by 5-15% in hepatocytes from hyperthyroid animals. Heat output was decreased by 4-9% with the glycoside in isolated liver parenchymal cells from the control animals and by 11% in the cells from the tri-iodothyronine-treated animals. These results do not support the hypothesis that hepatic (Na+ + K+)-ATPase plays a major role in increased heat production in hepatocytes from hyperthyroid rats.

scholarly journals Protein-Mediated DNA Loop Formation in Intrinsically Bent Substrates

2012 ◽  
Vol 102 (3) ◽  
pp. 71a
Author(s):  
Joel D. Revalee ◽  
Henry D. Wilson ◽  
Jens-Christian Meiners
Keyword(s):  
Loop Formation ◽  
Dna Loop

Effect of high salt intake on sodium, potassium-dependent adenosine triphosphatase activity in the erythrocytes of normotensive men

1988 ◽  
Vol 75 (2) ◽  
pp. 167-170 ◽  
Author(s):  
Antonio P. Quintanilla ◽  
Maria I. Weffer ◽  
Haengil Koh ◽  
Mohammed Rahman ◽  
Agostino Molteni ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Inorganic Phosphate ◽  
Adenosine Triphosphatase ◽  
Salt Intake ◽  
Control Period ◽  
Plasma Renin ◽  
Normal Diet ◽  
High Salt ◽  
Sodium Potassium ◽  
High Salt Intake

1. We measured ouabain-insensitive adenosine triphosphatase (ATPase), sodium, potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) and intracellular Na+ and K+ in the erythrocytes of 19 healthy volunteers, before and after supplementation of their normal diet with 6.0–8.9 g of salt (102–137 mmol of NaCl) per day, for 5 days. 2. The subjects had a small but significant gain in weight. Mean plasma renin activity decreased from 1.57 to 0.73 pmol of angiotensin I h−1 ml−1 and plasma aldosterone from 0.46 to 0.24 nmol/l. 3. Total ATPase activity fell from 197.9 nmol of inorganic phosphate h−1 mg−1 during the control period to 173.5 during the high-salt period (P < 0.0125). Na+,K+-ATPase activity fell from 162.2 to 141.4 nmol of inorganic phosphate h−1 mg−1 (P < 0.05). Intracellular Na + and intracellular K+ did not change. 4. These results are consistent with the hypothesis that salt-induced volume expansion causes the release of a factor inhibitory to the Na+ pump.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

scholarly journals Partial diploids of Escherichia coli carrying normal and mutant alleles affecting oxidative phosphorylation

1977 ◽  
Vol 162 (3) ◽  
pp. 665-670 ◽  
Author(s):  
F Gibson ◽  
G B Cox ◽  
J A Downie ◽  
J Radik
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Quenching ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Growth Yields ◽  
Normal Allele ◽  
Adenosine Triphosphatase Activity

A plasmid was isolated which included the region of the Escherichia coli chromosome carrying the known genes concerned with oxidative phosphorylation (unc genes). This plasmid was used to prepare partial diploids carrying normal unc alleles on the episome and one of the three mutant alleles (unc A401, uncB402 or unc-405) on the chromosome. These strains were compared with segregants from which the plasmid had been lost. Dominance of either normal ormutant unc alleles was determined by growth on succinate, growth yields on glucose, Mg-ATPase (Mg2+-stimulated adenosine triphosphatase) activity, atebrin-fluorescence quenching, ATP-dependent transhydrogenase activity and oxidative phosphorylation. In all the above tests, dominance of the normal allele was observed. However, in membranes from the diploid strains which carried a normal allele and either of the mutant alleles affecting Mg-ATPase activity (uncA401 or unc-405), the energy-linked functions were only partially restored.

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