Condensin compacts DNA in 15 nm steps and requires FACT for extrusion on chromatin

Abstract Condensin plays a central role in the organisation of chromosomes by compacting chromatin into loops during mitosis. Condensin achieves this through a loop extrusion mechanism that remains poorly understood. To identify the molecular steps of yeast condensin during loop formation, we used optical tweezers with fluorescence detection. We find that single yeast condensin complexes use ATP to extrude DNA through distinct 15 nm steps, thus advancing ~45 base pairs (bp) per step. Under increasing load, the condensin step size remains constant while step-dwell times increase, and stalls at forces >1 pN. We also show that nucleosome arrays hinder processive condensin extrusion and demonstrate that the histone chaperone FACT is required for compaction of nucleosomal arrays by condensin. Importantly, FACT-assisted compaction on nucleosomes also occurs through distinct 15 nm steps. Finally, we show that FACT is required for correct condensin localisation in vivo. Our results establish that loop extrusion by yeast condensin involves a 45 bp stroke that requires FACT for condensin function on chromatin.

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Condensin compacts DNA in 15 nm steps and requires FACT for extrusion on chromatin

10.1101/2021.09.15.460217 ◽

2021 ◽

Author(s):

Swathi Sudhakar ◽

Gemma LM Fisher ◽

Pilar Gutierrez Escribano ◽

Jonay Garcia Luis ◽

Erin Cutts ◽

...

Keyword(s):

Optical Tweezers ◽

Fluorescence Detection ◽

Histone Chaperone ◽

Loop Formation ◽

Step Size ◽

Base Pairs ◽

Extrusion Mechanism ◽

Increasing Load ◽

Nucleosome Arrays

Condensin plays a central role in the organisation of chromosomes by compacting chromatin into loops during mitosis. Condensin achieves this through a loop extrusion mechanism that remains poorly understood. To identify the molecular steps of yeast condensin during loop formation, we used optical tweezers with fluorescence detection. We find that single yeast condensin complexes use ATP to extrude DNA through distinct 15 nm steps, thus advancing ~45 base pairs (bp) per step. Under increasing load, the condensin step size remains constant while step-dwell times increase, and stalls at forces >1 pN. We also show that nucleosome arrays hinder processive condensin extrusion and demonstrate that the histone chaperone FACT is required for compaction of nucleosomal arrays by condensin. Importantly, FACT-assisted compaction on nucleosomes also occurs through distinct 15 nm steps. Finally, we show that FACT is required for correct condensin localisation in vivo. Our results establish that loop extrusion by yeast condensin involves a 45 bp stroke that requires FACT for condensin function on chromatin

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Single cardiac ventricular myosins are autonomous motors

Open Biology ◽

10.1098/rsob.170240 ◽

2018 ◽

Vol 8 (4) ◽

pp. 170240 ◽

Cited By ~ 12

Author(s):

Yihua Wang ◽

Chen-Ching Yuan ◽

Katarzyna Kazmierczak ◽

Danuta Szczesna-Cordary ◽

Thomas P. Burghardt

Keyword(s):

Actin Binding ◽

Control Force ◽

Cardiac Myosin ◽

Step Size ◽

Size Selection ◽

Essential Light Chain ◽

N Terminus ◽

Increasing Load

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.

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Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

Science Advances ◽

10.1126/sciadv.abd6030 ◽

2021 ◽

Vol 7 (11) ◽

pp. eabd6030

Author(s):

Isabel Strohkendl ◽

Fatema A. Saifuddin ◽

Bryan A. Gibson ◽

Michael K. Rosen ◽

Rick Russell ◽

...

Keyword(s):

Histone Modifications ◽

Dna Cleavage ◽

Genome Engineering ◽

Eukaryotic Cells ◽

Loop Formation ◽

Chromatin Compaction ◽

Dna Targeting ◽

Genomic Regions ◽

Dna Bendability ◽

Nucleosome Arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

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The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

Nature Communications ◽

10.1038/s41467-021-23505-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Evi Goulielmaki ◽

Maria Tsekrekou ◽

Nikos Batsiotos ◽

Mariana Ascensão-Ferreira ◽

Eleftheria Ledaki ◽

...

Keyword(s):

Dna Damage ◽

Rna Splicing ◽

Excision Repair ◽

Intron Retention ◽

Dna Lesions ◽

Splicing Factor ◽

Loop Formation ◽

Rna Targets ◽

Underlying Mechanisms

AbstractRNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

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Force generation by protein–DNA co-condensation

Nature Physics ◽

10.1038/s41567-021-01285-1 ◽

2021 ◽

Cited By ~ 1

Author(s):

Thomas Quail ◽

Stefan Golfier ◽

Maria Elsner ◽

Keisuke Ishihara ◽

Vasanthanarayan Murugesan ◽

...

Keyword(s):

Optical Tweezers ◽

Self Assembly ◽

Spider Silk ◽

Regulatory Elements ◽

Heterochromatin Formation ◽

First Order Phase Transition ◽

Dna Strand ◽

First Order Phase

AbstractInteractions between liquids and surfaces generate forces1,2 that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water3, modulation of the material properties of spider silk4 and self-assembly of microstructures5. Recent studies have shown that cells assemble biomolecular condensates via phase separation6. In the nucleus, these condensates are thought to drive transcription7, heterochromatin formation8, nucleolus assembly9 and DNA repair10. Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein–DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.

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siRNA, miRNA, and shRNA: in vivo Applications

Journal of Dental Research ◽

10.1177/154405910808701109 ◽

2008 ◽

Vol 87 (11) ◽

pp. 992-1003 ◽

Cited By ~ 48

Author(s):

P.N. Pushparaj ◽

J.J. Aarthi ◽

J. Manikandan ◽

S.D. Kumar

Keyword(s):

Metabolic Diseases ◽

Small Interfering Rnas ◽

Base Pairs ◽

Rna Molecules ◽

Rna Activation ◽

Research Findings ◽

Dental Diseases ◽

Short Hairpin Rnas ◽

Clinical Potential

RNA interference (RNAi), an accurate and potent gene-silencing method, was first experimentally documented in 1998 in Caenorhabditis elegans by Fire et al., who subsequently were awarded the 2006 Nobel Prize in Physiology/Medicine. Subsequent RNAi studies have demonstrated the clinical potential of synthetic small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) in dental diseases, eye diseases, cancer, metabolic diseases, neurodegenerative disorders, and other illnesses. siRNAs are generally from 21 to 25 base-pairs (bp) in length and have sequence-homology-driven gene-knockdown capability. RNAi offers researchers an effortless tool for investigating biological systems by selectively silencing genes. Key technical aspects—such as optimization of selectivity, stability, in vivo delivery, efficacy, and safety—need to be investigated before RNAi can become a successful therapeutic strategy. Nevertheless, this area shows a huge potential for the pharmaceutical industry around the globe. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs, could effectively activate a particular gene in a sequence-specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed ‘RNA activation’ (RNAa). In this review, we analyze these research findings and discussed the in vivo applications of siRNAs, miRNAs, and shRNAs.

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Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4557-4560.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4557-4560

Author(s):

O Bakker ◽

J N Philipsen ◽

B C Hennis ◽

G Ab

Keyword(s):

Dimethyl Sulfate ◽

Upstream Region ◽

Nuclear Factor ◽

Base Pairs ◽

Exonuclease Iii ◽

Factor I ◽

Nuclear Factor I ◽

Vitellogenin Gene

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

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In vivo analysis of the myosin heavy chain IIB promoter region

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.3.c681 ◽

1998 ◽

Vol 274 (3) ◽

pp. C681-C687 ◽

Cited By ~ 50

Author(s):

Steven J. Swoap

Keyword(s):

Reporter Gene ◽

Luciferase Activity ◽

Fiber Type ◽

Firefly Luciferase ◽

Renilla Luciferase ◽

Luciferase Reporter ◽

Base Pairs ◽

Specific Expression ◽

Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

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Phage lambda Cro protein and cI repressor use two different patterns of specific protein-DNA interactions to achieve sequence specificity in vivo.

Genetics ◽

10.1093/genetics/121.1.5 ◽

1989 ◽

Vol 121 (1) ◽

pp. 5-12

Author(s):

N Benson ◽

P Youderian

Keyword(s):

Specific Protein ◽

Specific Interactions ◽

Sequence Specificity ◽

Base Pairs ◽

Lambda Repressor ◽

Protein Dna Interactions ◽

Cro Protein ◽

Data Call ◽

Half Site

Abstract By assaying the binding of wild-type Cro to a set of 40 mutant lambda operators in vivo, we have determined that the 14 outermost base pairs of the 17 base pair, consensus lambda operator are critical for Cro binding. Cro protein recognizes 4 base pairs in a lambda operator half-site in different ways than cI repressor. The sequence determinants of Cro binding at these critical positions in vivo are nearly perfectly consistent with the model proposed by W. F. ANDERSON, D. H. OHLENDORF, Y. TAKEDA and B. W. MATTHEWS and modified by Y. TAKEDA, A. SARAI and V. M. RIVERA for the specific interactions between Cro and its operator, and explain the relative order of affinities of the six natural lambda operators for Cro. Our data call into question the idea that lambda repressor and Cro protein recognize the consensus lambda operator by nearly identical patterns of specific interactions.

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Stem–loop formation drives RNA folding in mechanical unzipping experiments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2025575119 ◽

2022 ◽

Vol 119 (3) ◽

pp. e2025575119

Author(s):

Paolo Rissone ◽

Cristiano V. Bizarro ◽

Felix Ritort

Keyword(s):

Optical Tweezers ◽

Rna Structure ◽

Nearest Neighbor ◽

Rna Folding ◽

General Mechanism ◽

Loop Formation ◽

Stem Loop ◽

Wide Range ◽

Irreversible Effects ◽

And Function

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force–distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping–rezipping process, we introduce a barrier energy landscape of the stem–loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem–loops that kinetically trap the long-lived intermediates observed in the FDC. Stem–loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

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