Visualizing biological reaction intermediates with DNA curtains

Zhao, Yiling; Jiang, Yanzhou; Qi, Zhi

doi:10.1088/1361-6463/aa59cf

Cited by 14 publications

(20 citation statements)

References 91 publications

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“…The working buffer used in DNA Curtains was: 40 mM Tris-HCl (pH 7.5), 150 mM KCl, 2 mM MgCl 2 , 1 mM DTT, 0.2 mg/ml BSA, and 0.5 nM YOYO1. For protein loading, both EWS-FLI1 and FUS-ERG were diluted to the indicated concentrations by the working buffer in 100 μl, and loaded into a 50 μl sample loop 25 . The blank working buffer was used to send the protein sample in the sample loop to the chamber 25 .…”

Section: Methodsmentioning

confidence: 99%

“…For protein loading, both EWS-FLI1 and FUS-ERG were diluted to the indicated concentrations by the working buffer in 100 μl, and loaded into a 50 μl sample loop 25 . The blank working buffer was used to send the protein sample in the sample loop to the chamber 25 . When proteins reached into the chamber, some of them interacted with and stayed on DNA, all other free proteins were washed out.…”

Section: Methodsmentioning

confidence: 99%

“…What are the biological functions of the DNA binding sites in this process? To unravel these underlying mechanistic principles, we use a high-throughput single-molecule technique called DNA Curtains 24 , 25 , in which arrays of individual DNA strands are arranged in the same orientation and visualized by total internal reflection fluorescence microscopy (TIRFM), to further investigate the role of FET fusion proteins in cancers by providing detailed biophysical evidence to link its biomolecular condensates with gene transcription in vitro.…”

Section: Introductionmentioning

confidence: 99%

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Loci-specific phase separation of FET fusion oncoproteins promotes gene transcription

et al. 2021

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Abnormally formed FUS/EWS/TAF15 (FET) fusion oncoproteins are essential oncogenic drivers in many human cancers. Interestingly, at the molecular level, they also form biomolecular condensates at specific loci. However, how these condensates lead to gene transcription and how features encoded in the DNA element regulate condensate formation remain unclear. Here, we develop an in vitro single-molecule assay to visualize phase separation on DNA. Using this technique, we observe that FET fusion proteins undergo phase separation at target binding loci and the phase separated condensates recruit RNA polymerase II and enhance gene transcription. Furthermore, we determine a threshold number of fusion-binding DNA elements that can enhance the formation of FET fusion protein condensates. These findings suggest that FET fusion oncoprotein promotes aberrant gene transcription through loci-specific phase separation, which may contribute to their oncogenic transformation ability in relevant cancers, such as sarcomas and leukemia.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Loci-specific phase separation of FET fusion oncoproteins promotes gene transcription

et al. 2021

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“…We further monitored VRN1 phase separation using DNA curtains, which is a high‐throughput single‐molecule technique for direct monitoring of protein–DNA interactions by fluorescence imaging. We established DNA curtains in a flow cell using Lambda DNA (N3011, New England Biolabs).…”

Section: Figurementioning

confidence: 99%

Mechanism of DNA‐Induced Phase Separation for Transcriptional Repressor VRN1

et al. 2019

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Phase separation of proteins/nucleic acids to form non‐membrane organelles is crucial in cellular gene‐expression regulation. However, little is known about transcriptional regulator phase separation and the underlying molecular mechanism. Vernalization 1 (VRN1) encodes a crucial transcriptional repressor involved in plant vernalization that contains two B3 DNA‐binding domains connected by an intrinsic disorder region (IDR) and nonspecifically binds DNA. We found that the Arabidopsis VRN1 protein undergoes liquid–liquid phase separation (LLPS) with DNA that is driven by multivalent protein–DNA interactions (LLPS), and that both B3 domains are required. The distribution of charged residues in the VRN1 IDR modulates the interaction strength between VRN1 and DNA, and changes in the charge pattern lead to interconversion between different states (precipitates, liquid droplets, and no phase separation). We further showed that VRN1 forms puncta in plant cell nuclei, suggesting that it may stabilize the vernalized state by repressing gene expression through LLPS.

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“…We further monitored VRN1 phase separation using DNAc urtains, [24][25][26] which is ah igh-throughput single-molecule technique for direct monitoring of protein-DNAi nteractions by fluorescence imaging.W ee stablished DNA curtains in af low cell using Lambda DNA( N3011, New England Biolabs). When VRN1 (50 nm)w as injected (Figure 2e), the DNAm olecules started to shrink, and bright fluorescent puncta were formed at the end of DNA ( Figure 2e,f).…”

mentioning

confidence: 99%

Mechanism of DNA‐Induced Phase Separation for Transcriptional Repressor VRN1

et al. 2019

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Phase separation of proteins/nucleic acids to form non-membrane organelles is crucial in cellular gene-expression regulation. However,l ittle is knowna bout transcriptional regulator phase separation and the underlying molecular mechanism. Vernalization 1( VRN1) encodes ac rucial transcriptional repressor involved in plant vernalization that contains two B3 DNA-binding domains connected by an intrinsic disorder region (IDR) and nonspecifically binds DNA. We found that the Arabidopsis VRN1 protein undergoes liquid-liquid phase separation (LLPS) with DNAt hat is driven by multivalent protein-DNAinteractions (LLPS), and that both B3 domains are required. The distribution of charged residues in the VRN1 IDR modulates the interaction strength between VRN1 and DNA, and changes in the charge pattern lead to interconversion between different states (precipitates, liquid droplets,a nd no phase separation). We further showed that VRN1 forms puncta in plant cell nuclei, suggesting that it may stabilizethe vernalized state by repressing gene expression through LLPS.

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Visualizing biological reaction intermediates with DNA curtains

Cited by 14 publications

References 91 publications

Loci-specific phase separation of FET fusion oncoproteins promotes gene transcription

Loci-specific phase separation of FET fusion oncoproteins promotes gene transcription

Mechanism of DNA‐Induced Phase Separation for Transcriptional Repressor VRN1

Mechanism of DNA‐Induced Phase Separation for Transcriptional Repressor VRN1

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