Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization

Bell, Nicholas A. W.; Haynes, Philip J.; Brunner, Katharina; Oliveira, T.M.; Flocco, Maria M.; Hoogenboom, Bart W.; Molloy, Justin E.

doi:10.1126/sciadv.abf3641

Cited by 30 publications

(20 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Prior to this study, the forces generated by co-condensation between DNA and proteins-such as FoxA1 and PARP1-were estimated to be on the order of sub-pN, placing them among the weakest nuclear forces alongside those generated by loop-extruding SMC complexes such as condensin and cohesin. 9,13,21 Here we show that Sox2, an abundant TF central to pluripotency and embryogenesis, can actively generate condensation forces up to 7 pN, one order of magnitude higher than previously reported values. It is worth noting that Klf4, another pluripotency TF, can also form condensates on DNA against a relatively high force (~8 pN) according to a recent report.…”

Section: Discussioncontrasting

confidence: 50%

“…[2][3][4][5] These nuclear compartments, or transcriptional hubs, connect enhancers to promoters, recruit the RNA polymerase and its regulators, and control gene expression in a dynamic fashion. 6,7 Notably, some TFs have been shown to form co-condensates with DNA, [8][9][10][11] which ensnare an amount of DNA into the condensates and thus exert tension on the outside free DNA. 12 The force generated by such protein-DNA co-condensation was reported to be in the sub-piconewton (pN) range.…”

Section: Introductionmentioning

confidence: 99%

“…12 The force generated by such protein-DNA co-condensation was reported to be in the sub-piconewton (pN) range. 8,9,13 Whether this mechanical effect driven by TF:DNA co-condensation is strong enough to be relevant in the nuclear milieu remains to be explored. Moreover, the genomic DNA in eukaryotic nuclei is spooled by histone proteins to form nucleosomes and further organized into higher-order chromatin structures.…”

Section: Introductionmentioning

confidence: 99%

“…We estimated the maximum condensation force that Sox2 can actively generate to be ~7 pN, an order of magnitude higher than those previously reported for other DNA-binding proteins. 9,13,16 Remarkably, when nucleosomes were present on DNA, the mechanical effects of Sox2:DNA condensation were drastically reduced. These results suggest that nucleosomes function more than just DNA packaging units, but also as a mechanical barrier to regulate the force generated by protein: DNA co-condensates.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Chromatin sequesters pioneer transcription factor Sox2 from exerting force on DNA

Nguyen

Chang

Watters

et al. 2022

Preprint

View full text Add to dashboard Cite

Formation of biomolecular condensates constitutes an emerging mechanism for transcriptional regulation. Recent studies suggest that the co-condensation between transcription factors (TFs) and DNA can generate mechanical forces driving genome rearrangements. However, the reported forces generated by such protein-DNA co-condensation are typically below one piconewton (pN), questioning its physiological significance. Moreover, the force-generating capacity of these condensates in the chromatin context remains unknown. Using single-molecule biophysical techniques, we show that Sox2, a nucleosome-binding pioneer TF, forms co-condensates with DNA, thereby exerting considerable mechanical tension on DNA strands both in cis and trans. Sox2 can generate forces up to 7 pN—similar in magnitude to other cellular forces. Sox2:DNA condensates are highly stable, withstanding disruptive forces high enough to melt DNA. We find that the disordered domains of Sox2 are required for maximum force generation but not condensate formation per se. Finally, we show that nucleosomes dramatically attenuate the mechanical stress exerted by Sox2 via sequestering it from coalescing on bare DNA. Our findings reveal that TF-mediated DNA condensation can exert significant mechanical stress which can nonetheless be alleviated by the chromatin organization, suggesting a new function of eukaryotic chromatin in protecting the genome from potentially deleterious nuclear forces.

show abstract

Section: Discussioncontrasting

confidence: 50%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Chromatin sequesters pioneer transcription factor Sox2 from exerting force on DNA

Nguyen

Chang

Watters

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…The PARP protein family mediates the PARylation of the translated substrate proteins involved in transcription and DNA damage repair, and PARP1 is a particularly important protein for detecting single-stranded DNA breaks (SSBs) [40]. A recent study showed that PARP1 can condenses DNA via loop stabilization [41]. The increased expression of PARP1 is also an important factor that mediates the drug resistance of tumor chemotherapy, especially the drug resistance related to DNA loss [42].…”

Section: Discussionmentioning

confidence: 99%

METTL3 promotes oxaliplatin resistance of gastric cancer CD133+ stem cells by promoting PARP1 mRNA stability

Wang

Lan

et al. 2022

Cell. Mol. Life Sci.

View full text Add to dashboard Cite

Oxaliplatin is the first-line regime for advanced gastric cancer treatment, while its resistance is a major problem that leads to the failure of clinical treatments. Tumor cell heterogeneity has been considered as one of the main causes for drug resistance in cancer. In this study, the mechanism of oxaliplatin resistance was investigated through in vitro human gastric cancer organoids and gastric cancer oxaliplatin-resistant cell lines and in vivo subcutaneous tumorigenicity experiments. The in vitro and in vivo results indicated that CD133+ stem cell-like cells are the main subpopulation and PARP1 is the central gene mediating oxaliplatin resistance in gastric cancer. It was found that PARP1 can effectively repair DNA damage caused by oxaliplatin by means of mediating the opening of base excision repair pathway, leading to the occurrence of drug resistance. The CD133+ stem cells also exhibited upregulated expression of N6-methyladenosine (m6A) mRNA and its writer METTL3 as showed by immunoprecipitation followed by sequencing and transcriptome analysis. METTTL3 enhances the stability of PARP1 by recruiting YTHDF1 to target the 3′-untranslated Region (3′-UTR) of PARP1 mRNA. The CD133+ tumor stem cells can regulate the stability and expression of m6A to PARP1 through METTL3, and thus exerting the PARP1-mediated DNA damage repair ability. Therefore, our study demonstrated that m6A Methyltransferase METTL3 facilitates oxaliplatin resistance in CD133+ gastric cancer stem cells by Promoting PARP1 mRNA stability which increases base excision repair pathway activity.

show abstract

An Introduction to Magnetic Tweezers

Dulin

2023

Methods in Molecular Biology

View full text Add to dashboard Cite

Magnetic tweezers are a single-molecule force and torque spectroscopy technique that enable the mechanical interrogation in vitro of biomolecules, such as nucleic acids and proteins. They use a magnetic field originating from either permanent magnets or electromagnets to attract a magnetic particle, thus stretching the tethering biomolecule. They nicely complement other force spectroscopy techniques such as optical tweezers and atomic force microscopy (AFM) as they operate as a very stable force clamp, enabling long-duration experiments over a very broad range of forces spanning from 10 fN to 1 nN, with 1–10 milliseconds time and sub-nanometer spatial resolution. Their simplicity, robustness, and versatility have made magnetic tweezers a key technique within the field of single-molecule biophysics, being broadly applied to study the mechanical properties of, e.g., nucleic acids, genome processing molecular motors, protein folding, and nucleoprotein filaments. Furthermore, magnetic tweezers allow for high-throughput single-molecule measurements by tracking hundreds of biomolecules simultaneously both in real-time and at high spatiotemporal resolution. Magnetic tweezers naturally combine with surface-based fluorescence spectroscopy techniques, such as total internal reflection fluorescence microscopy, enabling correlative fluorescence and force/torque spectroscopy on biomolecules. This chapter presents an introduction to magnetic tweezers including a description of the hardware, the theory behind force calibration, its spatiotemporal resolution, combining it with other techniques, and a (non-exhaustive) overview of biological applications.

show abstract

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization

Cited by 30 publications

References 71 publications

Chromatin sequesters pioneer transcription factor Sox2 from exerting force on DNA

Chromatin sequesters pioneer transcription factor Sox2 from exerting force on DNA

METTL3 promotes oxaliplatin resistance of gastric cancer CD133+ stem cells by promoting PARP1 mRNA stability

An Introduction to Magnetic Tweezers

Contact Info

Product

Resources

About