A Benchmark for Chromatin Binding Measurements in Live Cells

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2012 Jul 31

PMID 22844090

Citations 181

Authors

Davide Mazza

Alice Abernathy

Nicole Golob

Tatsuya Morisaki

James G McNally

Affiliations

Soon will be listed here.

Abstract

Live-cell measurement of protein binding to chromatin allows probing cellular biochemistry in physiological conditions, which are difficult to mimic in vitro. However, different studies have yielded widely discrepant predictions, and so it remains uncertain how to make the measurements accurately. To establish a benchmark we measured binding of the transcription factor p53 to chromatin by three approaches: fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single-molecule tracking (SMT). Using new procedures to analyze the SMT data and to guide the FRAP and FCS analysis, we show how all three approaches yield similar estimates for both the fraction of p53 molecules bound to chromatin (only about 20%) and the residence time of these bound molecules (∼1.8 s). We also apply these procedures to mutants in p53 chromatin binding. Our results support the model that p53 locates specific sites by first binding at sequence-independent sites.

Citing Articles

Increasingly efficient chromatin binding of cohesin and CTCF supports chromatin architecture formation during zebrafish embryogenesis.

Cossmann J, Kos P, Varamogianni-Mamatsi V, Assenheimer D, Bischof T, Kuhn T Nat Commun. 2025; 16(1):1833.

PMID: 39979259 PMC: 11842872. DOI: 10.1038/s41467-025-56889-5.

Thermodynamic principles link transcription factor affinities to single-molecule chromatin states in cells.

Schaepe J, Fries T, Doughty B, Crocker O, Hinks M, Marklund E bioRxiv. 2025; .

PMID: 39975040 PMC: 11838358. DOI: 10.1101/2025.01.27.635162.

Effective in vivo binding energy landscape illustrates kinetic stability of RBPJ-DNA binding.

Huynh D, Hoffmeister P, Friedrich T, Zhang K, Bartkuhn M, Ferrante F Nat Commun. 2025; 16(1):1259.

PMID: 39893191 PMC: 11787368. DOI: 10.1038/s41467-025-56515-4.

A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms.

McSwiggen D, Liu H, Tan R, Agramunt Puig S, Akella L, Berman R Elife. 2025; 12.

PMID: 39786807 PMC: 11717362. DOI: 10.7554/eLife.93183.

Cohesin positions the epigenetic reader Phf2 within the genome.

Tang W, Costantino L, Stocsits R, Wutz G, Ladurner R, Hudecz O EMBO J. 2025; 44(3):736-766.

PMID: 39748119 PMC: 11790891. DOI: 10.1038/s44318-024-00348-2.

References

Lahav G, Rosenfeld N, Sigal A, Geva-Zatorsky N, Levine A, Elowitz M . Dynamics of the p53-Mdm2 feedback loop in individual cells. Nat Genet. 2004; 36(2):147-50. DOI: 10.1038/ng1293. View

Smeenk L, van Heeringen S, Koeppel M, Gilbert B, Janssen-Megens E, Stunnenberg H . Role of p53 serine 46 in p53 target gene regulation. PLoS One. 2011; 6(3):e17574. PMC: 3048874. DOI: 10.1371/journal.pone.0017574. View

Elf J, Li G, Xie X . Probing transcription factor dynamics at the single-molecule level in a living cell. Science. 2007; 316(5828):1191-4. PMC: 2853898. DOI: 10.1126/science.1141967. View

Hinow P, Rogers C, Barbieri C, Pietenpol J, Kenworthy A, DiBenedetto E . The DNA binding activity of p53 displays reaction-diffusion kinetics. Biophys J. 2006; 91(1):330-42. PMC: 1479054. DOI: 10.1529/biophysj.105.078303. View

Darzacq X, Shav-Tal Y, de Turris V, Brody Y, Shenoy S, Phair R . In vivo dynamics of RNA polymerase II transcription. Nat Struct Mol Biol. 2007; 14(9):796-806. PMC: 4942130. DOI: 10.1038/nsmb1280. View

Matsuoka S, Shibata T, Ueda M . Statistical analysis of lateral diffusion and multistate kinetics in single-molecule imaging. Biophys J. 2009; 97(4):1115-24. PMC: 2726328. DOI: 10.1016/j.bpj.2009.06.007. View

Maiuri P, Knezevich A, de Marco A, Mazza D, Kula A, McNally J . Fast transcription rates of RNA polymerase II in human cells. EMBO Rep. 2011; 12(12):1280-5. PMC: 3245692. DOI: 10.1038/embor.2011.196. View

Kim H, Kim K, Choi J, Heo K, Baek H, Roeder R . p53 requires an intact C-terminal domain for DNA binding and transactivation. J Mol Biol. 2011; 415(5):843-54. PMC: 3267882. DOI: 10.1016/j.jmb.2011.12.001. View

Phair R, Scaffidi P, Elbi C, Vecerova J, Dey A, Ozato K . Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins. Mol Cell Biol. 2004; 24(14):6393-402. PMC: 434243. DOI: 10.1128/MCB.24.14.6393-6402.2004. View

10.

Hamard P, Lukin D, Manfredi J . p53 basic C terminus regulates p53 functions through DNA binding modulation of subset of target genes. J Biol Chem. 2012; 287(26):22397-407. PMC: 3381199. DOI: 10.1074/jbc.M111.331298. View

11.

Berkovich R, Wolfenson H, Eisenberg S, Ehrlich M, Weiss M, Klafter J . Accurate quantification of diffusion and binding kinetics of non-integral membrane proteins by FRAP. Traffic. 2011; 12(11):1648-57. DOI: 10.1111/j.1600-0854.2011.01264.x. View

12.

Phair R, Misteli T . High mobility of proteins in the mammalian cell nucleus. Nature. 2000; 404(6778):604-9. DOI: 10.1038/35007077. View

13.

Tokunaga M, Imamoto N, Sakata-Sogawa K . Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods. 2008; 5(2):159-61. DOI: 10.1038/nmeth1171. View

14.

Schroder J, Benink H, Dyba M, Los G . In vivo labeling method using a genetic construct for nanoscale resolution microscopy. Biophys J. 2009; 96(1):L01-3. PMC: 2710050. DOI: 10.1016/j.bpj.2008.09.032. View

15.

Sprague B, Pego R, Stavreva D, McNally J . Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys J. 2004; 86(6):3473-95. PMC: 1304253. DOI: 10.1529/biophysj.103.026765. View

16.

Michelman-Ribeiro A, Mazza D, Rosales T, Stasevich T, Boukari H, Rishi V . Direct measurement of association and dissociation rates of DNA binding in live cells by fluorescence correlation spectroscopy. Biophys J. 2009; 97(1):337-46. PMC: 2711375. DOI: 10.1016/j.bpj.2009.04.027. View

17.

Dundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum L, Phair R . A kinetic framework for a mammalian RNA polymerase in vivo. Science. 2002; 298(5598):1623-6. DOI: 10.1126/science.1076164. View

18.

Yechiel E, Edidin M . Micrometer-scale domains in fibroblast plasma membranes. J Cell Biol. 1987; 105(2):755-60. PMC: 2114748. DOI: 10.1083/jcb.105.2.755. View

19.

Stasevich T, Mueller F, Michelman-Ribeiro A, Rosales T, Knutson J, McNally J . Cross-validating FRAP and FCS to quantify the impact of photobleaching on in vivo binding estimates. Biophys J. 2010; 99(9):3093-101. PMC: 2966010. DOI: 10.1016/j.bpj.2010.08.059. View

20.

Capoulade J, Wachsmuth M, Hufnagel L, Knop M . Quantitative fluorescence imaging of protein diffusion and interaction in living cells. Nat Biotechnol. 2011; 29(9):835-9. DOI: 10.1038/nbt.1928. View