CHANGE IN CONFORMATION OF LINKER DNA UPON BINDING OF HISTONE H1.5 TO NUCLEOSOME: FLUORESCENT MICROSCOPY OF SINGLE COMPLEXES

The method of synthesis of fluorescently labeled DNA allowing assembly of mononucleosomes with 40 bp linkers was developed. Cy3 and Cy5 labels were introduced in the linkers at distances of 10 bp before the first and 15 bp after the last nucleotide of the nucleosome positioning DNA sequence, respectively. In the absence of histone H1.5 fluorescence microscopy of single complexes revealed the presence of two equally probable states of nucleosomes, differing in the conformation of linkers: open one with the energy transfer efficiency E between the labels being equal to 0.06 and closed one with E = 0.37. Binding of histone H1.5 with nucleosomes occurs in nanomolar range of concentrations, and the rate of complex formation is significantly higher than the dissociation rate. In the complexes, significant convergence of DNA linkers (E = 0.73) takes place, and their conformation in the region of labels becomes more uniform. Designed nucleosomal constructs are highly sensitive fluorescent sensors for the analysis of structural rearrangements of linkers and in combination with microscopy of single complexes allow studying the structure of complexes of nucleosomes with different chromatin architectural proteins.

1. Shrestha D., Jenei A., Nagy P., Vereb G., Szöllősi J. Understanding FRET as a research tool for cellular studies // Int. J. Mol. Sci. 2015. Vol.16. N 4. P. 6718–6756.

2. Sustarsic M.K. Taking the ruler to the jungle: singlemolecule FRET for understanding biomolecular structure and dynamics in live cells // Curr. Opin. Struct. Biol. 2015 Vol. 18. P. 52–59.

3. Arai Y.N. Extensive use of FRET in biological imaging // Microscopy (Oxf). 2013. Vol. 62. P. 419–428.

4. Sugawa M.A., Iwane A.H., Ishii Y., Yanagida T. Single molecule FRET for the study on structural dynamics of biomolecules // Biosystems. 2007. Vol. 88. N 3. P. 243–250.

5. Lee W., Obubuafo A., Lee Y.I., Davis L.M., Soper S.A. Single-pair fluorescence resonance energy transfer (spFRET) for the high sensitivity analysis of low-abundance proteins using aptamers as molecular recognition elements // J. Fluoresc. 2010. Vol. 20. N 1. P. 203–213.

6. Gansen A., Tóth K., Schwarz N., Langowski J. Structural variability of nucleosomes detected by single-pair Förster resonance energy transfer: histone acetylation, sequence variation, and salt effects // J. Phys. Chem. B. 2009. Vol. 113. N 9. P. 2604–2613.

7. Kudryashova K.S., Chertkov O.V., Nikitin D.V., Pestov N.A., Kulaeva O.I., Efremenko A.V., Solonin A.S., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET // Methods Mol. Biol. 2015. Vol. 1288. P. 395–412.

8. Ngo T.T., Ha T. Nucleosomes undergo slow spontaneous gaping // Nucleic Acids Res. 2015. Vol. 43. N 8. P. 3964– 3971.

9. Feofanov A.V., Kudryashova K.S., Chertkov O.V., Nikitin D.V., Pestov N.A., Kulaeva O.I., Studitsky V.M., Kirpichnikov M.P. Analysis of nucleosome transcription using singleparticle FRET // Springer Proc. Phys. 2015. Vol. 164. P. 255–260.

10. Ngo T.T., Zhang Q., Zhou R., Yodh J.G., Ha T. Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility // Cell. 2015. Vol.160. N 6. P. 1135–1144.

11. Kudryashova K.S., Nikitin D.V., Chertkov O.V., Gerasimova N.S., Valieva M.E., Studitsky V.M., Feofanov A.V. Development of fluorescently labeled mononucleosomes for the investigation of transcription mechanisms by single complex microscopy // Moscow Univ. Biol. Sci. Bull. 2015. Vol. 70. N 4. P. 189–193.

12. Luo Y., North J.A., Poirier M.G. Single molecule fluorescence methodologies for investigating transcription factor binding kinetics to nucleosomes and DNA // Methods. 2014. Vol. 70. N 2–3. P. 108–118.

13. Simon M.N., Shimko J.C., Forties R.A., Ferdinand M.B., Manohar M., Zhang M., Fishel, R., Ottesen J.J., Poirier M.G. Histone fold modifications control nucleosome unwrapping and disassembly // Proc. Natl. Acad. Sci. USA. 2011. Vol. 108. N 31. P. 12711–12716.

14. Syed S.H., Goutte-Gattat D., Becker N., Meyer S., Shukla M.S., Hayes J.J., Everaers R., Angelov D., Bednar J., Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome // Proc. Natl. Acad. Sci. USA. 2010. Vol. 107. N 21. P. 9620–9625.

15. Meyer S. B., Syed S.H., Goutte-Gattat D., Shukla M.S., Hayes J.J., Angelov D., Bednar J., Dimitrov S., Everaers R. From crystal and NMR structures, footprints and cryo-electron- micrographs to large and soft structures: nanoscale modeling of the nucleosomal stem // Nucleic Acids Res. 2011. Vol. 39. P. 9139–9154.

16. Zhou B.R., Feng H., Kato H., Dai L., Yang Y., Zhou Y., Bai Y. Structural insights into the histone H1-nucleosome complex // Proc. Natl. Acad. Sci. USA. 2013. Vol. 110. N 48. P. 19390–19395.

17. Gaykalova D.A., Kulaeva O.I., Bondarenko V.A., Studitsky V.M. Preparation and analysis of uniquely positioned mononucleosomes // Methods Mol. Biol. 2009. Vol. 523. P. 109–123.

18. Syed S.H., Goutte-Gattat D., Becker N., Meyer S., Shukla M.S., Hayes J.J., Everaers R., Angelov D., Bednar J., Dimitrov S. Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome // Proc. Natl. Acad. Sci. USA. 2010. Vol. 107. N 21. P. 9620–9625.

19. Meyer S., Becker N.B., Syed S.H., Goutte-Gattat D., Shukla M.S., Hayes J.J., Angelov D, Bednar J., Dimitrov S., Everaers R. From crystal and NMR structures, footprints and cryo-electron-micrographs to large and soft structures: nanoscale modeling of the nucleosomal stem // Nucleic Acids Res. 2011. Vol. 39. N 21. P. 9139–9154.

20. Song F., Chen P., Sun D., Wang M., Dong L., Liang D., Xu R.M., Zhu P., Li G. Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units // Science. 2014. Vol. 344. N 6182. P. 376–380.

21. Fan L., Roberts V.A. Complex of linker histone H5 with the nucleosome and its implications for chromatin packing // Proc. Natl. Acad. Sci. USA. 2006. Vol. 103. N 22. P. 8384–8289.

22. Cui F., Zhurkin V.B. Distinctive sequence patterns in metazoan and yeast nucleosomes: implications for linker histone binding to AT-rich and methylated DNA // Nucleic Acids Res. 2009. Vol. 37. N 9. P. 2818–2829.

23. Zhou B.R., Jiang J., Feng H., Ghirlando R., Xiao T.S., Bai Y. Structural mechanisms of nucleosome recognition by linker histones // Mol. Cell. 2015. Vol. 59. N 4. P. 628–638.

24. Bernier M., Luo Y., Nwokelo K.C., Goodwin M., Dreher S.J., Zhang P., Parthun M.R., Fondufe-Mittendorf Y., Ottesen J.J., Poirier M.G. Linker histone H1 and H3K56 acetylation are antagonistic regulators of nucleosome dynamics // Nat. Commun. 2015. Vol. 6. P. 10152.