Structural role of Pob3 CTD in FACT-mediated nucleosome uncoiling revealed by electron microscopy
https://doi.org/10.55959/MSU0137-0952-16-80-3S-17
Abstract
The histone chaperone FACT plays a key role in chromatin reorganization by mediating ATP-in dependent nucleosome unwinding. The yeast yFACT complex consists of the Spt16 and Pob3 subunits, which form a heterodimer functionally associated with the non-histone protein Nhp6. In this study, negative-stain transmission electron microscopy was used to investigate the inter action of the yFACT complex containing the Pob3 subunit with the C-terminal domain (CTD) removed with the nucleosome in the presence of Nhp6. As a result of CTD removal, the efficien cy of FACT binding to the nucleosome decreased by a factor of 2, and the ability to fully unfold the nucleosome was impaired: instead of the almost symmetrical, fully unfolded structures char acteristic of wild type yFACT, asymmetrical, partially unfolded structures were observed. The data obtained indicate the key role of the Pob3 CTD in ensuring FACT binding to the nucle osome, which is important for understanding the mechanisms of chromatin remodeling and transcription regulation.
Keywords
chaperon FACT,
CTD domain Pob3,
electron microscopy,
ATP-independent nucleosome unfolding,
chromatin transcription
Supporting agencies: This work was supported by the Russian Science Foundation, project no. 19-74-30003.
About the Authors
O. I. Volokh
Department of Bioenineering, School of Biology, Lomonosov Moscow State University
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
A. L. Sivkina
Department of Bioenineering, School of Biology, Lomonosov Moscow State University; Institute of Gene Biology, Russian Academy of Sciences
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
34/5 Vavilov Str., Moscow, 119334
V. M. Studitsky
Department of Bioenineering, School of Biology, Lomonosov Moscow State University; Fox Chase Cancer Center
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
333 Cottman Ave., Philadelphia, 19111, Pennsylvania, USA
O. S. Sokolova
Department of Bioenineering, School of Biology, Lomonosov Moscow State University; Faculty of Biology, MSU-BIT University
Russian Federation
Russian Federation
1–12 Leninskie gory, Moscow, 119234
1 International University Park Road, Shenzhen, Guangdong Province, 518172, China
References
1. Gurova K., Chang H.W., Valieva M.E., Sandlesh P., Studitsky V.M. Structure and function of the histone chap erone FACT–Resolving FACTual issues. Biochim. Biophys. Acta Gene Regul. Mech. 2018;1861(9):892–904.
2. Volokh O., Studitsky V.M., Sokolova O.S. Beyond chaperoning: The multifaceted role of FACT in chromatin transactions. Int. J. Mol. Sci. 2025;26(11):5176.
3. Hsieh F.K., Kulaeva O.I., Patel S.S., Dyer P.N., Luger K., Reinberg D., Studitsky V.M. Histone chaperone FACT ac tion during transcription through chromatin by RNA polymer ase II. Proc. Natl. Acad. Sci. U.S.A. 2013;110(19):7654–7659.
4. Sivkina A.L., Karlova M.G., Valieva M.E., Mc Cullough L.L., Formosa T., Shaytan A.K., Feofanov A.V., Kirpichnikov M.P., Sokolova O.S., Studitsky V.M. Electron microscopy analysis of ATP-independent nucleosome un folding by FACT. Commun. Biol. 2022;5(1):2.
5. Kemble D.J., McCullough L.L., Whitby F.G., For mosa T., Hill C.P. FACT Disrupts nucleosome structure by binding H2A-H2B with conserved peptide motifs. Mol. Cell. 2015;60(2):294–306.
6. Mayanagi K., Saikusa K., Miyazaki N., Akashi S., Iwasaki K., Nishimura Y., Morikawa K., Tsunaka Y. Struc tural visualization of key steps in nucleosome reorganization by human FACT. Sci. Rep. 2019;9(1):10183.
7. Liu Y., Zhou K., Zhang N., Wei H., Tan Y.Z., Zhang Z., Carragher B., Potter C.S., D’Arcy S., Luger K. FACT caught in the act of manipulating the nucleosome. Nature. 2020;577(7790):426–431.
8. Wittmeyer J., Joss L., Formosa T. Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodi mer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha. Biochemistry. 1999;38(28):8961–8971.
9. Volokh O.I., Sivkina A.L., Moiseenko A.V., Popi- nako A.V., Karlova M.G., Valieva M.E., Kotova E.Y., Kirpich nikov M.P., Formosa T., Studitsky V.M., Sokolova O.S. Mechanism of curaxin-dependent nucleosome unfolding by FACT. Front. Mol. Biosci. 2022;9:1048117.
10. Schorb M., Haberbosch I., Hagen W.J.H., Schwab Y., Mastronarde D.N. Software tools for automated transmission electron microscopy. Nat. Methods. 2019;16(6):471–477.
11. de la Cruz M.J., Martynowycz M.W., Hattne J., Gonen, T. MicroED data collection with SerialEM. Ultra microscopy. 2019;201:77–80.
12. Bell J.M., Chen M., Baldwin P.R., Ludtke S.J. High resolution single particle refinement in EMAN2.1. Methods. 2016;100:25–34.
Review
For citations:
Volokh O.I., Sivkina A.L., Studitsky V.M., Sokolova O.S. Structural role of Pob3 CTD in FACT-mediated nucleosome uncoiling revealed by electron microscopy. Vestnik Moskovskogo universiteta. Seriya 16. Biologiya. 2025;80(3S):113–117. (In Russ.) https://doi.org/10.55959/MSU0137-0952-16-80-3S-17
Views:
72
Email this article 