vestnik-bio-msuВестник Московского университета. Серия 16. БиологияVestnik Moskovskogo universiteta. Seriya 16. Biologiya0137-0952Lomonosov Moscow State University, School of Biologyvestnik-bio-msu-493Research ArticleМетодыMethodsАНАЛИЗ СТРУКТУРЫ НУКЛЕОСОМ В ПОЛИАКРИЛАМИДНОМ ГЕЛЕ МЕТОДОМ ФЁРСТЕРОВСКОГО РЕЗОНАНСНОГО ПЕРЕНОСА ЭНЕРГИИANALYSIS OF NUCLEOSOME STRUCTURE IN POLYACRYLAMIDE GEL USING FŐRSTER RESONANCE ENERGY TRANSFER METHODЧертковО. В.ChertkovO. V.

Кафедра биоинженерии, биологический факультет

Россия, 119234, г. Москва, Ленинские горы, д. 1, стр. 12

Россия, 117997, г. Москва, ул. Миклухо-Маклая, д. 16/10

вед. инженер кафедры биоинженерии биологического факультета МГУ. Тел.: 8-495-938-22-91

Bioengineering Department, School of Biology

Leninskiye Gory 1–12, Moscow, 119234, Russia

ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

o_chertkov@mail.ruВалиеваМ. Е.ValievaM. E.

Кафедра биоинженерии, биологический факультет

Россия, 119234, г. Москва, Ленинские горы, д. 1, стр. 12

аспирантка биологического факультета МГУ. Тел.: 8-495-938-22-91

Bioengineering Department, School of Biology

Leninskiye Gory 1–12, Moscow, 119234, Russia

durnopeyko-maria@gmail.ruМалюченкоН. В.MalyuchenkoN. V.

Кафедра биоинженерии, биологический факультет

Россия, 119234, г. Москва, Ленинские горы, д. 1, стр. 12

канд. биол. наук, доцент кафедры биоинженерии биологического факультета МГУ. Тел.: 8-495-938-00-05

Bioengineering Department, School of Biology

Leninskiye Gory 1–12, Moscow, 119234, Russia

mal_nat@mail.ruФеофановА. В.FeofanovA. V.

Кафедра биоинженерии, биологический факультет

Россия, 119234, г. Москва, Ленинские горы, д. 1, стр. 12

Россия, 117997, г. Москва, ул. Миклухо-Маклая, д. 16/10

докт. биол. наук, руководитель лаборатории оптической микроскопии и спектроскопии биомолекул ИБХ РАН, проф. кафедры биоинженерии биологического факультета МГУ. Тел. 8-495-938-22-91

Bioengineering Department, School of Biology

Leninskiye Gory 1–12, Moscow, 119234, Russia

ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

avfeofanov@yandex.ruМосковский государственный университет имени М.В. Ломоносова Институт биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова РАНРоссияLomonosov Moscow State University Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of SciencesRussian FederationМосковский государственный университет имени М.В. ЛомоносоваРоссияLomonosov Moscow State UniversityRussian Federation201720102017724229234Copyright © Чертков О.В., Валиева М.Е., Малюченко Н.В., Феофанов А.В., 20172017Чертков О.В., Валиева М.Е., Малюченко Н.В., Феофанов А.В.Chertkov O.V., Valieva M.E., Malyuchenko N.V., Feofanov A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestnik-bio-msu.elpub.ru/jour/article/view/493

На основе эффекта Фёрстеровского резонансного переноса энергии (FRET) разработана методика анализа структуры (Су3, Су5)-меченных нуклеосом в полиакриламидном геле после электрофореза в нативных условиях. Показано, что корректное применение этой методики требует контроля за неспецифическим межмолекулярным FRET и перепоглощением флуоресценции. Проведен сравнительный анализ результатов FRET-измерений двух типов нуклеосом и их комплексов с дрожжевым белком FACT, который подтвердил сходство структурных особенностей нуклеосом, выявляемых в геле и в водном растворе. Применение FRET-анализа в комбинации с электрофорезом позволяет не только разделить и визуализировать компоненты сложной смеси, оценив их относительное содержание, но и охарактеризовать структурные отличия этих компонентов in situ.

On the basis of Förster resonance energy transfer (FRET) effect a method to analyze the structure of (Cy3, Cy5)-labeled nucleosomes in polyacrylamide gel after electrophoresis in native conditions was developed. It is shown that the correct application of the method requires monitoring of nonspecific intermolecular FRET and fluorescence re-absorption. A comparative analysis of the results of the FRET measurements of two types of nucleosomes and their complexes with yeast protein FACT was performed, which confirmed the similarity of the structural features of nucleosomes detected in the gel and in aqueous solution. An application of FRET analysis in combination with electrophoresis allows one to separate and visualize components of a complex mixture, evaluate their relative content, and, in addition, to characterize the structural differences between these complexes in situ.

нуклеосомафлуоресценцияэлектрофорезперенос энергиимикроскопияэпигенетикаnucleosomefluorescenceelectrophoresisenergy transfermicroscopyepigeneticsReferencesValieva M.E., Armeev G.A., Kudryashova K.S., Gerasimova N.S., Shaytan A.K., Kulaeva O.I., McCullough L.L., Formosa T., Georgiev P.G., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. Large-scale ATP-independent nucleosome unfolding by a histone chaperone // Nat. Struct. Mol. Biol. 2016. Vol. 23. N 12. P. 1111–1116.Valieva M.E., Armeev G.A., Kudryashova K.S., Gerasimova N.S., Shaytan A.K., Kulaeva O.I., McCullough L.L., Formosa T., Georgiev P.G., Kirpichnikov M.P., Studitsky V.M., Feofanov A.V. Large-scale ATP-independent nucleosome unfolding by a histone chaperone // Nat. Struct. Mol. Biol. 2016. Vol. 23. N 12. P. 1111–1116.Dechassa M.L., Sabri A., Pondugula S., Kassabov S.R., Chatterjee N., Kladde M.P., Bartholomew B. SWI/SNF has intrinsic nucleosome disassembly activity that is dependent on adjacent nucleosomes // Mol. Cell. 2010. Vol. 38. N 4. P. 590–602.Dechassa M.L., Sabri A., Pondugula S., Kassabov S.R., Chatterjee N., Kladde M.P., Bartholomew B. SWI/SNF has intrinsic nucleosome disassembly activity that is dependent on adjacent nucleosomes // Mol. Cell. 2010. Vol. 38. N 4. P. 590–602.Kulaeva O.I., Hsieh F.K., Studitsky V.M. RNA polymerase complexes cooperate to relieve the nucleosomal barrier and evict histones // Proc. Natl. Acad. Sci. U.S.A. 2010. Vol. 107. N 25. P. 11325–11330.Kulaeva O.I., Hsieh F.K., Studitsky V.M. RNA polymerase complexes cooperate to relieve the nucleosomal barrier and evict histones // Proc. Natl. Acad. Sci. U.S.A. 2010. Vol. 107. N 25. P. 11325–11330.Chang H.W., Pandey M., Kulaeva O.I., Patel S.S., Studitsky V.M. Overcoming a nucleosomal barrier to replication // Sci. Adv. 2016. Vol. 2. N 11. e1601865.Chang H.W., Pandey M., Kulaeva O.I., Patel S.S., Studitsky V.M. Overcoming a nucleosomal barrier to replication // Sci. Adv. 2016. Vol. 2. N 11. e1601865.Pestov N.A., Gerasimova N.S., Kulaeva O.I., Studitsky V.M. Structure of transcribed chromatin is a sensor of DNA damage // Sci. Adv. 2015. Vol. 1. N 6. e1500021.Pestov N.A., Gerasimova N.S., Kulaeva O.I., Studitsky V.M. Structure of transcribed chromatin is a sensor of DNA damage // Sci. Adv. 2015. Vol. 1. N 6. e1500021.Luger K., Mäder A.W., Richmond R.K., Sargent D.F., Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 A resolution // Nature. 1997. Vol. 389. N 6648. P. 251–260.Luger K., Mäder A.W., Richmond R.K., Sargent D.F., Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 A resolution // Nature. 1997. Vol. 389. N 6648. P. 251–260.Gaykalova D.A., Kulaeva O.I., Volokh O., Shaytan A.K., Hsieh F.K., Kirpichnikov M.P., Sokolova O.S., Studitsky V.M. Structural analysis of nucleosomal barrier to transcription // Proc. Natl. Acad. Sci. U. S. A. 2015. Vol. 112. N 43. P. 5787– 5795.Gaykalova D.A., Kulaeva O.I., Volokh O., Shaytan A.K., Hsieh F.K., Kirpichnikov M.P., Sokolova O.S., Studitsky V.M. Structural analysis of nucleosomal barrier to transcription // Proc. Natl. Acad. Sci. U. S. A. 2015. Vol. 112. N 43. P. 5787– 5795.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.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.Buning R., Kropff W., Martens K., van Noort J. spFRET reveals changes in nucleosome breathing by neighboring nucleosomes // J. Phys. Condens. Matter. 2015. Vol. 27. N 6. 064103.Buning R., Kropff W., Martens K., van Noort J. spFRET reveals changes in nucleosome breathing by neighboring nucleosomes // J. Phys. Condens. Matter. 2015. Vol. 27. N 6. 064103.Gansen A., Tóth K., Schwarz N., Langowski J. Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure––a FRET study // Nucleic Acids Res. 2015. Vol. 43. N 3. P. 1433–1443.Gansen A., Tóth K., Schwarz N., Langowski J. Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure––a FRET study // Nucleic Acids Res. 2015. Vol. 43. N 3. P. 1433–1443.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.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.Sultanov D., Gerasimova N., Kudryashova K., Maluchenko N., Kotova E., Langelier M.-F., Pascal J., Kirpichnikov M., Feofanov A., Studitsky V. Unfolding of core nucleosomes by PARP- 1 revealed by spFRET microscopy // AIMS Genetics. 2017. Vol. 4. N 1. P. 21–31.Sultanov D., Gerasimova N., Kudryashova K., Maluchenko N., Kotova E., Langelier M.-F., Pascal J., Kirpichnikov M., Feofanov A., Studitsky V. Unfolding of core nucleosomes by PARP- 1 revealed by spFRET microscopy // AIMS Genetics. 2017. Vol. 4. N 1. P. 21–31.Valieva M.E., Gerasimova N.S., Kudryashova K.S., Kozlova A.L., Kirpichnikov M.P., Hu Q., Botuyan M.V., Mer G., Feofanov A.V., Studitsky V.M. Stabilization of nucleosomes by histone tails and by FACT revealed by spFRET microscopy // Cancers (Basel). 2017. Vol. 9. N 1, 3. DOI:10.3390/cancers9010003.Valieva M.E., Gerasimova N.S., Kudryashova K.S., Kozlova A.L., Kirpichnikov M.P., Hu Q., Botuyan M.V., Mer G., Feofanov A.V., Studitsky V.M. Stabilization of nucleosomes by histone tails and by FACT revealed by spFRET microscopy // Cancers (Basel). 2017. Vol. 9. N 1, 3. DOI:10.3390/cancers9010003.Shimahara H., Hirano T., Ohya K., Matsuta S., Seeram S.S., Tate S. Nucleosome structural changes induced by binding of non-histone chromosomal proteins HMGN1 and HMGN2 // FEBS Open Bio. 2013. Vol. 3. P. 184–191.Shimahara H., Hirano T., Ohya K., Matsuta S., Seeram S.S., Tate S. Nucleosome structural changes induced by binding of non-histone chromosomal proteins HMGN1 and HMGN2 // FEBS Open Bio. 2013. Vol. 3. P. 184–191.Kulaeva O.I., Gaykalova D.A., Pestov N.A., Golovastov V.V., Vassylyev D.G., Artsimovitch I., Studitsky V.M. Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II // Nat. Struct. Mol. Biol. 2009. Vol. 16. N 12. P. 1272–1278.Kulaeva O.I., Gaykalova D.A., Pestov N.A., Golovastov V.V., Vassylyev D.G., Artsimovitch I., Studitsky V.M. Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II // Nat. Struct. Mol. Biol. 2009. Vol. 16. N 12. P. 1272–1278.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.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.Xin H., Takahata S., Blanksma M., McCullough L., Stillman D.J., Formosa T. yFACT induces global accessibility of nucleosomal DNA without H2AH2B displacement // Mol. Cell. 2009. Vol. 35. N 3. P. 365–376.Xin H., Takahata S., Blanksma M., McCullough L., Stillman D.J., Formosa T. yFACT induces global accessibility of nucleosomal DNA without H2AH2B displacement // Mol. Cell. 2009. Vol. 35. N 3. P. 365–376.

The authors declare that there are no conflicts of interest present.