Experimental Analysis of hFACT Action During Pol II Transcription In Vitro

  • Fu-Kai Hsieh
  • Olga I. Kulaeva
  • Vasily M. Studitsky
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1276)

Abstract

FACT (facilitates chromatin transcription) is a histone chaperone that facilitates transcription through chromatin and promotes histone recovery during transcription. Here, we describe a highly purified experimental system that recapitulates many important properties of transcribed chromatin and the key aspects of hFACT action during this process in vitro. We present the protocols describing how to prepare different forms of nucleosomes, including intact nucleosome, covalently conjugated nucleosome, nucleosome missing one of the two H2A/2B dimers (hexasome) and tetrasome (a nucleosome missing both H2A/2B dimers). These complexes allow analysis of various aspects of FACT’s function. These approaches and other methods described below can also be applied to the study of other chromatin remodelers and chromatin-targeted factors.

Key words

Transcription Histone chaperone Histone cross-linking Chromatin assembly 

Notes

Acknowledgements

This work was supported by the National Institutes of Health RO1 grant GM58650 to V.M.S. and by RAS Presidium Program “Basic research for the development of biomedical technologies.”

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Fu-Kai Hsieh
    • 1
    • 2
  • Olga I. Kulaeva
    • 3
  • Vasily M. Studitsky
    • 1
    • 3
    • 4
  1. 1.Department of PharmacologyRutgers University-Robert Wood Johnson Medical SchoolPiscatawayUSA
  2. 2.Department of Molecular Biology, Harvard Medical SchoolMassachusetts General HospitalBostonUSA
  3. 3.Biology FacultyLomonosov Moscow State UniversityMoscowRussia
  4. 4.Cancer Epigenetics ProgramFox Chase Cancer CenterPhiladelphiaUSA

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