Chromatin assembly and in vitro transcription analyses for evaluation of individual protein activities in multicomponent transcriptional complexes

Takayuki Furumatsu, Hiroshi Asahara

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Eukaryotic DNA and core histones form the fundamental repeating units of chromatin. Condensed c hromatin, which has higher-order structures, prevents transcriptional complexes from accessing their target genes. Epigenetic regulation, including structural changes of chromatin, histone modification, and DNA methylation, strictly controls the pattern of gene expression and silencing. Recent studies have revealed that histone acetylation plays a crucial role in relaxing chromatin structure for initiation of transcription. Crosstalk between DNA-binding transcription factors and histone acetyltransferases (HATs) serves as a key mechanism for regulating gene expression and developmental processes. However, the precise roles of multicomponent transcriptional complexes have not been fully elucidated because of technical difficulties in using in vitro experimental systems. Previously we demonstrated that the DNA-binding transcription factor Sox9, HAT coactivator p300, and other regulatory factors (Smad3/4) cooperatively activate Sox9-dependent transcription on chromatin. Here, we describe an experimental approach to investigate the function of each component on reconstructed chromatin in vitro. Our methods offer a useful system for analyzing the additional effect of a third component in a transcriptional complex on chromatin structure.

Original languageEnglish
Title of host publicationGene Regulation
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages193-202
Number of pages10
ISBN (Print)9781627032834
DOIs
Publication statusPublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume977
ISSN (Print)1064-3745

Keywords

  • Chromatin assembly
  • Epigenetic regulation
  • Histone acetylation
  • In vitro transcription
  • P300
  • Smad3
  • Sox9

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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