Abstract
Eukaryotic DNA and core histones form the fundamental repeating units of chromatin. Condensed cÂhromatin, 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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jones PA, Takai D (2001) The role of DNA methylation in mammalian epigenetics. Science 293:1068–1070
Quina AS, Buschbeck M, Di Croce L (2006) Chromatin structure and epigenetics. Biochem Pharmacol 72:1563–1569
Wolffe AP, Hayes JJ (1999) Chromatin disruption and modification. Nucleic Acids Res 27:711–720
Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080
Furumatsu T, Ozaki T (2010) Epigenetic regulation in chondrogenesis. Acta Med Okayama 64:155–161
Akiyama H, Chaboissier MC, Martin JF et al (2002) The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 16:2813–2828
Shi Y, Massagué J (2003) Mechanisms of TGF-β signaling from cell membrane to the nucleus. Cell 113:685–700
Kamachi Y, Uchikawa M, Kondoh H (2000) Pairing SOX off: with partners in the regulation of embryonic development. Trends Genet 16:182–187
Tsuda M, Takahashi S, Takahashi Y et al (2003) Transcriptional co-activators CREB-binding protein and p300 regulate chondrocyte-specific gene expression via association with Sox9. J Biol Chem 278:27224–27229
Furumatsu T, Tsuda M, Yoshida K et al (2005) Sox9 and p300 cooperatively regulate chromatin-mediated transcription. J Biol Chem 280:35203–35208
Liu F (2003) Receptor-regulated Smads in TGF-β signaling. Front Biosci 8:s1280–s1303
Furumatsu T, Tsuda M, Taniguchi N et al (2005) Smad3 induces chondrogenesis through the activation of SOX9 via CREB-binding protein/p300 recruitment. J Biol Chem 280:8343–8350
Furumatsu T, Asahara H (2010) Histone acetylation influences the activity of Sox9-related transcriptional complex. Acta Med Okayama 64:351–357
Ferguson CM, Schwarz EM, Reynolds PR et al (2000) Smad2 and 3 mediate transforming growth factor-β1-induced inhibition of chondrocyte maturation. Endocrinology 141:4728–4735
Furumatsu T, Ozaki T, Asahara H (2009) Smad3 activates the Sox9-dependent transcription on chromatin. Int J Biochem Cell Biol 41:1198–1204
Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489
Ito T, Bulger M, Kobayashi R et al (1996) Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays. Mol Cell Biol 16:3112–3124
Ito T, Levenstein ME, Fyodorov DV et al (1999) ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev 13:1529–1539
Chan HM, La Thangue NB (2001) p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J Cell Sci 114:2363–2373
Asahara H, Santoso B, Guzman E et al (2001) Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB. Mol Cell Biol 21:7892–7900
Fyodorov DV, Kadonaga JT (2003) Chromatin assembly in vitro with purified recombinant ACF and NAP-1. Methods Enzymol 371:499–515
Konesky KL, Laybourn PJ (2007) Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context. Methods 41:259–270
Acknowledgments
We thank Dr. Takashi Ito and Dr. Toshifumi Ozaki for their kind assistance. This work was supported by Okayama Medical Foundation, Japan Orthopaedics and Traumatology Foundation (No. 225), and JSPS Fujita Memorial Fund for Medical Research.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Furumatsu, T., Asahara, H. (2013). Chromatin Assembly and In Vitro Transcription Analyses for Evaluation of Individual Protein Activities in Multicomponent Transcriptional Complexes. In: Bina, M. (eds) Gene Regulation. Methods in Molecular Biology, vol 977. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-284-1_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-284-1_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-283-4
Online ISBN: 978-1-62703-284-1
eBook Packages: Springer Protocols