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TECHNOLOGY DEVELOPMENT

Q²ChIP, a Quick and Quantitative Chromatin Immunoprecipitation Assay, Unravels Epigenetic Dynamics of Developmentally Regulated Genes in Human Carcinoma Cells

Institute of Basic Medical Sciences, Department of Biochemistry, Faculty of Medicine, University of Oslo, Oslo, Norway

Key Words. Chromatin immunoprecipitation • Differentiation • Embryonal carcinoma cell • Histone acetylation • Histone methylation

Correspondence: Philippe Collas, Ph.D., Institute of Basic Medical Sciences, Department of Biochemistry, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway. Telephone: +4722851066; Fax: +4722851058; e-mail: philc{at}medisin.uio.no

Received on July 13, 2006; accepted for publication on December 22, 2006.

First published online in STEM CELLS EXPRESS  February 1, 2007.


Chromatin immunoprecipitation (ChIP) is a key technique for studying protein-DNA interactions and mapping epigenetic histone modifications on DNA. Current ChIP protocols require extensive sample handling and large cell numbers. We developed a quick and quantitative (Q²)ChIP assay suitable for histone and transcription factor immunoprecipitation from chromatin amounts equivalent to as few as 100 cells. DNA-protein cross-linking in suspension in presence of butyrate, elimination of background chromatin through a tube shift after washes, and a combination of cross-link reversal, protein digestion, increased antibody-bead to chromatin ratio, and DNA elution into a single step considerably improve ChIP efficiency and shorten the procedure. We used Q²ChIP to monitor changes in histone H3 modifications on the 5' regulatory regions of the developmentally regulated genes OCT4, NANOG, LMNA, and PAX6 in the context of retinoic-acid-mediated human embryonal carcinoma cell differentiation. Quantitative polymerase chain reaction analysis of precipitated DNA unravels biphasic heterochromatin assembly on OCT4 and NANOG, involving H3 lysine (K)9 and K27 methylation followed by H3K9 deacetylation and additional H3K27 trimethylation. Di- and trimethylation of H3K4 remain relatively unaltered. In contrast, PAX6 displays histone modifications characteristic of repressed genes with potential for activation in undifferentiated cells. PAX6 undergoes H3K9 acetylation and enhanced H3K4 trimethylation upon transcriptional activation. Q²ChIP of the transcription factor Oct4 demonstrates its dissociation from the NANOG promoter upon differentiation. This study is, to our knowledge, the first to reveal histone modification changes on human OCT4 and NANOG regulatory sequences. The results demonstrate ordered chromatin rearrangement on developmentally regulated promoters upon differentiation.

Disclosure of potential conflicts of interest is found at the end of this article.

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