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J. Biol. Chem., Vol. 279, Issue 33, 35113-35120, August 13, 2004

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In Vitro Assembly of the Characteristic Chromatin Organization at the Yeast PHO5 Promoter by a Replication-independent Extract System^*

Philipp Korber and Wolfram Hörz

From the Adolf-Butenandt-Institut, University of Munich, Schillerstr. 44, 80336 München, Germany

An extensive set of analyses of the yeast PHO5 gene, mostly performed in vivo, has made this gene a model for the role of chromatin structure in gene regulation. In the repressed state, the PHO5 promoter shows a characteristic chromatin organization with four positioned nucleosomes and a short hypersensitive site. So far the basis for this nucleosome positioning has remained unresolved. We have therefore decided to complement the in vivo studies by an in vitro approach. As a first step, we have asked whether the characteristic PHO5 promoter chromatin structure depends on the cellular context including replication or higher order nuclear chromatin organization or whether it can be reconstituted in vitro in a cell-free system. To this end we have established an in vitro chromatin assembly system based on yeast extracts. It is capable of generating extensive regular nucleosomal arrays with physiological spacing. Assembly requires supplementation with exogenous histones and is dependent on energy leading to chromatin with dynamic properties due to ATP-dependent activities of the extract. Using the PHO5 promoter sequence as template in this replication independent system, we obtain a nucleosomal pattern over the PHO5 promoter region that is very similar to the in vivo pattern of the repressed state. This shows that the chromatin structure at the PHO5 promoter represents a self-organizing system in cell-free yeast extracts and provides a promising substrate for in vitro studies with a direct in vivo correlate.

Received for publication, May 17, 2004

This article is dedicated to the memory of Dr. Helmut Korber honoring his example of keen and unprejudiced observation and his genuine interest and curiosity, which he kept up for all his life.

^* This work was funded by the Deutsche Forschungsgemeinschaft (DFG), Transregio 5 and Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-89-218075420; Fax: 49-89-218075440; E-mail: hoerz{at}bio.med.unimuenchen.de.

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