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J. Biol. Chem., Vol. 277, Issue 34, 31038-31047, August 23, 2002

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Reconstitution of Nucleosome Positioning, Remodeling, Histone Acetylation, and Transcriptional Activation on the PHO5 Promoter^*

Andrea R. Terrell, Sriwan Wongwisansri^§¶, John L. Pilon, and Paul J. Laybourn^**

From the ^§ Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, the  School of Medicine, Washington University, St. Louis, Missouri 63110, and  Proligo, Boulder, Colorado 80301

The PHO5 gene promoter is an important model for the study of gene regulation in the context of chromatin. Upon PHO5 activation the chromatin structure is reconfigured, but the mechanism of this transition remains unclear. Using templates reconstituted into chromatin with purified recombinant yeast core histones, we have investigated the mechanism of chromatin structure reconfiguration on the PHO5 promoter, a prerequisite for transcriptional activation. Footprinting analyses show that intrinsic properties of the promoter DNA are sufficient for translational nucleosome positioning, which approximates that seen in vivo. We have found that both Pho4p and Pho2p can bind their cognate sites on chromatin-assembled templates without the aid of histone-modifying or nucleosome-remodeling factors. However, nucleosome remodeling by these transcriptional activators requires an ATP-dependent activity in a yeast nuclear extract fraction. Finally, transcriptional activation on chromatin templates requires acetyl-CoA in addition to these other activities and cofactors. The addition of acetyl-CoA results in significant core histone acetylation. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling, and nucleosome acetylation. Furthermore, we find that DNA binding, nucleosome remodeling, and transcriptional activation are separable steps, facilitating biochemical analysis of the PHO5 regulatory mechanism.

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