Reconstitution of Nucleosome Positioning, Remodeling, Histone Acetylation, and Transcriptional Activation on the PHO5Promoter

Paul J Laybourn,Andrea R Terrell,Sriwan Wongwisansri,John L Pilon

doi:10.1074/jbc.m204662200

Abstract

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.

Highlights

Chromatin functions to compact and organize DNA in the nucleus of eukaryotic cells in a manner that allows regulated access to genes for transcription and DNA replication
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
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

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The pPHO5-G-less plasmid was produced by subcloning the 526-bp BamHI to ApoI fragment (Ϫ542 to Ϫ16) of pMH313 [5] and a DNA fragment containing no guanine bases in the RNA-like strand into pUC19. Reactions were 12 ␮l and contained 50 ng of labeled probe, 225 ng of Pho2p competitor as described in the Fig. 3 legend, and EMSA binding buffer (12% glycerol, 20 mM Tris-Cl, pH 7.9, 100 mM KCl, 1 mM EDTA, 1 mM DTT, and 100 ␮g/ml BSA). Naked or reconstituted chromatin DNA (500 ng), 100 ng of poly(dI-dC), purified Pho4p and Pho2p (amounts indicated in Fig. 4 legend), 1% polyethylene glycol 3350, 50 mM HEPES-KOH, pH 7.6, 100 mM potassium glutamate, 5 mM EDTA, 10 mM magnesium acetate, 2.5 mM DTT, and 10% glycerol in a 20-␮l reaction volume were incubated at 30 °C for 15 min. The same blotted membrane was Western-blotted again using anti-acetylated H4 antibody (Upstate Biotechnology)

RESULTS

DISCUSSION

Methods