In vitro assembly of the characteristic chromatin organization at the yeast PHO5 promoter by a replication-independent extract system

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.     

The histone chaperone Asf1 increases the rate of histone eviction at the yeast PHO5 and PHO8 promoters

Eukaryotic gene expression starts off from a largely obstructive chromatin substrate that has to be rendered accessible by regulated mechanisms of chromatin remodeling. The yeast PHO5 promoter is a well known example for the contribution of positioned nucleosomes to gene repression and for extensive chromatin remodeling in the course of gene induction. Recently, the mechanism of this remodeling process was shown to lead to the disassembly of promoter nucleosomes and the eviction of the constituent histones in trans. This finding called for a histone acceptor in trans and thus made histone chaperones likely to be involved in this process. In this study we have shown that the histone chaperone Asf1 increases the rate of histone eviction at the PHO5 promoter. In the absence of Asf1 histone eviction is delayed, but the final outcome of the chromatin transition is not affected. The same is true for the coregulated PHO8 promoter where induction also leads to histone eviction and where the rate of histone loss is reduced in asf1 strains as well, although less severely. Importantly, the final extent of chromatin remodeling is not affected. We have also presented evidence that Asf1 and the SWI/SNF chromatin remodeling complex work in distinct parallel but functionally overlapping pathways, i.e. they both contribute toward the same outcome without being mutually strictly dependent.     

Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements.

The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions -469, -245 and -185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.     

Nucleosome stability at the yeast PHO5 and PHO8 promoters correlates with differential cofactor requirements for chromatin opening

The coregulated PHO5 and PHO8 genes in Saccharomyces cerevisiae provide typical examples for the role of chromatin in promoter regulation. It has been a long-standing question why the cofactors Snf2 and Gcn5 are essential for full induction of PHO8 but dispensable for opening of the PHO5 promoter. We show that this discrepancy may result from different stabilities of the two promoter chromatin structures. To test this hypothesis, we used our recently established yeast extract in vitro chromatin assembly system, which generates the characteristic PHO5 promoter chromatin. Here we show that this system also assembles the native PHO8 promoter nucleosome pattern. Remarkably, the positioning information for both native patterns is specific to the yeast extract. Salt gradient dialysis or Drosophila embryo extract does not support proper nucleosome positioning unless supplemented with yeast extract. By competitive assemblies in the yeast extract system we show that the PHO8 promoter has greater nucleosome positioning power and that the properly positioned nucleosomes are more stable than those at the PHO5 promoter. Thus we provide evidence for the correlation of inherently more stable chromatin with stricter cofactor requirements.     

Histones are incorporated in trans during reassembly of the yeast PHO5 promoter

In yeast, remodeling of PHO5 promoter chromatin upon activation is accompanied by transient hyperacetylation and subsequent eviction of histones from the promoter in trans. In the course of rerepression, nucleosomes have to be reassembled on the promoter. We have analyzed where the histones for reassembly of the inactive promoter chromatin come from. The use of a strain with two differently tagged and differently regulated versions of histone H3 allowed us to discriminate between histones originating from the chromatin fraction and histones arising from the soluble histone pool. In this way, we show that the incorporated histones originate from a source in trans. Promoter closure occurs very rapidly, and the histone chaperones Asf1 and Hir1 as well as the SWI/SNF nucleosome remodeling complex appear to be important for rapid reassembly of nucleosomes at the PHO5 promoter.