Induction of the yeast PHO5 and PHO8 genes leads to a prominent chromatin transition at their promoter regions as a prerequisite for transcription activation. Although induction of PHO8 is strictly dependent on Snf2 and Gcn5, there is no chromatin remodeler identified so far that would be essential for the opening of PHO5 promoter chromatin. Nonetheless, the nonessential but significant involvement of cofactors can be identified if the chromatin opening kinetics are delayed in the respective mutants. Using this approach, we have tested individually all 15 viable Snf2 type ATPase deletion mutants for their effect on PHO5 promoter induction and opening. Only the absence of Snf2 and Ino80 showed a strong delay in chromatin remodeling kinetics. The snf2 ino80 double mutation had a synthetic kinetic effect but eventually still allowed strong PHO5 induction. The same was true for the snf2 gcn5 and ino80 gcn5 double mutants. This strongly suggests a complex network of redundant and mutually independent parallel pathways that lead to the remodeling of the PHO5 promoter. Further, chromatin remodeling kinetics at a transcriptionally inactive TATA box-mutated PHO5 promoter were affected neither under wild type conditions nor in the absence of Snf2 or Gcn5. This demonstrates the complete independence of promoter chromatin opening from downstream PHO5 transcription processes. Finally, the histone variant Htz1 has no prominent role for the kinetics of PHO5 promoter chromatin remodeling.

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.The DNA of eukaryotic cells is compacted in the nucleus into a complex structure called chromatin. The first level of chromatin organization is formed by the nucleosome, which consists of a histone octamer core organizing ϳ1.7 turns of double-stranded DNA around its surface (1). DNA that is wound around a histone octamer in a canonical nucleosome is much less accessible for most DNA-interacting factors than DNA in the linker regions between nucleosomes.It is now widely accepted not only that nucleosomes serve a structural role for the compaction of eukaryotic DNA but also that the obstructive nature of the nucleosomal histone-DNA interactions is a means to regulate the expression of genetic information (2-4). This mode of regulation involves changes in chromatin structure at, for example, promoter or enhancer regions. A hallmark of such regulatory changes is the switch of DNA regions from a state that is protected from nucleases to a state that is sensitive, or even hypersensitive, to nucleases.To understand the process of regulation through chromatin structure it is therefore crucial to study the molecular mechanisms that lead to the inducible generation of hypersensitive sites. To this end, the PHO5 promoter in yeast became a classical model system (5). In its repressed state this promoter region is organized into four positioned nucleosomes with a short hypersensitive site in the middle. Upon activation by phosphate starvation this characteristic chromatin organization becomes remodeled into an extended hypersensitive region (6). The promoter nucleosomes in this induced state are completely disassembled as assayed by the loss of histone DNA c...

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The yeast PHO5 promoter is a model system for the role of chromatin in eukaryotic gene regulation. Four positioned nucleosomes in the repressed state give way to an extended DNase I hypersensitive site upon induction. Recently this hypersensitive site was shown to be devoid of histone DNA contacts. This raises the mechanistic question of how histones are removed from the promoter. A displacement in trans or movement in cis, the latter according to the well established nucleosome sliding mechanism, are the major alternatives. In this study, we embedded the PHO5 promoter into the context of a small plasmid which severely restricts the space for nucleosome sliding along the DNA in cis. Such a construct would either preclude the chromatin transition upon induction altogether, were it to occur in cis, or gross changes in chromatin around the plasmid would be the consequence. We observed neither. Instead, promoter opening on the plasmid was indistinguishable from opening at the native chromosomal locus. This makes a sliding mechanism for the chromatin transition at the PHO5 promoter highly unlikely and points to histone eviction in trans.All DNA-related processes like replication, transcription, recombination, and repair are mediated by protein factors and machines which use DNA as their substrate. It is therefore crucial that these proteins have access to the DNA for direct binding interaction, often in a sequence-specific manner. Restriction of this access is one mechanism by which cellular functions involving DNA are regulated. One way to achieve this goal is the packaging of the eukaryotic genome into a complex DNA-protein structure called chromatin, and modulation of the structure of chromatin is recognized as a key step in the cascade of DNA-related processes.The basic unit of chromatin is the nucleosome, which is made up of a core of eight histone proteins with about 1.7 turns of DNA wound around this octamer. The nucleosomes form a beads-on-a-string structure along the DNA and are further organized into a so-far ill-defined hierarchy of higher order structures involving several nonhistone chromatin proteins. The influence of higher order structure on DNA-related processes is not well understood so far. At the level of the nucleosome, however, the tight interaction of the DNA with the surface of the histone octamer imposes a significant barrier for the access of proteins to their binding sites.Our laboratory has been interested in the influence of chromatin structure on gene regulation for a long time. We employ the PHO genes in yeast as a model system which shows striking chromatin transitions upon the activation of several of the genes related to phosphate metabolism. In particular, under repressive conditions (with P i [ϩP i ]) the promoter region of the PHO5 gene is organized into four clearly positioned nucleosomes with a short hypersensitive site of about 60-bp length between nucleosomes Ϫ2 and Ϫ3 (34) (Fig. 1A). Upon PHO5 activation by phosphate starvation (without P i [ϪP i ]), these four nucleosomes be...