Nucleosome disruption at the yeast PHO5 promoter upon PHO5 induction occurs in the absence of DNA replication.

Activation of the PHO5 gene in S. cerevisiae by phosphate starvation was previously shown to be accompanied by the disappearance of four positioned nucleosomes from the promoter. To investigate the mechanism, we replaced the PHO80 gene, a negative regulator of PHO5, by a temperature-sensitive allele. As a consequence, PHO5 can be activated in the presence of phosphate by a temperature shift from 24 degrees C to 37 degrees C. Under these conditions, the promoter undergoes the same chromatin transition as in phosphate-starved cells. Disruption of the nucleosomes by the temperature shift also occurs when DNA replication is prevented. Nucleosomes re-form when the temperature is shifted from 37 degrees C back to 24 degrees C in nondividing cells. Glucose is required for the disruption of the nucleosomes during the temperature upshift, not for their re-formation during the temperature downshift. These experiments prove that DNA replication is not required for the transition between the nucleosomal and the non-nucleosomal state at the PHO5 promoter.     

Evidence for histone eviction in trans upon induction of the yeast PHO5 promoter

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.     

Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast.

The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed. Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3. Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments. The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases. Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant. Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern. This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays.     

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.     

A transient histone hyperacetylation signal marks nucleosomes for remodeling at the PHO8 promoter in vivo.

Chromatin remodeling of the yeast PHO8 promoter requires the SAGA histone acetyltransferase complex. We report here that SAGA is necessary and sufficient to establish an activator-dependent hyperacetylation peak over the PHO8 promoter that is restricted to those nucleosomes that are remodeled upon activation. This local hyperacetylated state is observed upon activation in the absence of the SWI/SNF complex when the remodeling process is frozen subsequent to activator binding. Hyperacetylation is lost, however, if remodeling is permitted to go to completion. Thus, a transient histone hyperacetylation signal is shown to be a prerequisite for, and determinant of, the domain of nucleosome remodeling in vivo.     

A functional role for nucleosomes in the repression of a yeast promoter.

Induction of the PHO5 gene in S. cerevisiae was previously shown to be accompanied by the removal of four positioned nucleosomes from the promoter. In order to assess the role of nucleosomes in the cascade of gene activation, DNA corresponding to one of these nucleosomes was excised. In its place two foreign DNA segments of the same length were inserted: a fragment from the African green monkey alpha-satellite DNA which is known to associate with histones in a highly specific fashion to give a uniquely positioned nucleosome or, alternatively, a fragment derived from pBR322 DNA. The promoter constructs were fused to the lacZ gene on centromere plasmids and transformed into yeast cells. The satellite fragment formed a nucleosome which persisted under inducing conditions. At the same time the inducibility of the PHO5 promoter was virtually abolished. When various subfragments containing between 35 and 100 bp of the satellite segment were tested, they were all found to decrease the inducibility of the promoter, full repression required the full length molecule, however. In contrast, the pBR fragment made the promoter weakly constitutive, and induction proceeded to levels even higher than with a promoter lacking an insert. Analysis of the chromatin structure reveals a nucleosome on the pBR segment at noninducing conditions which is removed upon induction. It is concluded that the quality of the histone-DNA interactions at the promoter makes an intrinsic contribution to the regulation of the gene.