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.     

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.     

Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter

We have analyzed the histone modification status of the PHO5 promoter from yeast by the ChIP technology and have focused on changes occurring upon activation. Using various acetylation-specific antibodies, we found a dramatic loss of the acetylation signal upon induction of the promoter. This turned out to be due, however, to the progressive loss of histones altogether. The fully remodeled promoter appears to be devoid of histones as judged by ChIP analyses. Local histone hyperacetylation does indeed occur, however, prior to remodeling. This can explain the delay in chromatin remodeling in the absence of histone acetyltransferase activity of the SAGA complex that was previously documented for the PHO5 promoter. Our findings shed new light on the nucleosomal structure of fully remodeled chromatin. At the same time, they point out the need for novel controls when the ChIP technique is used to study histone modifications in the context of chromatin remodeling in vivo.     

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.     

Chromatin structural transitions in Drosophila embryo cell-free extract result in a high conformational flexibility of nucleosomal DNA.

DNA within chromatin has considerably more restricted flexibility in comparison with naked DNA. This raises the main question of how the functioning multi-enzyme complexes overcome the nucleosomal level of DNA packaging. We studied the DNA conformational flexibility of reconstituted chromatin in a cell-free system derived from Drosophila embryo extracts. Using this system, we have found evidence for a energy-independent chromatin remodelling process that efficiently destabilizes the nucleosome structure resulting in a high conformational flexibility of nucleosomal DNA. The described chromatin remodelling process may lay on the basis of defined molecular principles governing the molecular heterogeneity of chromatin structures in vivo.     

Nucleosomes positioned by ORC facilitate the initiation of DNA replication

The packaging of eukaryotic DNA into nucleosomes is a critical regulator of nuclear events. To address the interplay between chromatin and replication initiation, we have assessed the determinants and function of the nucleosomal configuration of S. cerevisiae replication origins. Using in vitro and in vivo assays, we demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins. Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin. Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation. These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function.     

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.