The RSC chromatin remodeling complex has a crucial role in the complete remodeler set for yeast PHO5 promoter opening

Although yeast PHO5 promoter chromatin opening is a founding model for chromatin remodeling, the complete set of involved remodelers remained unknown for a long time. The SWI/SNF and INO80 remodelers cooperate here, but nonessentially, and none of the many tested single or combined remodeler gene mutations could prevent PHO5 promoter opening. RSC, the most abundant and only remodeler essential for viability, was a controversial candidate for the unrecognized remodeling activity but unassessed in vivo. Now we show that remodels the structure of chromatin (RSC) is crucially involved in PHO5 promoter opening. Further, the isw1 chd1 double deletion also delayed chromatin remodeling. Strikingly, combined absence of RSC and Isw1/Chd1 or Snf2 abolished for the first time promoter opening on otherwise sufficient induction in vivo. Together with previous findings, we recognize now a surprisingly complex network of five remodelers (RSC, SWI/SNF, INO80, Isw1 and Chd1) from four subfamilies (SWI/SNF, INO80, ISWI and CHD) as involved in PHO5 promoter chromatin remodeling. This is likely the first described complete remodeler set for a physiological chromatin transition. RSC was hardly involved at the coregulated PHO8 or PHO84 promoters despite cofactor recruitment by the same transactivator and RSC’s presence at all three promoters. Therefore, promoter-specific chromatin rather than transactivators determine remodeler requirements.     

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.     

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.     

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.     

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.     

Insulator: from chromatin domain boundary to gene regulation

An insulator or boundary element is a novel class of regulatory sequence and has properties consistent with a role in limiting enhancer activity; it helps to define the boundary between differentially regulated loci. Originally, an insulator was defined operationally by its ability to protect against positional effects and/or to block distal enhancer activity in transgenic assays, whereas recent experiments have focused on the role of insulators or boundaries in their native context. Many new features have been added to our concept of insulators, thereby revealing the complexity of their function. All these need to be accounted for by successful models. The research of insulator elements should provide new insights into the nature of enhancer function and gene regulation and provide an effective tool in applied gene therapy.     

Growth hormone gene regulation: a paradigm for cell-type-specific gene activation

The growth hormone (GH) gene is specifically expressed within specialized cells of the anterior pituitary. The central role in GH gene activation is played by GHF-1, a homeodomain protein that is itself specifically expressed in the anterior pituitary.     

The chromatin structure of an actively expressed, single copy yeast gene.

When the yeast galactokinase gene is not active (repressed, not expressed, quiescent), there is an exceptionally regular nucleosome array on coding sequence galactokinase chromatin, as shown by both denaturing and non-denaturing gel analysis of staphylococcal nuclease digests. Expression of the gene results in a limited smearing of the nucleosome repeat peaks and an increase in interpeak DNA, appearing as a regular ladder of DNA bands on denaturing gels. On non-denaturing gels the pattern is more complex and molecular weight dependent. These data suggest an increase in intracore particle DNA accessibility, allowing staphylococcal nuclease to digest throughout the nucleosome in expressed chromatin. Comparison to bulk chromatin and to an operationally inactive gene (35S rDNA) show that the alteration is specific to expressed chromatin. In contrast, DNase I shows no differences in the digestion of the gene specific chromatin in expressed or inactive states.     

Computational modeling of the EGF-receptor system: a paradigm for systems biology

Computational models have rarely been used as tools by biologists but, when models provide experimentally testable predictions, they can be extremely useful. The epidermal growth factor receptor (EGFR) is probably the best-understood receptor system, and computational models have played a significant part in its elucidation. For many years, models have been used to analyze EGFR dynamics and to interpret mutational studies, and are now being used to understand processes including signal transduction, autocrine loops and developmental patterning. The success of EGFR modeling can be a guide to combining models and experiments productively to understand complex biological processes as integrated systems.     

The nucleotide sequence of the yeast PHO5 gene: a putative precursor of repressible acid phosphatase contains a signal peptide.

The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.