Biomedical subjects

Wolfram Hörz

Publications and source records attributed to Wolfram Hörz.

11 recordsLinked to original sources

PubMed2006

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.

Acid Phosphatase↗

PubMed2005

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.

Acid Phosphatase↗

PubMed2005

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.

Acid Phosphatase↗

PubMed2004

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.

Adenosine Triphosphate↗

PubMed2004

SWRred not shaken; mixing the histones.

The recently isolated SWR1 complex catalyzes an ATP-dependent histone exchange with specificity for the histone variant H2A.Z. This provides a new theme in chromatin remodeling mechanisms and an explanation of how histone variants are incorporated into the nucleosome outside of S phase. In connection with the isolation of histone variant specific chaperone complexes, we are beginning to see the mechanisms that mix the histone octamer with intriguing implications for epigenetic inheritance.

Adenosine Triphosphatases↗

PubMed2004

Anatomy of a hypersensitive site.

The 600-bp accessible region at the activated PHO5 promoter in S. cerevisiae has become a paradigm for hypersensitive sites. In this review, we summarize the various experimental strategies used to characterize chromatin at the active promoter and point out their virtues and their limitations. We describe the properties of chromatin at the active PHO5 promoter and what we currently know about the transition from the inactive to the active state. The implications for generating a hypersensitive region in chromatin are discussed.

Acetylation↗

PubMed2004

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.

Chromatin↗

PubMed2003

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.

Acetylation↗

PubMed2003

Multiple mechanistically distinct functions of SAGA at the PHO5 promoter.

Our previous studies have shown that the rate of chromatin remodeling and consequently the rate of PHO5 activation are strongly decreased in the absence of Gcn5 histone acetyltransferase activity. Using chromatin immunoprecipitation, we demonstrate that SAGA is physically recruited to the PHO5 promoter. Recruitment is dependent on the specific activator Pho4 and occurs only under inducing conditions. Spt3, another subunit of SAGA, also plays a role in PHO5 activation but has a function that is completely different from that of Gcn5. An SPT3 deletion severely compromises the PHO5 promoter and reduces the extent of transcriptional activation by diminishing the binding of the TATA binding protein to the promoter without, however, affecting the rate or the extent of chromatin remodeling. A gcn5 spt3 double mutant shows a synthetic phenotype almost as severe as that observed for an spt7 or spt20 mutant. The latter two mutations are known to prevent the assembly of the complex and consequently lead to the loss of all SAGA functions. The absence of the Ada2 subunit causes a strong delay in chromatin remodeling and promoter activation that closely resembles the delay observed in the absence of Gcn5. A deletion of only the Ada2 SANT domain has exactly the same effect, strongly suggesting that Ada2 controls Gcn5 activity by virtue of its SANT domain. Finally, the Gcn5 bromodomain also contributes to but is not essential for Gcn5 function at the PHO5 promoter. Taken together, the results provide a detailed and differentiated description of the role of SAGA as a coactivator at the PHO5 promoter.

Carrier Proteins↗

PubMed2001

ATP-dependent nucleosome remodeling.

It has been a long-standing challenge to decipher the principles that enable cells to both organize their genomes into compact chromatin and ensure that the genetic information remains accessible to regulatory factors and enzymes within the confines of the nucleus. The discovery of nucleosome remodeling activities that utilize the energy of ATP to render nucleosomal DNA accessible has been a great leap forward. In vitro, these enzymes weaken the tight wrapping of DNA around the histone octamers, thereby facilitating the sliding of histone octamers to neighboring DNA segments, their displacement to unlinked DNA, and the accumulation of patches of accessible DNA on the surface of nucleosomes. It is presumed that the collective action of these enzymes endows chromatin with dynamic properties that govern all nuclear functions dealing with chromatin as a substrate. The diverse set of ATPases that qualify as the molecular motors of the nucleosome remodeling process have a common history and are part of a superfamily. The physiological context of their remodeling action builds on the association with a wide range of other proteins to form distinct complexes for nucleosome remodeling. This review summarizes the recent progress in our understanding of the mechanisms underlying the nucleosome remodeling reaction, the targeting of remodeling machines to selected sites in chromatin, and their integration into complex regulatory schemes.

Adenosine Triphosphatases↗

PubMed2001

Neither Reb1p nor poly(dA*T) elements are responsible for the highly specific chromatin organization at the ILV1 promoter.

Analysis of the chromatin structure at the yeast ILV1 locus revealed highly positioned nucleosomes covering the entire locus except for a hypersensitive site in the promoter region. All previously identified cis-acting elements required for GCN4-independent ILV1 basal level transcription, including a binding site for the REB1 protein (Reb1p), and a poly(dA*dT) element (26 As out of 32 nucleotides) situated 15 base pairs downstream of the Reb1p-binding site, reside within this hypersensitive site. The existence of a second A*T-rich element (25 As out of 33 nucleotides) present six base pairs upstream of the Reb1p-binding site, suggested that nucleosome exclusion from the hypersensitive site in the ILV1 promoter region might be dictated by synergistic action of the two poly(dA*dT) elements. Replacing one or both of them had, however, no effect on the chromatin structure of the ILV1 promoter, although drastically reduced basal transcription. Similarly, deletion of the Reb1p-binding site, albeit affecting ILV1 expression, had no detectable effect on chromatin at the ILV1 promoter. The absence of a good correlation between effects of these elements on gene activity and on chromatin structure at the ILV1 promoter indicates that the chromatin organization present at the ILV1 promoter is independent of the known regulatory elements and most likely dictated directly by the DNA sequence.

Bacterial Proteins↗