Biomedical subjects

W Hörz

Publications and source records attributed to W Hörz.

At least 19 recordsLinked to original sources

PubMed1997

RNA polymerase II holoenzyme recruitment is sufficient to remodel chromatin at the yeast PHO5 promoter.

We examine transcriptional activation and chromatin remodeling at the PHO5 promoter in yeast by fusion proteins that are thought to act by recruiting the RNA polymerase II holoenzyme to DNA in the absence of a classic activating region. These hybrid proteins (e.g., Gal11+Pho4 or Gal4(58-97)+Pho4 in the presence of a GAL11P allele) efficiently activated transcription and remodeled chromatin. Similar chromatin remodeling was observed at a PHO5 promoter deleted for TATA and thus unable to support transcription. We conclude that recruitment of the holoenzyme or associated proteins suffices for chromatin remodeling. We also show that the SWI/SNF complex is required neither for efficient transcription of the wild-type PHO5 nor the GAL1 promoters, and we observe nearly complete chromatin remodeling at PHO5 in the absence of Snf2.

Chromatin↗

PubMed1997

Transcription factors vs nucleosomes: regulation of the PHO5 promoter in yeast.

Activation of the Saccharomyces cerevisiae PHO5 gene is accompanied by the disruption of four positioned nucleosomes at the promoter. The chromatin transition requires a DNA-binding protein, Pho4, and its transactivation domain. The mechanism of nucleosome disruption and the contribution of the nucleosomes to PHO5 regulation are reviewed.

Chromatin↗

PubMed1996

The homeodomain protein Pho2 and the basic-helix-loop-helix protein Pho4 bind DNA cooperatively at the yeast PHO5 promoter.

Two transcription factors, the bHLH protein Pho4 and the homeodomain protein Pho2, are required for transcriptional activation of the PHO5 promoter in Saccharomyces cerevisiae. There are two essential Pho4 binding sites, corresponding to the regulatory elements UASp1 and UASp2 at the PHO5 promoter, but only a single, dispensable Pho2 binding site had previously been identified. We have reinvestigated binding of Pho2 to the PHO5 promoter using purified recombinant protein and have found multiple Pho2 binding sites of different affinities along the promoter. One of the high affinity Pho2 sites largely overlaps the Pho4 binding site at UASp1. Cooperative DNA binding of the two proteins to their overlapping sites, resulting in a high-affinity ternary complex, was demonstrated. Pho2 and Pho4 also bind DNA cooperatively at UASp2 where two Pho2 sites flank the Pho4 site. Finally, Pho2 facilitates binding of Pho4 to a third, cryptic Pho4 binding site which binds Pho4 with lower affinity than UASp1 or UASp2. These results suggest that cooperative DNA binding with Pho4 is integral to the mechanism by which Pho2 regulates transcription of the PHO5 gene.

Base Sequence↗

PubMed1996

Regulation of gene expression by nucleosomes.

During the past year, the characterization of mechanisms and factors capable of disrupting nucleosomes during transcriptional activation has been a recurrent theme in studies which address the contribution of nucleosome structure to gene regulation. In vivo studies using yeast and Drosophila together with biochemical purification schemes using nucleosome perturbation assays have provided evidence for the existence of multiprotein complexes that are able to alleviate nucleosome repression. At the same time, new insights into the mechanism of heterochromatin formation have been gained, which have direct links to nucleosome structure.

Animals↗

PubMed1995

Interplay between nucleosomes and transcription factors at the yeast PHO5 promoter.

In this review, we summarize experiments which have used the yeast PHO5 gene to determine the functional consequences of nucleosome structure in the promoter region. In the PHO5 system, nucleosomes participate in promoter repression by interfering with factor binding. Therefore, disruption of nucleosome structure is likely a prerequisite for promoter activation. There still remain several important questions regarding the assembly and disassembly of chromatin repression. Recent experiments have shown that the PHO5 chromatin transition is replication and transcription independent. Nucleosome disruption does, however, depend upon binding of a transactivator, Pho4, to the PHO5 promoter. Moreover, the activation domain of Pho4 appears to play a critical role in chromatin disruption.

Chromatin↗

PubMed1994

A nucleosome precludes binding of the transcription factor Pho4 in vivo to a critical target site in the PHO5 promoter.

Activation of the Saccharomyces cerevisiae PHO5 gene by phosphate starvation is accompanied by the disappearance of two pairs of positioned nucleosomes that flank a short hypersensitive region in the promoter. The transcription factor Pho4 is the key regulator of this transition. By in vitro footprinting it was previously shown that there is a low affinity site (UASp1) which is contained in the short hypersensitive region in the inactive promoter, and a high affinity site (UASp2) which is located in the adjacent nucleosome. To investigate the interplay between nucleosomes and Pho4, we have performed in vivo footprinting experiments with dimethylsulfate. Pho4 was found to bind to both sites in the active promoter. In contrast, it binds to neither site in the repressed promoter. Lack of binding under repressing conditions is largely due to the low affinity of Pho4 for its binding sites under these conditions. Despite the increased affinity of Pho4 for its target sites under activating conditions, binding to UASp2 is prevented by the presence of the nucleosome and can only occur after prior disruption of this nucleosome in a process that requires UASp1. Protection of the PHO5 UASp2 by the nucleosome is not absolute, however, since overexpression of Pho4 can disrupt this nucleosome even when UASp1 is deleted. Also under these conditions, with only UASp2 present, all four nucleosomes at the PHO5 promoter are disrupted, whereas no chromatin change at all is observed when both UAS elements are destroyed.

Allosteric Regulation↗

PubMed1994

The transactivation domain of Pho4 is required for nucleosome disruption at the PHO5 promoter.

The chromatin structure of the PHO5 promoter is disrupted when the promoter is derepressed by phosphate starvation. The transactivator, Pho4, is primarily responsible for this change. We have used deletion mutations of Pho4 in order to determine which protein domains are involved in nucleosome dissolution. Our results show that the DNA binding domain by itself is not sufficient to trigger chromatin disruption, even when overexpressed. In vivo footprinting reveals that Pho4 derivatives lacking the N-terminal activation domain can bind to UASp1, which resides in a constitutively nucleosome-free region, but not to UASp2, which lies within a nucleosome in the repressed PHO5 promoter. The acidic activation domain of Pho4 appears to be involved in nucleosome disruption. Substitution of the native transactivation domain of Pho4 with that from VP16 results in substantial chromatin disruption. In every case, the ability of the Pho4 mutants to activate transcription correlates with their ability to disrupt nucleosome structure in the PHO5 promoter. Therefore, we conclude that the Pho4 activation domain has at least two roles: (i) to trigger disruption of nucleosome structure over the promoter, thereby facilitating the binding of transcription factors, and (ii) to interact with the transcriptional apparatus at the proximal promoter.

Amino Acid Sequence↗

PubMed1994

Analysis of the promoter of the human prostatic acid phosphatase gene.

From the analysis of two overlapping cosmid clones prepared from human genomic DNA libraries, a contig of 44 kb containing a 5' portion of the PAP gene and 17 kb of the upstream region was established. It was characterized by restriction mapping and sequence analysis of 2.5 kb upstream of the initiation codon. Two major transcription initiation sites were found to be located around 56 and 91 bp upstream of the initiation codon, as determined by nuclease S1 and primer extension mapping. Expression of the PAP gene was measured by Northern blots in the androgen responsive LNCaP cell line. It was found to be induced 2-3-fold by the addition of the synthetic androgen mibolerone to the cells. The induced mRNA levels were approx. 10-times lower than those for the prostate-specific antigen (PSA) in LNCaP cells.

Acid Phosphatase↗

PubMed1994

[Chromatin structure and gene regulation].

It is becoming increasingly clear that the nucleosome, the basic repeat unit of eukaryotic chromatin, is involved also in gene regulation. In particular, the study of inducible genes has shown that nucleosomes contribute to the repressed basal state, and that they can be rearranged in response to induction. The role of the nucleosomes in gene regulation and possible mechanisms for their structural modulation are discussed.

Acid Phosphatase↗

PubMed1993

Structural and functional requirements for the chromatin transition at the PHO5 promoter in Saccharomyces cerevisiae upon PHO5 activation.

The PHO5 promoter from Saccharomyces cerevisiae can exist in two chromatin configurations depending on its state of activity. In the repressed promoter a short hypersensitive site containing a binding site for the transcription factor PHO4 is flanked by specifically positioned nucleosomes. After induction two nucleosomes upstream and two downstream of the hypersensitive site are disrupted, and the entire promoter becomes accessible. We have investigated mechanisms responsible for setting up the structure of the repressed state and for the transition. Episomal centromeric plasmids bearing the PHO5 promoter show the same chromatin structure as the endogenous chromosomal copy arguing that the chromosomal context is not essential and that the nucleosomal organization is not set up from a distance. Deleting most of the hypersensitive region including the PHO4 binding site also leaves the positioning of the adjacent nucleosomes in the repressed promoter unchanged indicating that histone-DNA interactions play an important role in setting up nucleosome positions. However, when half of the DNA of a nucleosome is deleted a new nucleosome forms at the same location with respect to the neighboring nucleosome indicating that boundary effects also contribute to nucleosome positioning in the native promoter. Disruption of the nucleosomes under activating conditions is shown to require interaction of PHO4 with its binding site located within the hypersensitive region. This disruption takes place also in two independent constructs in which the TATA box had been deleted and as a result the gene was not transcribed. This result shows for the first time that the generation of active chromatin at a regulated promoter is not the result of gene expression but occurs prior to transcription.

Base Sequence↗

PubMed1993

Histones, nucleosomes and transcription.

It is becoming increasingly clear that nucleosome structure is integrally involved in gene regulation. In particular, the study of inducible genes has shown that nucleosomes not only contribute to a repressed basal state, but can also be rearranged in response to induction. The mechanism of this process is just beginning to be elucidated, and genetic studies have implicated several proteins in the modulation of nucleosome structure.

Animals↗

PubMed1993

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work, the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nM of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease S1 analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcription 96 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to cells results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

Androgens↗

PubMed1992

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.

Acid Phosphatase↗

PubMed1992

Activation of the weakly regulated PHO8 promoter in S. cerevisiae: chromatin transition and binding sites for the positive regulatory protein PHO4.

PHO8 encodes an alkaline phosphatase in Saccharomyces cerevisiae whose transcription is regulated by the phosphate concentration in the medium. This occurs through the action of several positive and negative regulatory proteins, also involved in the regulation of other members of the phosphatase gene family. A central role is played by PHO4, the gene encoding a DNA binding regulatory protein. Digestion experiments with DNasel, micrococcal nuclease and 20 different restriction nucleases show that under conditions of PHO8 repression, there is a highly ordered chromatin structure at the promoter consisting of three hypersensitive regions, approximately 820 to 690, 540 to 510, and 230 to 160 bp upstream of the initiation codon. These hypersensitive sites are surrounded by DNA organized in nucleosomes. Gel shift analysis and in vitro footprinting revealed the presence of two PHO4 binding sites at the PHO8 promoter: a low affinity site at -728 and a high affinity site at -532. Each one is located within a hypersensitive site. Upon derepression of PHO8, the chromatin structure changes significantly: The two upstream hypersensitive sites containing the PHO4 binding sites merge, resulting in a long region of hypersensitivity. This transition is PHO4 dependent. However, not all of the promoter becomes nucleosome free. Instead, as a novel feature, regions of intermediate accessibility are generated upstream and downstream of the third hypersensitive site, the latter region encompassing the TATA-box. The available data fit best into a concept that these regions are organized in unstable or partly unfolded nucleosomes.

Alkaline Phosphatase↗

PubMed1991

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.

Base Sequence↗

PubMed1991

[In vitro culture of pulp cells].

The purpose of our study was the culture of cellular elements of the pulp. Migration and proliferation of fibroblast-like cells can be observed during the in vitro culture of pulpal tissue. However, no mitotic acticity of the odontoblasts could be induced by nutritive variations of the culture medium. The proliferation of the fibroblastlike cells varied during the primary culture and the first subculture. By contrast, during the second subculture there is a homogeneous process of growth. The growth rate of the second subcultures is therefore suitable for a biological evaluation of dental materials.

Animals↗

PubMed1990

Role of trans-activating proteins in the generation of active chromatin at the PHO5 promoter in S. cerevisiae.

Induction of the PHO5 gene in Saccharomyces cerevisiae by phosphate starvation was previously shown to be accompanied by the removal of four positioned nucleosomes from the promoter. We have now investigated the role of two trans-activating proteins, encoded by PHO2 and PHO4, which bind to the PHO5 promoter. Both proteins are absolutely required for the chromatin transition to occur as shown by analysis of null mutants of the two genes. Transformation of these mutant strains with plasmids containing the respective genes restores the wild type chromatin response. Increasing the gene dosage of PHO2 and of PHO4 makes it possible to differentiate functionally between the two proteins. From over-expressing PHO4 in a wild type and also in a pho2 null mutant strain and complementary experiments with PHO2, it is concluded that the PHO4 protein is the primary trigger for the chromatin transition, consistent with one of its two binding sites being located between positioned nucleosomes in repressed chromatin and thereby accessible. PHO2, the binding site of which is located within a nucleosome under conditions of PHO5 repression, contributes to the chromatin transition either by destabilizing histone-DNA interactions or by under-going interactions with PHO4.

Acid Phosphatase↗

PubMed1990

An experimental study on the influence of the derived casts on the accuracy of different recording materials. Part I: Plaster, impression compound, and wax.

An experimental study was conducted to analyze the accuracy of transferring jaw relations with recording materials and the derived casts. A specific measuring system was designed to determine three-dimensional deviations of the condyles of an articulator. Four interocclusal recording materials were analyzed after various storage periods. The results indicated that the accuracy of the derived cast had a critical influence on the accuracy of the transferred jaw relations. None of the materials were reliable; the most accurate material generated deviations of approximately 300 microns.

Analysis of Variance↗