Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

Bergman, Lawrence W.; Stranathan, M C; Preis, L H

doi:10.1128/mcb.6.1.38

Cited by 18 publications

(14 citation statements)

References 17 publications

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“…Minichromosomes, or circular plasmids packaged into chromatin, have been purified from S. cerevisiae for the analyses of transcription (48), retroviral integration (41), centromere function (27), and chromatin structure (7,53). We modified a 1.5-kb TRP1/ARS1 circle by inserting the PHO5 promoter and open reading frame to form pTA-PHO5 (Fig.…”

Section: Resultsmentioning

confidence: 99%

An In Vitro System Recapitulates Chromatin Remodeling at the PHO5 Promoter

Haswell

O’Shea

1999

Molecular and Cellular Biology

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The Saccharomyces cerevisiae gene PHO5 is an excellent system with which to study regulated changes in chromatin structure. The PHO5 promoter is packaged into four positioned nucleosomes under repressing conditions; upon induction, the structure of these nucleosomes is altered such that the promoter DNA becomes accessible to nucleases. We report here the development and characterization of an in vitro system in which partially purified PHO5 minichromosomes undergo promoter chromatin remodeling. Several hallmarks of the PHO5 chromatin transition in vivo were reproduced in this system. Chromatin remodeling of PHO5 minichromosomes required the transcription factors Pho4 and Pho2, was localized to the promoter region of PHO5, and was independent of the chromatin-remodeling complex Swi-Snf. In vitro chromatin remodeling also required the addition of fractionated nuclear extract and hydrolyzable ATP. This in vitro system should serve as a useful tool for identifying the components required for this reaction and for elucidating the mechanism by which the PHO5 promoter chromatin structure is changed.The packaging of eukaryotic DNA into nucleosomes presents a barrier to cellular processes that require specific contacts with DNA. During transcription, sequence-specific DNA binding proteins and the basal transcription apparatus must recognize and bind to appropriate promoter elements. Biochemical and genetic analyses demonstrate that the packaging of DNA into nucleosomes inhibits its stable association with transcription factors (2,29,37,57). A number of cellular activities capable of facilitating factor binding to chromatin have been identified (3). These activities are thought to function by directly modifying chromatin structure.The Saccharomyces cerevisiae gene PHO5 is a well-characterized system with which to study regulated gene expression. PHO5 encodes a secreted acid phosphatase whose transcription is regulated in response to environmental phosphate levels (for a review, see reference 32). When phosphate is plentiful, PHO5 expression is repressed; when phosphate is limiting, PHO5 expression is induced. Activation of PHO5 transcription requires two transcription factors: Pho4, a basic helix-loophelix protein, and Pho2, a homeodomain protein (58). In vitro, Pho2 enhances the binding of Pho4 to two regulatory sequences in the PHO5 promoter, UASp1 and UASp2 (4, 5).When transcription of PHO5 is activated, its promoter undergoes a dramatic change in chromatin structure (for a review, see reference 51). When yeast cells are grown in highphosphate medium, two pairs of positioned nucleosomes flank a DNase I-hypersensitive site, which contains UASp1 (Fig. 1, ϩP i ). UASp2 and the TATA box are packaged into nucleosomes Ϫ2 and Ϫ1, respectively. In vivo footprinting experiments indicate that Pho4 does not bind the PHO5 promoter under repressing conditions (57). When environmental phosphate is limiting, the positioned nucleosomes no longer protect the PHO5 promoter, and Pho4 binds to UASp1 and UASp2 (Fig. 1, ϪP i ). In vivo foo...

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Section: Resultsmentioning

confidence: 99%

An In Vitro System Recapitulates Chromatin Remodeling at the PHO5 Promoter

Haswell

O’Shea

1999

Molecular and Cellular Biology

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“…In budding yeast, deletion of either the PHO85 CDK or its interacting cyclin partner PHO80 leads to constitutive expression of P i acquisition genes including PHO5 (an acid phosphatase gene) in P i -sufficient medium (43)(44)(45)(46)(47). In contrast, phoA, the counterpart of PHO85 in A. nidulans, has no apparent role in regulation of acid phosphatase expression (6).…”

Section: Deletion Of An-pho80 Causes Constitutive Induction Of Phosphmentioning

confidence: 99%

The Pho80-like Cyclin of Aspergillus nidulans Regulates Development Independently of Its Role in Phosphate Acquisition

Dou

Hashmi

et al. 2004

Journal of Biological Chemistry

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In Saccharomyces cerevisiae, phosphate acquisition enzymes are regulated by a cyclin-dependent kinase (Pho85), a cyclin (Pho80), the cyclin-dependent kinase inhibitor Pho81, and the helix-loop-helix transcription factor Pho4 (the PHO system). Previous studies in Aspergillus nidulans indicate that a Pho85-like kinase, PHOA, does not regulate the classic PHO system but regulates development in a phosphate-dependent manner. A Pho80-like cyclin has now been isolated through its interaction with PHOA. Surprisingly, unlike PHOA, An-PHO80 does play a negative role in the PHO system. Similarly, an ortholog of Pho4 previously identified genetically as palcA also regulates the PHO system. However, An-PHO81, a putative cyclin-dependent kinase inhibitor, does not regulate the PHO system. Therefore, there are significant differences between the classic PHO system conserved between S. cerevisiae and Neurospora crassa compared with that which has evolved in A. nidulans. Most interestingly, under low phosphate conditions, the An-PHO80 cyclin also promotes sexual development while having a negative effect on asexual development. These effects are independent of the role An-PHO80 has in the classic PHO system. However, in high phosphate medium, An-PHO80 affects development because of deregulation of the PHO system as loss of palcA Pho4 function negates the developmental defects caused by lack of An-pho80. Therefore, under low phosphate conditions the An-PHO80 cyclin regulates development independently of the PHO system, whereas in high phosphate it affects development through the PHO system. The data indicate that a single cyclin can control various aspects of growth and development in a multicellular organism.

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“…We have compared different in vivo data, obtained by deletion mapping (7,19,24), with our in vitro data. The UASJ (-382 to -391) and UASI (-332 to -341) proposed by Bergman et al (6) do not coincide with our UASs obtained by DNase I and exonuclease III footprinting (Fig.…”

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confidence: 99%

The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

Vogel¹,

Hörz²,

Hinnen³

1989

Mol. Cell. Biol.

150

136

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The repressible acid phosphatase gene PHOS of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHOS gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHOS promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PH04 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASpl) and the other located at -239 to -262 (UASp2). Exonuclease m footprint experiments revealed stops at -349 and -368 (UASpl) as well as at -245 and -260 (UASp2). Gel retardation assays with the PH02 protein revealed a binding region that lay between the two PH04-binding sites. DNase I footprint analysis suggested a PH02-binding site covering the region between -277 and -296.The promoter region of the strongly regulated acid phosphatase gene PH05 (4) has been recently analyzed by deletion mapping (6,19,24). In addition to the TATA box, two regions were found (24) to be essential for activation of the PHOS promoter. Sequence comparisons defined four elements from which a 19-base-pair dyad consensus sequence was deduced. Three of the elements, called UASp, were located within deletions showing reduced transcription of PHOS (24), at -367, -245, and -185 relative to the translational start.Not much is known about the physical interaction of regulatory proteins with the PHOS promoter. Since mutations defective in PH02 or PH04 are epistatic to all other regulatory mutations and show a negative phenotype, it is likely that the PHO2 and PHO4 proteins are positive transcription factors that interact with the PHOS promoter DNA. The finding that a pho2 mutation can be complemented by overexpression of the PHO4 protein (20) is a further indication that the PHO2 and PHO4 proteins exert their functions at the same late hierarchical level of PH05 activation.Extensive studies of the chromatin fine structure of the PH05 gene (1, 2, 5, 7) have been done. Interestingly, the element at -367, which is essential for gene induction (24), resides in the center of a hypersensitive region under conditions of PH05 repression (1, 2).In press). Interestingly, the pho2 mutation showed no influence on the basal level of TRP4 expression but rather modulated the general control response of TRP4.MATERIALS AND METHODS Escherichia coli vector for PH04 and PH02 expression. The PH04 gene (13, 15) was reisolated from a S288C background by screening an ordered centromere library (25). An NcoI site at the ATG of PH04 was introduced by site-specific mutagenesis (14). The PH04 gene (NcoI-BamHI fragment) was placed under control of the inducible PL promoter by using the vector pPLmu, a derivative of PLc24 (23) containing the phage Mu Ner gene ribosome-binding site with an NcoI site at its ATG start codon (obtained from Biogen).The PH02 gene (25) was fused to the same vector but carrying a differ...

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Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

Cited by 18 publications

References 17 publications

An In Vitro System Recapitulates Chromatin Remodeling at the PHO5 Promoter

An In Vitro System Recapitulates Chromatin Remodeling at the PHO5 Promoter

The Pho80-like Cyclin of Aspergillus nidulans Regulates Development Independently of Its Role in Phosphate Acquisition

The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions.

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