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Epigenetic inheritance of an inducibly nucleosome-depleted promoter and its associated transcriptional state in the apparent absence of transcriptional activators.

Ohsawa R, Adkins M, Tyler JK - Epigenetics Chromatin (2009)

Bottom Line: Dynamic changes to the chromatin structure play a critical role in transcriptional regulation.Notably, the epigenetic inheritance of the nucleosome-depleted PHO5 promoter through DNA replication does not require ongoing transcription.Our results suggest that there may be a memory or an epigenetic mark on the nucleosome-depleted PHO5 promoter that is independent of the transcription apparatus and maintains the promoter in a nucleosome-depleted state through DNA replication.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, USA. ryosuke.ohsawa@ucdenver.edu

ABSTRACT

Background: Dynamic changes to the chromatin structure play a critical role in transcriptional regulation. This is exemplified by the Spt6-mediated histone deposition on to histone-depleted promoters that results in displacement of the general transcriptional machinery during transcriptional repression.

Results: Using the yeast PHO5 promoter as a model, we have previously shown that blocking Spt6-mediated histone deposition on to the promoter leads to persistent transcription in the apparent absence of transcriptional activators in vivo. We now show that the nucleosome-depleted PHO5 promoter and its associated transcriptionally active state can be inherited through DNA replication even in the absence of transcriptional activators. Transcriptional reinitiation from the nucleosome-depleted PHO5 promoter in the apparent absence of activators in vivo does not require Mediator. Notably, the epigenetic inheritance of the nucleosome-depleted PHO5 promoter through DNA replication does not require ongoing transcription.

Conclusion: Our results suggest that there may be a memory or an epigenetic mark on the nucleosome-depleted PHO5 promoter that is independent of the transcription apparatus and maintains the promoter in a nucleosome-depleted state through DNA replication.

No MeSH data available.


Related in: MedlinePlus

Mediator is not required for PHO5 transcription in the absence of activators in vivo. (a) Mediator is required for transcriptional initiation of PHO5 in vivo. Strains JKT0010 (WT), ROY010 (pho80), and ROY011 (pho80 srb4) growing in phosphate containing media (PHO5 repressed) were shifted to 39°C in low phosphate media, followed by analysis of PHO5 induction via the phosphatase activity assay. (b) Mediator is not required for continued transcription from the nucleosome-depleted promoter in the absence of activators. Strains JKT0010 (WT), JMY002 (spt6), and SKW067 (spt6 srb4) were shifted to 39°C while PHO5 transcription was occurring in low phosphate media, followed by the addition of phosphate as the signal to repress PHO5 transcription and analysis of PHO5 induction via the phosphatase activity assay.
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Figure 3: Mediator is not required for PHO5 transcription in the absence of activators in vivo. (a) Mediator is required for transcriptional initiation of PHO5 in vivo. Strains JKT0010 (WT), ROY010 (pho80), and ROY011 (pho80 srb4) growing in phosphate containing media (PHO5 repressed) were shifted to 39°C in low phosphate media, followed by analysis of PHO5 induction via the phosphatase activity assay. (b) Mediator is not required for continued transcription from the nucleosome-depleted promoter in the absence of activators. Strains JKT0010 (WT), JMY002 (spt6), and SKW067 (spt6 srb4) were shifted to 39°C while PHO5 transcription was occurring in low phosphate media, followed by the addition of phosphate as the signal to repress PHO5 transcription and analysis of PHO5 induction via the phosphatase activity assay.

Mentions: Next, we set out to identify the nature of the epigenetic mark or factor on the PHO5 promoter that signals for it to remain nucleosome-depleted even after DNA replication in the apparent absence of activators. Given that the histones are mostly depleted from the promoter, we focused on the transcription machinery rather than chromatin modifications or chromatin remodelers per se. First, we asked whether Mediator is required for continued transcription from the nucleosome-depleted promoter in the absence of activators, because Mediator is known to be required for transcriptional reinitiation in vitro [18,19]. To inactivate Mediator, we used a conditional mutant of the Mediator subunit Srb4. We first confirmed that Mediator is required for transcriptional initiation from the PHO5 promoter in vivo. As inactivation of Srb4 by a shift to 39°C blocks cell growth, we performed these experiments in the context of a temperature-sensitive mutation of PHO80, which allowed us to bypass the need for cell growth (in order to use up cellular phosphate stores) that is normally required for PHO5 induction. In this experiment, we clearly see that PHO5 is induced upon inactivation of Pho80 as expected, and that this induction of PHO5 requires Srb4 because it does not occur in the pho80 srb4 double mutant (Figure 3(a)). As such, Mediator is required for initiation of transcription from the PHO5 gene.


Epigenetic inheritance of an inducibly nucleosome-depleted promoter and its associated transcriptional state in the apparent absence of transcriptional activators.

Ohsawa R, Adkins M, Tyler JK - Epigenetics Chromatin (2009)

Mediator is not required for PHO5 transcription in the absence of activators in vivo. (a) Mediator is required for transcriptional initiation of PHO5 in vivo. Strains JKT0010 (WT), ROY010 (pho80), and ROY011 (pho80 srb4) growing in phosphate containing media (PHO5 repressed) were shifted to 39°C in low phosphate media, followed by analysis of PHO5 induction via the phosphatase activity assay. (b) Mediator is not required for continued transcription from the nucleosome-depleted promoter in the absence of activators. Strains JKT0010 (WT), JMY002 (spt6), and SKW067 (spt6 srb4) were shifted to 39°C while PHO5 transcription was occurring in low phosphate media, followed by the addition of phosphate as the signal to repress PHO5 transcription and analysis of PHO5 induction via the phosphatase activity assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2749832&req=5

Figure 3: Mediator is not required for PHO5 transcription in the absence of activators in vivo. (a) Mediator is required for transcriptional initiation of PHO5 in vivo. Strains JKT0010 (WT), ROY010 (pho80), and ROY011 (pho80 srb4) growing in phosphate containing media (PHO5 repressed) were shifted to 39°C in low phosphate media, followed by analysis of PHO5 induction via the phosphatase activity assay. (b) Mediator is not required for continued transcription from the nucleosome-depleted promoter in the absence of activators. Strains JKT0010 (WT), JMY002 (spt6), and SKW067 (spt6 srb4) were shifted to 39°C while PHO5 transcription was occurring in low phosphate media, followed by the addition of phosphate as the signal to repress PHO5 transcription and analysis of PHO5 induction via the phosphatase activity assay.
Mentions: Next, we set out to identify the nature of the epigenetic mark or factor on the PHO5 promoter that signals for it to remain nucleosome-depleted even after DNA replication in the apparent absence of activators. Given that the histones are mostly depleted from the promoter, we focused on the transcription machinery rather than chromatin modifications or chromatin remodelers per se. First, we asked whether Mediator is required for continued transcription from the nucleosome-depleted promoter in the absence of activators, because Mediator is known to be required for transcriptional reinitiation in vitro [18,19]. To inactivate Mediator, we used a conditional mutant of the Mediator subunit Srb4. We first confirmed that Mediator is required for transcriptional initiation from the PHO5 promoter in vivo. As inactivation of Srb4 by a shift to 39°C blocks cell growth, we performed these experiments in the context of a temperature-sensitive mutation of PHO80, which allowed us to bypass the need for cell growth (in order to use up cellular phosphate stores) that is normally required for PHO5 induction. In this experiment, we clearly see that PHO5 is induced upon inactivation of Pho80 as expected, and that this induction of PHO5 requires Srb4 because it does not occur in the pho80 srb4 double mutant (Figure 3(a)). As such, Mediator is required for initiation of transcription from the PHO5 gene.

Bottom Line: Dynamic changes to the chromatin structure play a critical role in transcriptional regulation.Notably, the epigenetic inheritance of the nucleosome-depleted PHO5 promoter through DNA replication does not require ongoing transcription.Our results suggest that there may be a memory or an epigenetic mark on the nucleosome-depleted PHO5 promoter that is independent of the transcription apparatus and maintains the promoter in a nucleosome-depleted state through DNA replication.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO, USA. ryosuke.ohsawa@ucdenver.edu

ABSTRACT

Background: Dynamic changes to the chromatin structure play a critical role in transcriptional regulation. This is exemplified by the Spt6-mediated histone deposition on to histone-depleted promoters that results in displacement of the general transcriptional machinery during transcriptional repression.

Results: Using the yeast PHO5 promoter as a model, we have previously shown that blocking Spt6-mediated histone deposition on to the promoter leads to persistent transcription in the apparent absence of transcriptional activators in vivo. We now show that the nucleosome-depleted PHO5 promoter and its associated transcriptionally active state can be inherited through DNA replication even in the absence of transcriptional activators. Transcriptional reinitiation from the nucleosome-depleted PHO5 promoter in the apparent absence of activators in vivo does not require Mediator. Notably, the epigenetic inheritance of the nucleosome-depleted PHO5 promoter through DNA replication does not require ongoing transcription.

Conclusion: Our results suggest that there may be a memory or an epigenetic mark on the nucleosome-depleted PHO5 promoter that is independent of the transcription apparatus and maintains the promoter in a nucleosome-depleted state through DNA replication.

No MeSH data available.


Related in: MedlinePlus