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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

Spt6-mediated chromatin assembly and transcriptional repression at the PHO5 promoter. (a) UASp1 and UASp2 are binding sites for the Pho2 and Pho4 transactivators. During activation, chromatin disassembly of the four yellow nucleosomes is promoted by Asf1 to allow access of the general transcription machinery to the promoter. During repression, chromatin is reassembled over the PHO5 promoter by the histone H3/H4 chaperone Spt6 to compete with the general transcription machinery for DNA binding. (b) Strain JKT0010 (WT), JMY0002 (spt6-140), and MAY0067 (spt6-1004) were grown in phosphate-depleted media to activate PHO5 transcription. Following a 4 hour shift to 39°C to inactivate Spt6, phosphate was added as a signal for PHO5 repression. Samples were assayed for phosphatase activity at the indicated times after addition of phosphate. (c) Strain JKT0010 (WT) and MAY0067 (spt6-1004) were initially grown in phosphate-rich media (+Pi) where PHO5 is repressed, then shifted to phosphate-depleted media to activate PHO5 transcription (-Pi). Following a 2 hour shift to 39°C, phosphate was added. Samples were taken at the indicated times, and analyzed for histone occupancy at PHO5 UASp2 by chromatin immunoprecipitation (ChIP) analysis. The amount of immunoprecipitated DNA was determined by quantitative PCR. As a control, primer sets were used for TELVIR. Quantitation of H3 levels over the UASp2 region is a ratio of immunoprecipitated UASp2 product relative to the immunoprecipitated TELVIR product divided by the ratio of input UASp2 product relative to the ratio of input TELVIR product. Averages of three independent experiments are shown; error bars indicate the 95% confidence interval.
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Figure 1: Spt6-mediated chromatin assembly and transcriptional repression at the PHO5 promoter. (a) UASp1 and UASp2 are binding sites for the Pho2 and Pho4 transactivators. During activation, chromatin disassembly of the four yellow nucleosomes is promoted by Asf1 to allow access of the general transcription machinery to the promoter. During repression, chromatin is reassembled over the PHO5 promoter by the histone H3/H4 chaperone Spt6 to compete with the general transcription machinery for DNA binding. (b) Strain JKT0010 (WT), JMY0002 (spt6-140), and MAY0067 (spt6-1004) were grown in phosphate-depleted media to activate PHO5 transcription. Following a 4 hour shift to 39°C to inactivate Spt6, phosphate was added as a signal for PHO5 repression. Samples were assayed for phosphatase activity at the indicated times after addition of phosphate. (c) Strain JKT0010 (WT) and MAY0067 (spt6-1004) were initially grown in phosphate-rich media (+Pi) where PHO5 is repressed, then shifted to phosphate-depleted media to activate PHO5 transcription (-Pi). Following a 2 hour shift to 39°C, phosphate was added. Samples were taken at the indicated times, and analyzed for histone occupancy at PHO5 UASp2 by chromatin immunoprecipitation (ChIP) analysis. The amount of immunoprecipitated DNA was determined by quantitative PCR. As a control, primer sets were used for TELVIR. Quantitation of H3 levels over the UASp2 region is a ratio of immunoprecipitated UASp2 product relative to the immunoprecipitated TELVIR product divided by the ratio of input UASp2 product relative to the ratio of input TELVIR product. Averages of three independent experiments are shown; error bars indicate the 95% confidence interval.

Mentions: The yeast PHO5 gene encodes an acid phosphatase and its expression is tightly regulated by intracellular phosphate levels. In low phosphate conditions, the sequence-specific transactivator Pho4 is dephosphorylated causing it to localize to the nucleus where it binds the PHO5 promoter [14,15]. Pho4 binding to the DNA initiates depletion of the four positioned nucleosomes that normally reside over the PHO5 promoter including the Pho4 binding site termed UASp2 and the TATA box [16]. In repressing conditions (high phosphate), Pho4 is phosphorylated by Pho80-Pho85, which causes its export to the cytoplasm [14,17]. Loss of Pho4 from the promoter leads to the histones being reassembled by Spt6 at the PHO5 promoter [16] (Figure 1). We have previously shown that inactivation of Spt6 prior to addition of the signal for repression (phosphate) results in the PHO5 promoter remaining nucleosome-depleted and transcriptionally active in vivo, even though the activators no longer occupy the promoter [13]. This indicates that the main role of some transcriptional activators is to maintain promoters in a nucleosome-depleted state, which in turn indirectly allows the binding of the general transcription machinery to the core promoter.


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)

Spt6-mediated chromatin assembly and transcriptional repression at the PHO5 promoter. (a) UASp1 and UASp2 are binding sites for the Pho2 and Pho4 transactivators. During activation, chromatin disassembly of the four yellow nucleosomes is promoted by Asf1 to allow access of the general transcription machinery to the promoter. During repression, chromatin is reassembled over the PHO5 promoter by the histone H3/H4 chaperone Spt6 to compete with the general transcription machinery for DNA binding. (b) Strain JKT0010 (WT), JMY0002 (spt6-140), and MAY0067 (spt6-1004) were grown in phosphate-depleted media to activate PHO5 transcription. Following a 4 hour shift to 39°C to inactivate Spt6, phosphate was added as a signal for PHO5 repression. Samples were assayed for phosphatase activity at the indicated times after addition of phosphate. (c) Strain JKT0010 (WT) and MAY0067 (spt6-1004) were initially grown in phosphate-rich media (+Pi) where PHO5 is repressed, then shifted to phosphate-depleted media to activate PHO5 transcription (-Pi). Following a 2 hour shift to 39°C, phosphate was added. Samples were taken at the indicated times, and analyzed for histone occupancy at PHO5 UASp2 by chromatin immunoprecipitation (ChIP) analysis. The amount of immunoprecipitated DNA was determined by quantitative PCR. As a control, primer sets were used for TELVIR. Quantitation of H3 levels over the UASp2 region is a ratio of immunoprecipitated UASp2 product relative to the immunoprecipitated TELVIR product divided by the ratio of input UASp2 product relative to the ratio of input TELVIR product. Averages of three independent experiments are shown; error bars indicate the 95% confidence interval.
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Related In: Results  -  Collection

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Figure 1: Spt6-mediated chromatin assembly and transcriptional repression at the PHO5 promoter. (a) UASp1 and UASp2 are binding sites for the Pho2 and Pho4 transactivators. During activation, chromatin disassembly of the four yellow nucleosomes is promoted by Asf1 to allow access of the general transcription machinery to the promoter. During repression, chromatin is reassembled over the PHO5 promoter by the histone H3/H4 chaperone Spt6 to compete with the general transcription machinery for DNA binding. (b) Strain JKT0010 (WT), JMY0002 (spt6-140), and MAY0067 (spt6-1004) were grown in phosphate-depleted media to activate PHO5 transcription. Following a 4 hour shift to 39°C to inactivate Spt6, phosphate was added as a signal for PHO5 repression. Samples were assayed for phosphatase activity at the indicated times after addition of phosphate. (c) Strain JKT0010 (WT) and MAY0067 (spt6-1004) were initially grown in phosphate-rich media (+Pi) where PHO5 is repressed, then shifted to phosphate-depleted media to activate PHO5 transcription (-Pi). Following a 2 hour shift to 39°C, phosphate was added. Samples were taken at the indicated times, and analyzed for histone occupancy at PHO5 UASp2 by chromatin immunoprecipitation (ChIP) analysis. The amount of immunoprecipitated DNA was determined by quantitative PCR. As a control, primer sets were used for TELVIR. Quantitation of H3 levels over the UASp2 region is a ratio of immunoprecipitated UASp2 product relative to the immunoprecipitated TELVIR product divided by the ratio of input UASp2 product relative to the ratio of input TELVIR product. Averages of three independent experiments are shown; error bars indicate the 95% confidence interval.
Mentions: The yeast PHO5 gene encodes an acid phosphatase and its expression is tightly regulated by intracellular phosphate levels. In low phosphate conditions, the sequence-specific transactivator Pho4 is dephosphorylated causing it to localize to the nucleus where it binds the PHO5 promoter [14,15]. Pho4 binding to the DNA initiates depletion of the four positioned nucleosomes that normally reside over the PHO5 promoter including the Pho4 binding site termed UASp2 and the TATA box [16]. In repressing conditions (high phosphate), Pho4 is phosphorylated by Pho80-Pho85, which causes its export to the cytoplasm [14,17]. Loss of Pho4 from the promoter leads to the histones being reassembled by Spt6 at the PHO5 promoter [16] (Figure 1). We have previously shown that inactivation of Spt6 prior to addition of the signal for repression (phosphate) results in the PHO5 promoter remaining nucleosome-depleted and transcriptionally active in vivo, even though the activators no longer occupy the promoter [13]. This indicates that the main role of some transcriptional activators is to maintain promoters in a nucleosome-depleted state, which in turn indirectly allows the binding of the general transcription machinery to the core promoter.

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