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The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1.

Liang CY, Hsu PH, Chou DF, Pan CY, Wang LC, Huang WC, Tsai MD, Lo WS - Nucleic Acids Res. (2011)

Bottom Line: Overexpression of Rph1 reduced the expression of PHR1 and increased UV sensitivity.The catalytically deficient mutant (H235A) of Rph1 diminished the repressive transcriptional effect on PHR1 expression, which indicates that histone demethylase activity contributes to transcriptional repression.Notably, overexpression of Rph1 and H3K36A mutant reduced histone acetylation at the URS, which implies a crosstalk between histone demethylation and acetylation at the PHR1 promoter.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
The dynamics of histone methylation have emerged as an important issue since the identification of histone demethylases. We studied the regulatory function of Rph1/KDM4 (lysine demethylase), a histone H3K36 demethylase, on transcription in Saccharomyces cerevisiae. Overexpression of Rph1 reduced the expression of PHR1 and increased UV sensitivity. The catalytically deficient mutant (H235A) of Rph1 diminished the repressive transcriptional effect on PHR1 expression, which indicates that histone demethylase activity contributes to transcriptional repression. Chromatin immunoprecipitation analysis demonstrated that Rph1 was associated at the upstream repression sequence of PHR1 through zinc-finger domains and was dissociated after UV irradiation. Notably, overexpression of Rph1 and H3K36A mutant reduced histone acetylation at the URS, which implies a crosstalk between histone demethylation and acetylation at the PHR1 promoter. In addition, the crucial checkpoint protein Rad53 acted as an upstream regulator of Rph1 and dominated the phosphorylation of Rph1 that was required for efficient PHR1 expression and the dissociation of Rph1. The release of Rph1 from chromatin also required the phosphorylation at S652. Our study demonstrates that the histone demethylase Rph1 is associated with a specific chromatin locus and modulates histone modifications to repress a DNA damage responsive gene under control of damage checkpoint signaling.

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Rph1 is dissociated from PHR1 promoter in response to UV irradiation. (A) The indicated strains were irradiated (UV: 20 mJ/cm2) and harvested for ChIP with anti-HA antibody. IB with anti-HA antibody showed the expression of Rph1. Anti-Pgk1 was the loading control and the ratio of Rph1/Pgk1 is indicated. (B) The quantitative result is shown from comparable samples in (A). (C) ChIP assay from samples in (A) with anti-H3K36me3 and anti-acH3 antibodies was followed by qPCR to monitor the URS region signals. Data are from three biological repeats. *P < 0.05.
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Figure 4: Rph1 is dissociated from PHR1 promoter in response to UV irradiation. (A) The indicated strains were irradiated (UV: 20 mJ/cm2) and harvested for ChIP with anti-HA antibody. IB with anti-HA antibody showed the expression of Rph1. Anti-Pgk1 was the loading control and the ratio of Rph1/Pgk1 is indicated. (B) The quantitative result is shown from comparable samples in (A). (C) ChIP assay from samples in (A) with anti-H3K36me3 and anti-acH3 antibodies was followed by qPCR to monitor the URS region signals. Data are from three biological repeats. *P < 0.05.

Mentions: In response to DNA damage, transcriptional induction of PHR1 should require a priori de-repression. Previously, Rph1 was named photolyase regulatory protein (PRP), which bound to the PHR1 URS and regulated the induction of PHR1 transcription after DNA damage (24). We performed ChIP assays to study the dynamics of Rph1 in response to DNA damage in vivo. After UV irradiation, the association of both Rph1 and the rph1-H235A mutant was significantly decreased, which suggests that Rph1 was released from the promoter of PHR1 after DNA damage (Figure 4A). To determine whether the protein level of Rph1 was affected by DNA damage, Rph1-HA expression was analyzed by immunoblotting (IB; Figure 4A). Rph1 levels were only slightly decreased after UV irradiation, which suggests that the dissociation from the promoter cannot simply be attributed to protein expression levels of Rph1 and rph1-H235A. The quantitative-ChIP results also confirmed the association and dissociation of Rph1 at the PHR1 promoter in vivo (Figure 4B).Figure 4.


The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1.

Liang CY, Hsu PH, Chou DF, Pan CY, Wang LC, Huang WC, Tsai MD, Lo WS - Nucleic Acids Res. (2011)

Rph1 is dissociated from PHR1 promoter in response to UV irradiation. (A) The indicated strains were irradiated (UV: 20 mJ/cm2) and harvested for ChIP with anti-HA antibody. IB with anti-HA antibody showed the expression of Rph1. Anti-Pgk1 was the loading control and the ratio of Rph1/Pgk1 is indicated. (B) The quantitative result is shown from comparable samples in (A). (C) ChIP assay from samples in (A) with anti-H3K36me3 and anti-acH3 antibodies was followed by qPCR to monitor the URS region signals. Data are from three biological repeats. *P < 0.05.
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Figure 4: Rph1 is dissociated from PHR1 promoter in response to UV irradiation. (A) The indicated strains were irradiated (UV: 20 mJ/cm2) and harvested for ChIP with anti-HA antibody. IB with anti-HA antibody showed the expression of Rph1. Anti-Pgk1 was the loading control and the ratio of Rph1/Pgk1 is indicated. (B) The quantitative result is shown from comparable samples in (A). (C) ChIP assay from samples in (A) with anti-H3K36me3 and anti-acH3 antibodies was followed by qPCR to monitor the URS region signals. Data are from three biological repeats. *P < 0.05.
Mentions: In response to DNA damage, transcriptional induction of PHR1 should require a priori de-repression. Previously, Rph1 was named photolyase regulatory protein (PRP), which bound to the PHR1 URS and regulated the induction of PHR1 transcription after DNA damage (24). We performed ChIP assays to study the dynamics of Rph1 in response to DNA damage in vivo. After UV irradiation, the association of both Rph1 and the rph1-H235A mutant was significantly decreased, which suggests that Rph1 was released from the promoter of PHR1 after DNA damage (Figure 4A). To determine whether the protein level of Rph1 was affected by DNA damage, Rph1-HA expression was analyzed by immunoblotting (IB; Figure 4A). Rph1 levels were only slightly decreased after UV irradiation, which suggests that the dissociation from the promoter cannot simply be attributed to protein expression levels of Rph1 and rph1-H235A. The quantitative-ChIP results also confirmed the association and dissociation of Rph1 at the PHR1 promoter in vivo (Figure 4B).Figure 4.

Bottom Line: Overexpression of Rph1 reduced the expression of PHR1 and increased UV sensitivity.The catalytically deficient mutant (H235A) of Rph1 diminished the repressive transcriptional effect on PHR1 expression, which indicates that histone demethylase activity contributes to transcriptional repression.Notably, overexpression of Rph1 and H3K36A mutant reduced histone acetylation at the URS, which implies a crosstalk between histone demethylation and acetylation at the PHR1 promoter.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
The dynamics of histone methylation have emerged as an important issue since the identification of histone demethylases. We studied the regulatory function of Rph1/KDM4 (lysine demethylase), a histone H3K36 demethylase, on transcription in Saccharomyces cerevisiae. Overexpression of Rph1 reduced the expression of PHR1 and increased UV sensitivity. The catalytically deficient mutant (H235A) of Rph1 diminished the repressive transcriptional effect on PHR1 expression, which indicates that histone demethylase activity contributes to transcriptional repression. Chromatin immunoprecipitation analysis demonstrated that Rph1 was associated at the upstream repression sequence of PHR1 through zinc-finger domains and was dissociated after UV irradiation. Notably, overexpression of Rph1 and H3K36A mutant reduced histone acetylation at the URS, which implies a crosstalk between histone demethylation and acetylation at the PHR1 promoter. In addition, the crucial checkpoint protein Rad53 acted as an upstream regulator of Rph1 and dominated the phosphorylation of Rph1 that was required for efficient PHR1 expression and the dissociation of Rph1. The release of Rph1 from chromatin also required the phosphorylation at S652. Our study demonstrates that the histone demethylase Rph1 is associated with a specific chromatin locus and modulates histone modifications to repress a DNA damage responsive gene under control of damage checkpoint signaling.

Show MeSH
Related in: MedlinePlus