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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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Histone H3K36 demethylase Rph1 regulates the transcription of PHR1. (A) Left: The rph1Δ strains carrying constitutively overexpressed RPH1 or control 2-µ vector were streaked on selective plate. Right: The UV-sensitivity test was performed with indicated strains. Cells were spotted on plates containing galactose or glucose with 5-fold serial dilution and subjected to UV irradiation. Asterisks denotes the constitutive expression of RPH1 with its own promoter in 2-µ vector. (B) UV sensitivity was tested with indicated strains. Yeast strains (WT, histone mutants, rph1Δ and set2Δ) grown in glucose in log-phase or induced with galactose for 4 h were spotted on selective plates. (C) PHR1 expression in WT, rph1Δ and set2Δ strains. The cells were cultured to early log phase and subjected to 20 mj/cm2 UV irradiation. RT–qPCR was performed and transcription of PHR1 of each strain was normalized to ACT1. Error bars indicate the SD from three biological repeats. (D) PHR1 level in WT or rph1Δ yeast containing control vector (Vector), WT RPH1 or H235A-mutated RPH1 under different promoters before or after 20 mJ/cm2 UV irradiation (right). Error bars are the SD of five biological replicates. *P < 0.05.
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Figure 1: Histone H3K36 demethylase Rph1 regulates the transcription of PHR1. (A) Left: The rph1Δ strains carrying constitutively overexpressed RPH1 or control 2-µ vector were streaked on selective plate. Right: The UV-sensitivity test was performed with indicated strains. Cells were spotted on plates containing galactose or glucose with 5-fold serial dilution and subjected to UV irradiation. Asterisks denotes the constitutive expression of RPH1 with its own promoter in 2-µ vector. (B) UV sensitivity was tested with indicated strains. Yeast strains (WT, histone mutants, rph1Δ and set2Δ) grown in glucose in log-phase or induced with galactose for 4 h were spotted on selective plates. (C) PHR1 expression in WT, rph1Δ and set2Δ strains. The cells were cultured to early log phase and subjected to 20 mj/cm2 UV irradiation. RT–qPCR was performed and transcription of PHR1 of each strain was normalized to ACT1. Error bars indicate the SD from three biological repeats. (D) PHR1 level in WT or rph1Δ yeast containing control vector (Vector), WT RPH1 or H235A-mutated RPH1 under different promoters before or after 20 mJ/cm2 UV irradiation (right). Error bars are the SD of five biological replicates. *P < 0.05.

Mentions: To characterize the biological function of histone demethylases, including Rph1, Jhd1 and Jhd2, we first performed phenotypic analyses and observed that deletion of any histone demethylase caused no overt growth phenotype (15), which may be due to overlapping activities of the multiple histone lysine demethylases in yeast. In an independent approach, we used overexpression to probe the function of individual histone demethylases. Of these, only Rph1 overexpression showed elevated sensitivity to UV-induced damage (15). However, the constitutive overexpression of Rph1 with its own promoter in a multi-copy (2μ) plasmid caused a severe defect in cell growth (Figure 1A, left panel and 2 lower rows in right panel). Thus, we used an inducible expression strategy to study the biological phenotype resulting from temporally increased Rph1 levels. Cells with galactose-inducible overexpression of RPH1 grew well without UV treatment yet displayed hypersensitivity to UV irradiation at 25 mj/cm2 (Figure 1A, 2 upper rows in right panel). Because previous experiments suggested that Rph1 is a histone demethylase specific to tri-methylated-H3K36 (15,38), we surmised that the demethylation at H3K36 may be linked to UV sensitivity. To test the possibility, we used alanine-substituted mutants blocking methylation at histone H3 K4, K36 and K79 in UV-sensitivity assays. Interestingly, only histone H3K36A and H3K79A mutants showed increased UV sensitivity (Figure 1B, upper panel). Dot1-mediated H3K79 methylation is linked with H2B ubiquitination and is involved in the DNA damage response (39). However, H3K36 methylation in the UV damage response has not been well-established. Furthermore, we found that the deletion of the H3K36 methyl-transferase Set2 (set2Δ) also enhanced the UV sensitivity (Figure 1B, middle panel). Moreover, overexpression of Rph1 in the WT conferred high sensitivity to UV irradiation, whereas overexpression of Rph1 combined with set2Δ caused an additive effect (Figure 1B, lower panel), which was more significant at a higher dosage. These observations suggest that Set2 and Rph1 likely work in parallel pathways to control UV sensitivity.Figure 1.


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)

Histone H3K36 demethylase Rph1 regulates the transcription of PHR1. (A) Left: The rph1Δ strains carrying constitutively overexpressed RPH1 or control 2-µ vector were streaked on selective plate. Right: The UV-sensitivity test was performed with indicated strains. Cells were spotted on plates containing galactose or glucose with 5-fold serial dilution and subjected to UV irradiation. Asterisks denotes the constitutive expression of RPH1 with its own promoter in 2-µ vector. (B) UV sensitivity was tested with indicated strains. Yeast strains (WT, histone mutants, rph1Δ and set2Δ) grown in glucose in log-phase or induced with galactose for 4 h were spotted on selective plates. (C) PHR1 expression in WT, rph1Δ and set2Δ strains. The cells were cultured to early log phase and subjected to 20 mj/cm2 UV irradiation. RT–qPCR was performed and transcription of PHR1 of each strain was normalized to ACT1. Error bars indicate the SD from three biological repeats. (D) PHR1 level in WT or rph1Δ yeast containing control vector (Vector), WT RPH1 or H235A-mutated RPH1 under different promoters before or after 20 mJ/cm2 UV irradiation (right). Error bars are the SD of five biological replicates. *P < 0.05.
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Figure 1: Histone H3K36 demethylase Rph1 regulates the transcription of PHR1. (A) Left: The rph1Δ strains carrying constitutively overexpressed RPH1 or control 2-µ vector were streaked on selective plate. Right: The UV-sensitivity test was performed with indicated strains. Cells were spotted on plates containing galactose or glucose with 5-fold serial dilution and subjected to UV irradiation. Asterisks denotes the constitutive expression of RPH1 with its own promoter in 2-µ vector. (B) UV sensitivity was tested with indicated strains. Yeast strains (WT, histone mutants, rph1Δ and set2Δ) grown in glucose in log-phase or induced with galactose for 4 h were spotted on selective plates. (C) PHR1 expression in WT, rph1Δ and set2Δ strains. The cells were cultured to early log phase and subjected to 20 mj/cm2 UV irradiation. RT–qPCR was performed and transcription of PHR1 of each strain was normalized to ACT1. Error bars indicate the SD from three biological repeats. (D) PHR1 level in WT or rph1Δ yeast containing control vector (Vector), WT RPH1 or H235A-mutated RPH1 under different promoters before or after 20 mJ/cm2 UV irradiation (right). Error bars are the SD of five biological replicates. *P < 0.05.
Mentions: To characterize the biological function of histone demethylases, including Rph1, Jhd1 and Jhd2, we first performed phenotypic analyses and observed that deletion of any histone demethylase caused no overt growth phenotype (15), which may be due to overlapping activities of the multiple histone lysine demethylases in yeast. In an independent approach, we used overexpression to probe the function of individual histone demethylases. Of these, only Rph1 overexpression showed elevated sensitivity to UV-induced damage (15). However, the constitutive overexpression of Rph1 with its own promoter in a multi-copy (2μ) plasmid caused a severe defect in cell growth (Figure 1A, left panel and 2 lower rows in right panel). Thus, we used an inducible expression strategy to study the biological phenotype resulting from temporally increased Rph1 levels. Cells with galactose-inducible overexpression of RPH1 grew well without UV treatment yet displayed hypersensitivity to UV irradiation at 25 mj/cm2 (Figure 1A, 2 upper rows in right panel). Because previous experiments suggested that Rph1 is a histone demethylase specific to tri-methylated-H3K36 (15,38), we surmised that the demethylation at H3K36 may be linked to UV sensitivity. To test the possibility, we used alanine-substituted mutants blocking methylation at histone H3 K4, K36 and K79 in UV-sensitivity assays. Interestingly, only histone H3K36A and H3K79A mutants showed increased UV sensitivity (Figure 1B, upper panel). Dot1-mediated H3K79 methylation is linked with H2B ubiquitination and is involved in the DNA damage response (39). However, H3K36 methylation in the UV damage response has not been well-established. Furthermore, we found that the deletion of the H3K36 methyl-transferase Set2 (set2Δ) also enhanced the UV sensitivity (Figure 1B, middle panel). Moreover, overexpression of Rph1 in the WT conferred high sensitivity to UV irradiation, whereas overexpression of Rph1 combined with set2Δ caused an additive effect (Figure 1B, lower panel), which was more significant at a higher dosage. These observations suggest that Set2 and Rph1 likely work in parallel pathways to control UV sensitivity.Figure 1.

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