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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 binds to the upstream repression sequence (URS) of PHR1 through ZF domains. (A) Top panel: The schematic representation of primers specific to different regions on PHR1 for PCR. +1 indicates the transcription start site of PHR1. The primer sequences are in Supplementary Table S2. Lower panel: The specificity of H3K36me3 at the PHR1 promoter and coding region were detected by ChIP in WT, rph1Δ, set2Δ and H3K36A mutants. Bar graph represents the quantified results from three biological repeats. (B) ChIP with anti-HA and anti-H3K36me3 antibodies were performed with the indicated strains. The right panels show the fold change relative to the control (vector), which was normalized by input. (C) The ZF domains are required for transcriptional repression on PHR1 and for specific association with URSPHR1. Left panel: PHR1 expression in rph1Δ (vector), induced WT RPH1 or ZF-deleted RPH1 (ZFΔ). ***P < 0.001 compared with vector. Right panel: ChIP with anti-HA antibody at the UAS or URS regions. Data are from three different biological samples.
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Figure 2: Rph1 binds to the upstream repression sequence (URS) of PHR1 through ZF domains. (A) Top panel: The schematic representation of primers specific to different regions on PHR1 for PCR. +1 indicates the transcription start site of PHR1. The primer sequences are in Supplementary Table S2. Lower panel: The specificity of H3K36me3 at the PHR1 promoter and coding region were detected by ChIP in WT, rph1Δ, set2Δ and H3K36A mutants. Bar graph represents the quantified results from three biological repeats. (B) ChIP with anti-HA and anti-H3K36me3 antibodies were performed with the indicated strains. The right panels show the fold change relative to the control (vector), which was normalized by input. (C) The ZF domains are required for transcriptional repression on PHR1 and for specific association with URSPHR1. Left panel: PHR1 expression in rph1Δ (vector), induced WT RPH1 or ZF-deleted RPH1 (ZFΔ). ***P < 0.001 compared with vector. Right panel: ChIP with anti-HA antibody at the UAS or URS regions. Data are from three different biological samples.

Mentions: We have previously demonstrated that Rph1 plays a major role in transcriptional repression of PHR1 (Figure 1). We next used ChIP assays to evaluate whether the Rph1-mediated repression of PHR1 affects the chromatin structure. The primary protein-coding gene structure of PHR1 is illustrated in Figure 2A (upper panel). We first determined whether deletion of RPH1 changed the H3K36 tri-methylation at the PHR1 gene region (Figure 2A, lower panel). To confirm the specificity of the H3K36me3 signal, we used set2Δ and H3K36A mutants for H3K36me3-ChIP and found extremely low levels of H3K36me3 in set2Δ and H3K36A mutants as compared with that in WT and rph1Δ. Interestingly, rph1Δ showed an increased level at the promoter of PHR1 but not the 3′ coding region of PHR1, suggesting that Rph1-mediated demethylation participates in the regulation of PHR1 promoter activity. We next investigated whether Rph1 is physically associated with chromatin in vivo. To determine the temporal regulation of PHR1 expression by Rph1, we used the inducible GAL1 promoter to analyze the immediate effect of overexpressed Rph1 on the transcriptional regulation of PHR1. ChIP assays were performed to detect the relative abundance of HA-tagged Rph1 and H3K36me3 at the PHR1 promoter region containing a 300 bp 5′-upstream sequence (Figure 2B). In agreement with in vitro electrophoretic mobility shift assay (EMSA) and footprinting analyses, which demonstrated that Rph1 binds to a specific PHR1 promoter sequence (23), our ChIP analysis also revealed that Rph1 binding was enriched at the PHR1 promoter by at least 4-folds in both the WT (RPH1) and mutant rph1-H235A as when compared with that ine rph1Δ mutant (vector alone) (Figure 2B, HA-IP). However, only the WT Rph1 but not the rph1-H235A reduced H3K36 tri-methylation (Figure 2B, H3K36me3-IP), which indicates that the enzymatic activity of Rph1 is required for H3K36 demethylation at the promoter of PHR1. This result was not merely due to the induced overexpression of Rph1 by the GAL1 promoter because we also found similar results by using the GPD1 promoter to drive a constitute expression of Rph1 in a low-copy (CEN) plasmid (Supplementary Figure S2). Therefore, Rph1 is associated with the promoter of PHR1 resulting in a decreased H3K36 methylation to influence transcriptional repression.Figure 2.


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 binds to the upstream repression sequence (URS) of PHR1 through ZF domains. (A) Top panel: The schematic representation of primers specific to different regions on PHR1 for PCR. +1 indicates the transcription start site of PHR1. The primer sequences are in Supplementary Table S2. Lower panel: The specificity of H3K36me3 at the PHR1 promoter and coding region were detected by ChIP in WT, rph1Δ, set2Δ and H3K36A mutants. Bar graph represents the quantified results from three biological repeats. (B) ChIP with anti-HA and anti-H3K36me3 antibodies were performed with the indicated strains. The right panels show the fold change relative to the control (vector), which was normalized by input. (C) The ZF domains are required for transcriptional repression on PHR1 and for specific association with URSPHR1. Left panel: PHR1 expression in rph1Δ (vector), induced WT RPH1 or ZF-deleted RPH1 (ZFΔ). ***P < 0.001 compared with vector. Right panel: ChIP with anti-HA antibody at the UAS or URS regions. Data are from three different biological samples.
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Figure 2: Rph1 binds to the upstream repression sequence (URS) of PHR1 through ZF domains. (A) Top panel: The schematic representation of primers specific to different regions on PHR1 for PCR. +1 indicates the transcription start site of PHR1. The primer sequences are in Supplementary Table S2. Lower panel: The specificity of H3K36me3 at the PHR1 promoter and coding region were detected by ChIP in WT, rph1Δ, set2Δ and H3K36A mutants. Bar graph represents the quantified results from three biological repeats. (B) ChIP with anti-HA and anti-H3K36me3 antibodies were performed with the indicated strains. The right panels show the fold change relative to the control (vector), which was normalized by input. (C) The ZF domains are required for transcriptional repression on PHR1 and for specific association with URSPHR1. Left panel: PHR1 expression in rph1Δ (vector), induced WT RPH1 or ZF-deleted RPH1 (ZFΔ). ***P < 0.001 compared with vector. Right panel: ChIP with anti-HA antibody at the UAS or URS regions. Data are from three different biological samples.
Mentions: We have previously demonstrated that Rph1 plays a major role in transcriptional repression of PHR1 (Figure 1). We next used ChIP assays to evaluate whether the Rph1-mediated repression of PHR1 affects the chromatin structure. The primary protein-coding gene structure of PHR1 is illustrated in Figure 2A (upper panel). We first determined whether deletion of RPH1 changed the H3K36 tri-methylation at the PHR1 gene region (Figure 2A, lower panel). To confirm the specificity of the H3K36me3 signal, we used set2Δ and H3K36A mutants for H3K36me3-ChIP and found extremely low levels of H3K36me3 in set2Δ and H3K36A mutants as compared with that in WT and rph1Δ. Interestingly, rph1Δ showed an increased level at the promoter of PHR1 but not the 3′ coding region of PHR1, suggesting that Rph1-mediated demethylation participates in the regulation of PHR1 promoter activity. We next investigated whether Rph1 is physically associated with chromatin in vivo. To determine the temporal regulation of PHR1 expression by Rph1, we used the inducible GAL1 promoter to analyze the immediate effect of overexpressed Rph1 on the transcriptional regulation of PHR1. ChIP assays were performed to detect the relative abundance of HA-tagged Rph1 and H3K36me3 at the PHR1 promoter region containing a 300 bp 5′-upstream sequence (Figure 2B). In agreement with in vitro electrophoretic mobility shift assay (EMSA) and footprinting analyses, which demonstrated that Rph1 binds to a specific PHR1 promoter sequence (23), our ChIP analysis also revealed that Rph1 binding was enriched at the PHR1 promoter by at least 4-folds in both the WT (RPH1) and mutant rph1-H235A as when compared with that ine rph1Δ mutant (vector alone) (Figure 2B, HA-IP). However, only the WT Rph1 but not the rph1-H235A reduced H3K36 tri-methylation (Figure 2B, H3K36me3-IP), which indicates that the enzymatic activity of Rph1 is required for H3K36 demethylation at the promoter of PHR1. This result was not merely due to the induced overexpression of Rph1 by the GAL1 promoter because we also found similar results by using the GPD1 promoter to drive a constitute expression of Rph1 in a low-copy (CEN) plasmid (Supplementary Figure S2). Therefore, Rph1 is associated with the promoter of PHR1 resulting in a decreased H3K36 methylation to influence transcriptional repression.Figure 2.

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