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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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Demethylation at H3K36 coexists with a reduction of histone acetylations specifically at the URS of PHR1. (A) Histone H3/H4 acetylation was altered at the URS and coding region of PHR1. ChIP-qPCR from the indicated strains was performed with anti-AcH3 and anti-AcH4. *P < 0.05. Data are from three biological repeats. (B) RPD3 deletion restored the reduction of H3 acetylation at the URS. Indicated HDAC deletion strains with overexpressed Rph1 were harvested for acH3-ChIP. (C) Rpd3-Myc is associated at URS region in the presence of Rph1. The rph1Δ cells containing control vector or overexpressed Rph1 with Myc-tagged Rpd3 were harvested for Myc-ChIP and qPCR. Error bars represents the SD from two biological repeats. (D) Left: H3K36A showed the reduction of H3 acetylation at the URS of PHR1. Cells carrying WT or H3K36 mutated (H3K36A) histones were subject to acH3-ChIP followed by qPCR. Right: PHR1 expression in WT or H3K36A mutant. (E) ChIP with anti-Rpb3 at URS region of PHR1 in WT, rph1Δ, set2Δ and H3K36A mutants. Error bars shows the SD from two biological samples.
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Figure 3: Demethylation at H3K36 coexists with a reduction of histone acetylations specifically at the URS of PHR1. (A) Histone H3/H4 acetylation was altered at the URS and coding region of PHR1. ChIP-qPCR from the indicated strains was performed with anti-AcH3 and anti-AcH4. *P < 0.05. Data are from three biological repeats. (B) RPD3 deletion restored the reduction of H3 acetylation at the URS. Indicated HDAC deletion strains with overexpressed Rph1 were harvested for acH3-ChIP. (C) Rpd3-Myc is associated at URS region in the presence of Rph1. The rph1Δ cells containing control vector or overexpressed Rph1 with Myc-tagged Rpd3 were harvested for Myc-ChIP and qPCR. Error bars represents the SD from two biological repeats. (D) Left: H3K36A showed the reduction of H3 acetylation at the URS of PHR1. Cells carrying WT or H3K36 mutated (H3K36A) histones were subject to acH3-ChIP followed by qPCR. Right: PHR1 expression in WT or H3K36A mutant. (E) ChIP with anti-Rpb3 at URS region of PHR1 in WT, rph1Δ, set2Δ and H3K36A mutants. Error bars shows the SD from two biological samples.

Mentions: The synergistic crosstalk of histone modifications has been suggested as an important regulatory mechanism in gene expression (33,42,43). We hypothesized that gene-specific synergistic histone modifications also take place on the PHR1 promoter by interacting with DNA-bound transcription factors. Because Rph1 bound to the URSPHR1 but not UES + UASPHR1 region (Figure 2C) and consequently decreased H3K36 methylation in the absence of UV irradiation, we examined whether histone H3K36 demethylation affected histone acetylations. Results from ChIP assays with anti-acetylated histone H3K9/14 (acH3) and H4K5/8/12/16 (acH4) revealed that Rph1 altered histone acetylation in different regions (Figure 3A). The levels of histone H3/H4 acetylations were reduced by about 50% only at the URSPHR1 but not the UASPHR1 or coding region (Figure 3A). Thus, the association of WT Rph1 at URSPHR1 reduced the histone acetylations in this region. Furthermore, the catalytic-deficient rph1-H235A mutant displayed patterns similar to those of the rph1Δ (vector) in histone acetylations, which indicates that Rph1 demethylase activity is critical for the crosstalk of histone modifications at the PHR1 promoter. The reduced histone acetylation implies an involvement of histone deacetylase(s). To examine this possibility, we performed acH3-ChIP using various histone deacetylase (HDAC)-deleted strains, including HDA1, HDA2 (Type II) and RPD3 (Type I), in the presence of overexpressed RPH1. Lack of RPD3 restored the H3 acetylation level at the URSPHR1 in the presence of Rph1 (Figure 3B). Moreover, results from ChIP experiments revealed the significant association of Rpd3 at the URSPHR1 but not the UASPHR1 region in the presence of Rph1 (Figure 3C), which indicates that Rpd3 specifically deacetylated histones at the URSPHR1 region responsible for the transcriptional repression of PHR1.Figure 3.


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)

Demethylation at H3K36 coexists with a reduction of histone acetylations specifically at the URS of PHR1. (A) Histone H3/H4 acetylation was altered at the URS and coding region of PHR1. ChIP-qPCR from the indicated strains was performed with anti-AcH3 and anti-AcH4. *P < 0.05. Data are from three biological repeats. (B) RPD3 deletion restored the reduction of H3 acetylation at the URS. Indicated HDAC deletion strains with overexpressed Rph1 were harvested for acH3-ChIP. (C) Rpd3-Myc is associated at URS region in the presence of Rph1. The rph1Δ cells containing control vector or overexpressed Rph1 with Myc-tagged Rpd3 were harvested for Myc-ChIP and qPCR. Error bars represents the SD from two biological repeats. (D) Left: H3K36A showed the reduction of H3 acetylation at the URS of PHR1. Cells carrying WT or H3K36 mutated (H3K36A) histones were subject to acH3-ChIP followed by qPCR. Right: PHR1 expression in WT or H3K36A mutant. (E) ChIP with anti-Rpb3 at URS region of PHR1 in WT, rph1Δ, set2Δ and H3K36A mutants. Error bars shows the SD from two biological samples.
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Figure 3: Demethylation at H3K36 coexists with a reduction of histone acetylations specifically at the URS of PHR1. (A) Histone H3/H4 acetylation was altered at the URS and coding region of PHR1. ChIP-qPCR from the indicated strains was performed with anti-AcH3 and anti-AcH4. *P < 0.05. Data are from three biological repeats. (B) RPD3 deletion restored the reduction of H3 acetylation at the URS. Indicated HDAC deletion strains with overexpressed Rph1 were harvested for acH3-ChIP. (C) Rpd3-Myc is associated at URS region in the presence of Rph1. The rph1Δ cells containing control vector or overexpressed Rph1 with Myc-tagged Rpd3 were harvested for Myc-ChIP and qPCR. Error bars represents the SD from two biological repeats. (D) Left: H3K36A showed the reduction of H3 acetylation at the URS of PHR1. Cells carrying WT or H3K36 mutated (H3K36A) histones were subject to acH3-ChIP followed by qPCR. Right: PHR1 expression in WT or H3K36A mutant. (E) ChIP with anti-Rpb3 at URS region of PHR1 in WT, rph1Δ, set2Δ and H3K36A mutants. Error bars shows the SD from two biological samples.
Mentions: The synergistic crosstalk of histone modifications has been suggested as an important regulatory mechanism in gene expression (33,42,43). We hypothesized that gene-specific synergistic histone modifications also take place on the PHR1 promoter by interacting with DNA-bound transcription factors. Because Rph1 bound to the URSPHR1 but not UES + UASPHR1 region (Figure 2C) and consequently decreased H3K36 methylation in the absence of UV irradiation, we examined whether histone H3K36 demethylation affected histone acetylations. Results from ChIP assays with anti-acetylated histone H3K9/14 (acH3) and H4K5/8/12/16 (acH4) revealed that Rph1 altered histone acetylation in different regions (Figure 3A). The levels of histone H3/H4 acetylations were reduced by about 50% only at the URSPHR1 but not the UASPHR1 or coding region (Figure 3A). Thus, the association of WT Rph1 at URSPHR1 reduced the histone acetylations in this region. Furthermore, the catalytic-deficient rph1-H235A mutant displayed patterns similar to those of the rph1Δ (vector) in histone acetylations, which indicates that Rph1 demethylase activity is critical for the crosstalk of histone modifications at the PHR1 promoter. The reduced histone acetylation implies an involvement of histone deacetylase(s). To examine this possibility, we performed acH3-ChIP using various histone deacetylase (HDAC)-deleted strains, including HDA1, HDA2 (Type II) and RPD3 (Type I), in the presence of overexpressed RPH1. Lack of RPD3 restored the H3 acetylation level at the URSPHR1 in the presence of Rph1 (Figure 3B). Moreover, results from ChIP experiments revealed the significant association of Rpd3 at the URSPHR1 but not the UASPHR1 region in the presence of Rph1 (Figure 3C), which indicates that Rpd3 specifically deacetylated histones at the URSPHR1 region responsible for the transcriptional repression of PHR1.Figure 3.

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