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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 regulates transcription of PHR1 mediated by Rad53 in response to DNA damage. (A) The expression of PHR1 and HUG1 in sml1Δ (WT), sml1Δ rad53Δ (rad53Δ) and sml1Δ dun1Δ (dun1Δ) mutants responding to different doses of UV irradiation. HUG1 was used as an indicator of intact DNA damage signaling. *P < 0.05. (B) UV-sensitivity test of indicated strains in the rph1Δ rad53Δ background with different combinations of control vector, overexpressed RAD53 or kinase-dead (KD) and WT RPH1. (C) PHR1 expression of strains as in (B). Cells harvested from early-log phase underwent 30 mJ/cm2 irradiation. *, #, §P < 0.05. (D) ChIP with anti-Rph1 (HA) at URS from comparable samples as in Figure 4C. Bar graphs show qPCR results in URS of PHR1 promoter. IB indicated the protein expression of Rph1, and Pgk1 is a loading control. All RT-qPCR data are at least from three different biological samples. Results of ChIP are from 2 biological samples. *P < 0.05.
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Figure 5: Rph1 regulates transcription of PHR1 mediated by Rad53 in response to DNA damage. (A) The expression of PHR1 and HUG1 in sml1Δ (WT), sml1Δ rad53Δ (rad53Δ) and sml1Δ dun1Δ (dun1Δ) mutants responding to different doses of UV irradiation. HUG1 was used as an indicator of intact DNA damage signaling. *P < 0.05. (B) UV-sensitivity test of indicated strains in the rph1Δ rad53Δ background with different combinations of control vector, overexpressed RAD53 or kinase-dead (KD) and WT RPH1. (C) PHR1 expression of strains as in (B). Cells harvested from early-log phase underwent 30 mJ/cm2 irradiation. *, #, §P < 0.05. (D) ChIP with anti-Rph1 (HA) at URS from comparable samples as in Figure 4C. Bar graphs show qPCR results in URS of PHR1 promoter. IB indicated the protein expression of Rph1, and Pgk1 is a loading control. All RT-qPCR data are at least from three different biological samples. Results of ChIP are from 2 biological samples. *P < 0.05.

Mentions: It has been suggested that Rph1 phosphorylation is under the control of the Mec1-Rad53 damage checkpoint pathway but distinct from the Dun1-Crt1 pathway (48). In budding yeast, Rad53, the ortholog of mammalian Chk2, is a crucial checkpoint protein, and Dun1 is considered the main downstream kinase of Rad53 responding to DNA damage (49,50). However, the connection between the expression of PHR1, a DNA repair gene and Rad53 is still unknown. To examine whether Rad53 plays a regulatory role in PHR1 expression in DNA damage signaling, we analyzed the PHR1 transcription level in a rad53Δ strain. Deletion of RAD53 is lethal. Therefore, we evaluated the role of RAD53 by using a rad53Δ sml1Δ strain, which loss of SML1 suppresses the lethality of RAD53 deletion (51). By using RT–qPCR analysis, we found the expression level of PHR1 in the absence of UV irradiation decreased by 30% in the rad53Δ mutant but not in the dun1Δ mutant (Figure 5A). With UV irradiation (30 mj/cm2), the induction of PHR1 was reduced by 35% in the rad53Δ mutant but only slightly in the dun1Δ strain. The Rad53-Dun1-regulated gene HUG1 was not induced in either the rad53Δ or dun1Δ mutant (Figure 5A, right panel). Therefore, our data demonstrate that an efficient PHR1 expression requires RAD53 but is less dependent on DUN1.Figure 5.


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 regulates transcription of PHR1 mediated by Rad53 in response to DNA damage. (A) The expression of PHR1 and HUG1 in sml1Δ (WT), sml1Δ rad53Δ (rad53Δ) and sml1Δ dun1Δ (dun1Δ) mutants responding to different doses of UV irradiation. HUG1 was used as an indicator of intact DNA damage signaling. *P < 0.05. (B) UV-sensitivity test of indicated strains in the rph1Δ rad53Δ background with different combinations of control vector, overexpressed RAD53 or kinase-dead (KD) and WT RPH1. (C) PHR1 expression of strains as in (B). Cells harvested from early-log phase underwent 30 mJ/cm2 irradiation. *, #, §P < 0.05. (D) ChIP with anti-Rph1 (HA) at URS from comparable samples as in Figure 4C. Bar graphs show qPCR results in URS of PHR1 promoter. IB indicated the protein expression of Rph1, and Pgk1 is a loading control. All RT-qPCR data are at least from three different biological samples. Results of ChIP are from 2 biological samples. *P < 0.05.
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Figure 5: Rph1 regulates transcription of PHR1 mediated by Rad53 in response to DNA damage. (A) The expression of PHR1 and HUG1 in sml1Δ (WT), sml1Δ rad53Δ (rad53Δ) and sml1Δ dun1Δ (dun1Δ) mutants responding to different doses of UV irradiation. HUG1 was used as an indicator of intact DNA damage signaling. *P < 0.05. (B) UV-sensitivity test of indicated strains in the rph1Δ rad53Δ background with different combinations of control vector, overexpressed RAD53 or kinase-dead (KD) and WT RPH1. (C) PHR1 expression of strains as in (B). Cells harvested from early-log phase underwent 30 mJ/cm2 irradiation. *, #, §P < 0.05. (D) ChIP with anti-Rph1 (HA) at URS from comparable samples as in Figure 4C. Bar graphs show qPCR results in URS of PHR1 promoter. IB indicated the protein expression of Rph1, and Pgk1 is a loading control. All RT-qPCR data are at least from three different biological samples. Results of ChIP are from 2 biological samples. *P < 0.05.
Mentions: It has been suggested that Rph1 phosphorylation is under the control of the Mec1-Rad53 damage checkpoint pathway but distinct from the Dun1-Crt1 pathway (48). In budding yeast, Rad53, the ortholog of mammalian Chk2, is a crucial checkpoint protein, and Dun1 is considered the main downstream kinase of Rad53 responding to DNA damage (49,50). However, the connection between the expression of PHR1, a DNA repair gene and Rad53 is still unknown. To examine whether Rad53 plays a regulatory role in PHR1 expression in DNA damage signaling, we analyzed the PHR1 transcription level in a rad53Δ strain. Deletion of RAD53 is lethal. Therefore, we evaluated the role of RAD53 by using a rad53Δ sml1Δ strain, which loss of SML1 suppresses the lethality of RAD53 deletion (51). By using RT–qPCR analysis, we found the expression level of PHR1 in the absence of UV irradiation decreased by 30% in the rad53Δ mutant but not in the dun1Δ mutant (Figure 5A). With UV irradiation (30 mj/cm2), the induction of PHR1 was reduced by 35% in the rad53Δ mutant but only slightly in the dun1Δ strain. The Rad53-Dun1-regulated gene HUG1 was not induced in either the rad53Δ or dun1Δ mutant (Figure 5A, right panel). Therefore, our data demonstrate that an efficient PHR1 expression requires RAD53 but is less dependent on DUN1.Figure 5.

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