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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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The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. (A) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ32P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. (B) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 (RPH1) or phospho-defective Rph1 mutants. (C and D) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. *P < 0.05.
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Figure 6: The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. (A) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ32P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. (B) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 (RPH1) or phospho-defective Rph1 mutants. (C and D) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. *P < 0.05.

Mentions: To test whether Rph1 is a substrate of Rad53, we performed an in vitro kinase assay by incubating IP-activated Rad53 (WT or KD) with recombinant Rph1. By autoradiography, we detected a specific signal of Rph1 phosphorylation in the presence of WT Rad53 but not rad53-KD, which established that Rad53 kinase dominated the phosphorylation of Rph1 (Figure 6A). Previous proteomic studies had revealed that Rph1 was phosphorylated at multiple serine residues induced by DNA damage or cell cycle arrest (52–55). To determine the functional role of phosphorylation, we generated a series of alanine-substituted mutations on putative phosphorylated serine residues (S412, S459, S557, S561, S652 and S689) to analyze the UV sensitivity of these mutants. Because the Rph1 phosphorylation triggered by, UV irradiation may reflect a transient response, we attempted to examine the immediate response by use of the GAL inducible expression system. However, we found severe growth defects in the WT and phospho-mutants of Rph1 (Figure 1A and Supplementary Figure S6) cultured on synthetic complete selective medium in the presence of galactose (SCM-URA + galactose) but not glucose (SCM-URA + glucose), presumably because of constitutively overexpressed Rph1. To avoid this potential issue to obscure the growth phenotype, we modified our experimental protocol to use the GAL1 promoter to induce the overexpression of the WT and phospho-mutants of Rph1 4 h before UV irradiation, then scored the phenotype 2 days later (see ‘Methods and Materials’ section). When the UV dose was increased to 30 mJ/cm2, we found that the rph1-S652A mutant began to show a hypersensitivity to UV irradiation, even greater than that of Rph1 overexpression (Figure 6B, Supplementary Figure S7). From bioinformatics studies (Scansite, http://scansite.mit.edu/and GPS2.1, http://gps.biocuckoo.org/), we selected S459 (embedded within the putative bipartite nuclear localization region) and S652 (a consensus phosphorylated site detected in genome-wide LC/MS analysis) (52–55) to further characterize the functional role of Rph1 phosphorylation in PHR1 expression. The rph1-S459A mutant displayed a similar UV-sensitivity phenotype to WT Rph1, whereas rph1-S652A and rph1-S459AS652A mutants were hypersensitive to UV irradiation (Figure 6B), which indicates that phosphorylation at S652 may play a critical role in Rph1 function responding to DNA damage. We subsequently measured the expression of PHR1 and association of Rph1 phospho-mutants with URSPHR1. The expression levels of PHR1 were comparable in rph1-S459A and WT Rph1 but were reduced to 30% (P < 0.05) in rph1-S652A and rph1-S459AS652A mutants, regardless of UV treatment (Figure 6C). Remarkably, results from HA (Rph1)-ChIP assays showed that S652A mutation did not affect the Rph1 association with URSPHR1 but greatly impaired the dissociation from URSPHR1 on UV treatment (Figure 6D), which indicates that phosphorylation at S652 is important for Rph1 to dissociate from URSPHR1 in the presence of UV irradiation. These data support that chromatin association and dissociation of Rph1 on the PHR1 promoter mediated by protein phosphorylation is the major regulatory mechanism for PHR1 expression responding to DNA damage.Figure 6.


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)

The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. (A) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ32P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. (B) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 (RPH1) or phospho-defective Rph1 mutants. (C and D) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. *P < 0.05.
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Figure 6: The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. (A) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ32P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. (B) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 (RPH1) or phospho-defective Rph1 mutants. (C and D) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. *P < 0.05.
Mentions: To test whether Rph1 is a substrate of Rad53, we performed an in vitro kinase assay by incubating IP-activated Rad53 (WT or KD) with recombinant Rph1. By autoradiography, we detected a specific signal of Rph1 phosphorylation in the presence of WT Rad53 but not rad53-KD, which established that Rad53 kinase dominated the phosphorylation of Rph1 (Figure 6A). Previous proteomic studies had revealed that Rph1 was phosphorylated at multiple serine residues induced by DNA damage or cell cycle arrest (52–55). To determine the functional role of phosphorylation, we generated a series of alanine-substituted mutations on putative phosphorylated serine residues (S412, S459, S557, S561, S652 and S689) to analyze the UV sensitivity of these mutants. Because the Rph1 phosphorylation triggered by, UV irradiation may reflect a transient response, we attempted to examine the immediate response by use of the GAL inducible expression system. However, we found severe growth defects in the WT and phospho-mutants of Rph1 (Figure 1A and Supplementary Figure S6) cultured on synthetic complete selective medium in the presence of galactose (SCM-URA + galactose) but not glucose (SCM-URA + glucose), presumably because of constitutively overexpressed Rph1. To avoid this potential issue to obscure the growth phenotype, we modified our experimental protocol to use the GAL1 promoter to induce the overexpression of the WT and phospho-mutants of Rph1 4 h before UV irradiation, then scored the phenotype 2 days later (see ‘Methods and Materials’ section). When the UV dose was increased to 30 mJ/cm2, we found that the rph1-S652A mutant began to show a hypersensitivity to UV irradiation, even greater than that of Rph1 overexpression (Figure 6B, Supplementary Figure S7). From bioinformatics studies (Scansite, http://scansite.mit.edu/and GPS2.1, http://gps.biocuckoo.org/), we selected S459 (embedded within the putative bipartite nuclear localization region) and S652 (a consensus phosphorylated site detected in genome-wide LC/MS analysis) (52–55) to further characterize the functional role of Rph1 phosphorylation in PHR1 expression. The rph1-S459A mutant displayed a similar UV-sensitivity phenotype to WT Rph1, whereas rph1-S652A and rph1-S459AS652A mutants were hypersensitive to UV irradiation (Figure 6B), which indicates that phosphorylation at S652 may play a critical role in Rph1 function responding to DNA damage. We subsequently measured the expression of PHR1 and association of Rph1 phospho-mutants with URSPHR1. The expression levels of PHR1 were comparable in rph1-S459A and WT Rph1 but were reduced to 30% (P < 0.05) in rph1-S652A and rph1-S459AS652A mutants, regardless of UV treatment (Figure 6C). Remarkably, results from HA (Rph1)-ChIP assays showed that S652A mutation did not affect the Rph1 association with URSPHR1 but greatly impaired the dissociation from URSPHR1 on UV treatment (Figure 6D), which indicates that phosphorylation at S652 is important for Rph1 to dissociate from URSPHR1 in the presence of UV irradiation. These data support that chromatin association and dissociation of Rph1 on the PHR1 promoter mediated by protein phosphorylation is the major regulatory mechanism for PHR1 expression responding to DNA damage.Figure 6.

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