The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1

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.

Rph1/KDM4 Mediates Nutrient-Limitation Signaling that Leads to the Transcriptional Induction of Autophagy

“…A ChIP approach showed a 2-fold enrichment of Rph1-PA at the ATG7 promoter compared to Rph1 ΔZ -PA (Figure 5G). A similar enrichment was found at the PHR1 promoter, which was used as a positive control [30], but not at the non-coding region located at the tip of chromosome VI (ChrVI-260K), which was used as a negative control. This indicates that Rph1 directly binds the promoter region of ATG7 and that this binding is significantly reduced upon deletion of the zing-finger domains of the protein.…”

“…Previous studies showed that the transcriptional repression of some Rph1 targets is dependent on its histone demethylase activity [30]. We therefore proposed that Rph1 may repress ATG genes transcription, and thereby autophagy, in a similar manner.…”

“…Next, we analyzed the previously described Rph1 H235A mutant in which the replacement of histidine 235 with alanine inhibits the protein’s histone demethylase activity (Figure S3A,B; [30]). Protein analysis verified the stability of Rph1 H235A compared to wild-type Rph1 (Figure 5C).…”

Histone modifications in DNA damage response

“…Other studies show that Set2-dependent H3K36me3 is critical for appropriate resection and HR through recruiting repair factors CtIP (retinoblastoma binding protein 8), RPA (replication protein A1) and Rad51 (Jha and Strahl, 2014;Pfister et al, 2014). In the end, H3K36 demethylase Rph1 functions as a transcriptional repressor and inhibits the expression of DNA repair enzyme gene PHR1 and other stress-response genes by H3K36 demethylation, while DNA damage and environmental stress induce Rph1 phosphorylation and dissociation from chromatin to facilitate gene expression (Liang et al, 2011;Liang et al, 2013), indicating that H3K36 methylation is essential for DNA repair.…”

“…Additionally, the polycomb repressive complex 1, which contains Bmi1, Ring1, and Ring2, is required for H2A/H2AX ubiquitination (Bergink et al, 2006;Cao et al, 2005). Although the role of Ring1-mediated H2A ubiquitination in DNA damage response is not clear, Bmi1 and Ring2 are recruited to sites of DNA damage where they contribute to the monoubiquitylation of H2A/H2AX at Lys119/Lys120 (Ginjala et al, 2011;Pan et al, 2011). Bmi1/Ring2-dependent H2A/H2AX ubiquitination is critical for the localization of 53BP1, BRCA1 and other repair factors (Facchino et al, 2010;Ismail et al, 2010).…”